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Cell-Free Protein Expression and Functional Assay in Nanowell Chip Format
Abstract
The expression and characterization of large protein libraries requires high-throughput tools for rapid and cost-effective expression and screening. A promising tool to meet these requirements is miniaturized high-density plates in chip format, consisting of an array of wells with submicroliter volumes. Here, we show the combination of nanowell chip technology and cell-free transcription and translation of proteins. Using piezoelectric dispensers, we transferred proteins into nanowells down to volumes of 100 nL and successfully detected fluorescence using confocal laser scanning. Moreover, we showed cell-free expression of proteins on a nanoliter scale using commercially available coupled transcription and translation systems. To reduce costs, we demonstrated the feasibility of diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present an enzymatic inhibition assay in nanowells to anticipate further applications, such as the high-throughput screening of drug candidates or the identification of novel enzymes for biotechnology.
... I) Cell-free expression platforms are able to synthesize proteins from PCR-based templates without time-consuming cloning procedures, thereby facilitating high-throughput protein synthesis strategies (Beebe et al., 2011;Kanter et al., 2007;Zawada et al., 2011). Highthroughput methods are further favored by short reaction times and the scalability of reactions down to small volumes (Angenendt et al., 2004). As a result, cell-free protein synthesis has emerged as a valuable tool for the application in microarrays and biochips for biomedical research (Berrade et al., 2011;Nand et al., 2012;Whittaker, 2012). ...
... As cell-free protein expression gives the fastest access to the protein of interest, the established method may be a powerful tool for confirming data previously obtained from computational protocols. Since cell-free protein expression systems are scalable down to small volumes (Angenendt et al., 2004) and are thus high-throughput compatible (Whittaker, 2012), various putative ISPs obtained from the computational screening protocols may be analyzed in a parallel manner using in vitro expression techniques which saves time and costs (Katzen et al., 2005). ...
... Transcription-translation reactions of 25-50 µl were used throughout the work. However, referring to other cell-free expression systems, reactions potentially can be scaled up to 100 liter and downscaled to the nl range depending on the desired application (Angenendt et al., 2004;Swartz, 2006;Voloshin and Swartz, 2008;Zawada et al., 2011). Miniaturization of the CHO-based cell-free reactions may enable the implementation of the transcriptiontranslation reaction mixtures in protein microarrays and biochips which is more difficult to accomplish using cell-based approaches (Angenendt et al., 2004). ...
... These goals have been accomplished in the form of droplets, [21][22][23][24] protein-producing gel, 25 and microfluidic devices. 14,[26][27][28] With the droplets, a pseudo-filtration membrane is formed through oil-in-water emulsions, and the platform offers an ultra-high-throughput screening method because a large number of droplets can be easily and rapidly created. [21][22][23][24] For the protein-producing gel, the isolation of the genetic template and reaction solution is achieved through a hydrogel, and the large surface-area-to-volume ratio of the reaction vessel enhances protein synthesis yield. ...
... By scaling the devices for additional protein expression units, high-throughput protein synthesis is possible. 14,[26][27][28] In this work, we designed and fabricated two miniaturized array devices for continuous-exchange CFPS and investigated the effect of membrane orientation on protein synthesis yield. Compared with the devices reported previously, 14 the membrane in this work is oriented vertically in reference to the table surface to reduce or eliminate possible membrane clogging (due to possible sedimentation of large molecules such as aggregated proteins or ribosomes). ...
Cell-free protein synthesis (CFPS) has been used as an alternative to cell-based recombinant technology for protein production in academic and industrial labs. The continuous-exchange format generally has higher expression yield by constantly supplying a nutrient solution and removing inhibitory by-products through a porous membrane. Because of the concern of possible membrane clogging by large molecules in the CFPS solution, we investigated the effects of membrane orientation on protein synthesis. We fabricated a miniaturized array device called Vertical-I with its membrane oriented vertically in reference to the table surface and found that the protein synthesis yield in the Vertical-I device was 144% higher than the Horizontal Device reported previously. The reaction time was also faster; β-glucuronidase reached the synthesis yield plateau after 2 h in the Vertical-I device versus 4 h in the Horizontal Device. Possible clogging of membrane pores was confirmed by fluorescein diffusion measurement. Using these results, we designed a device called Vertical-II that would fit into a 96 well plate holder for compatibility with commercial reagent dispensers and microplate readers. The experimentally optimized device increased protein expression 406% over the Horizontal Device and consumes 5 times fewer reagents than a commercial device, showing the potential for high-throughput protein synthesis.
... His-tag [61] Large-scale synthesis of proteins suggested [12] Use of tags for large scale synthesis suggested [13] Cell-free synthesis in nanowells [22,23] Screening in nanowells [24] PISA Selfassembly Nanowells [14] [ as self-assembly template as described a year earlier by Weng et al. [17]. ...
... Historically seen, Takulapalli et al. have not been the first using nanowells. Already in 2004, Angenendt et al. [22] expressed GFP premixed with cell-free mix in 100 nL nanowells on a microstructured chip and detected it via fluorescence. But they did not immobilize the nascent proteins on the surface of their wells to generate microarrays. ...
Today, whole genomes are analysed by Next Generation Sequencing systems, or displayed by DNA microarray in situ synthesis. However, genomic data are mainly static and do not exactly reveal the complexity of protein-interaction networks of any organism or cell. To investigate protein interactions, the generation of protein microarrays, displaying whole proteomes is a prerequisite. But traditional protein microarray generation is time-consuming, costly and is restricted by a wide range of technical difficulties concerning cell culturing, protein purification and transfer onto microarray. Some of these obstacles can be bypassed by application of cell-free expression systems, enabling fast in situ synthesis of protein microarrays without need for cell culturing. This review provides a historical timeline of the different methods to generate protein microarrays by cell-free expression, highlights differences and similarities and reports the current state of the different approaches.This article is protected by copyright. All rights reserved
... The authors of this study demonstrated high-throughput screening of the enzyme β-galactosidase in nanowells and generated proteins in sufficient quantity for detection by confocal laser scanning. (Angenendt, Nyarsik et al. 2004). ...
... A couple of years after the development of the NAPPA method, a technique called MIST emerged ( Figure 4C). The authors applied a multiple spotting technique (Angenendt, Glökler et al. 2003) for the detection of the fluorescent signal and protein expression in nanoliter volume (Angenendt, Nyarsik et al. 2004). Later, they used this methodology to develop in situ synthesized protein arrays (Angenendt, Kreutzberger et al. 2006). ...
The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution, and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.
... Likewise, high yield production (≈2 mg/ml concentration) and purification of a single therapeutic dose of a model protein (superfolder GFP) can be achieved on portable microfluidic bioreactors in which nanoporous membrane were adopted for swift and continuous exchange of small molecules (i.e., nucleotides, inorganic phosphates) and waste products for on-demand, point-of-care applications (Timm, Shankles, Foster, Doktycz, & Retterer, 2016). Similarly, to envision a high-throughput instrument similar to that of protein microarrays, microfluidic devices coupled with CFPS can be adopted for characterization of the expression, immobilization, display, and screening of massive protein libraries in an agile and cost-effective manner (Angenendt et al., 2004). Moreover, "cell mimics" capable of controlling the flux of a natural cell can be replicated by coupling cell-free protein production systems inside nanoporous picolitre volume containers. ...
Flexibility in the exchange of genetic material takes place between different organisms of the same or different species. This phenomenon is known to play a key role in the genetic, physiological and ecological performance of the host. Exchange of genetic materials can cause both beneficial and/or adverse biological consequences. Horizontal gene transfer (HGT) or lateral gene transfer (LGT) as a general mechanism leads to biodiversity and biological innovations in nature. HGT mediators are among the genetic engineering tools for selective introduction of desired changes in the genome for gene/cell therapy purposes. HGT, however, is crucial in development, emergence and recurrence of various human-related diseases, such as cancer, genetic-, metabolic-, and neurodegenerative disorders and can negatively affect the therapeutic outcome, by promoting resistant forms or disrupting the performance of …
... We typically fill in the well with 80 µL of a feeding solution; this volume is again more than 2 orders of magnitude less than 10 mL of a feeding solution in RTS 500. The decrease in the volume of both reaction mix and feeding resolution will significantly reduce reagent consumption when using IVT for high-throughput assays 170 Although Figures 3-2 shows a 2 x 3 array device, an array of a higher number (e.g., 8 x 12) can be implemented as is readily appreciated by those in the field. As explained above, such an nested-well device has the following advantages: (1) less reagent consumption due to miniaturization; (2) higher protein expression yield due to incorporation of a mechanism for fluid manipulation; and (3) a potential to implement protein synthesis in a high-throughput format. ...
... Developing cell-free protein synthesis systems: a focus on mammalian cells Review mid, cultivation of positive colonies, plasmid isolation, sequencing, cell transfection and cell cultivation for protein production can be avoided. High-throughput CFPS methods are further favored by short reaction times and their potential for miniaturization and automation [60,61]. These benefits turn CFPS into a valuable tool to speed up the entire process and product development timeline. ...
Sophisticated cell-free protein synthesis (CFPS) systems have been developed as an alternative to recombinant expression in cultured cells. In this review, we present advances in the field of mammalian-based CFPS by highlighting recently established systems derived from mouse fibroblasts, HeLa, hybridoma, CHO and K562 cells. We further highlight ongoing challenges in the field of mammalian-based CFPS, such as the optimization of already established platforms and the development of novel systems in order to further increase protein yields and reduce manufacturing costs while facilitating the synthesis of a huge number of biologically active target proteins. Advances in mammalian-based CFPS shall expand the number of future applications of CFPS in the area of pharmaceutical research and development.
... The microarray technology was extended to allow ELISA to be performed in this high-density format using conventional hardware [6]. We have demonstrated cell-free protein expression in nanowell plates in chip format and have monitored the enzymatic activity of those in vitro expressed proteins [7]. We have previously demonstrated the use of proteins in proteomics [8] and in large-scale selection systems [9]. ...
External quality assessment schemes are evaluated by method-dependent consensus values or by method independent target values, determined with reference measurement procedures. The reference measurement procedures yield values having a high trueness and precision with a defined uncertainty of measurement. The great advantage of the reference measurement procedure is that the comparability of results in the different medical laboratories is promoted and improved intending in the same reference interval for an analyte in all laboratories. At the same time the manufacturers are motivated to calibrate their analytical systems in such a way, that the best comparability of values in the external quality assessment schemes is given. The European regulation in that field is the ''In-vitro-diagnostic medical device directive''. This directive gives an additional role to the external quality assessment schemes that is the vigilance of the market. The vigilance procedure of the diagnostic market intends to regis-trate and inform the manufacturers and the competent authorities of any incidence in the diagnostic system. To perform this vigilance procedure in an appropriate way a European standard has been developed (EN 14136). The interaction between EQAS-organizers the manufacturers and the routine laboratories will improve the quality of measurements in the medical laboratories in favour of the patients. One typical reference method measurement procedure will be presented to determine the concentration of digoxin and digitoxin in EQA-samples. The reference measurement procedures have already proved their significance within epidemiological studies.
... The rapidly widening gap between exploding sequence information and knowledge about the encoded proteins can be effectively filled when the "software" of cell-free protein synthesis is integrated with the "hardware" of microfluidic systems. Nanowell array formats are used to express individual proteins in extremely small reaction volumes (≤100 nL) [32,33]. The miniaturization of reaction volumes expands the throughput of protein synthesis and conserves costly compounds. ...
Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms.
... Glass MCPs are designed for high-sensitivity detection, including fluorescent and luminescent detection and scintillation counting, where extremely low backgrounds with no cross-talk are required; this material is chemically stable and biologically inert. These plates are excellent for evaporation and long-term storage [9,10,11]. Glass MCPs are suitable for LC with MS detection. ...
Microplates are routinely used in Radio- or Immuno-assays. Recently, microplates have found use not only in analytical but also in the pre-analytical phase in bioanalyses (sample storage, sample preparation). New connection of this technology to liquid chromatography could be economical, fast and simple solution for many routine laboratories handling large sequences of biological samples. This review summarises the application of microplates in bioanalytical laboratories. Different types of sorbents, materials and shapes of microplates are discussed, and the main advantages and disadvantages of microplates used in clinical research are presented.
... Cell-free transcription and translation system on solid surface has been developed and utilized in many genomic studies. [6][7][8] As a new research tool, cell-free gene expression has been applied to DNA-based biochips, which showed possibilities of developing synthetic biological systems and biochemical materials at nanoscale. [8][9][10][11] The biosynthetic reaction on surface of microchips was first realized by Buxboim et al. and they developed and utilized the microchips assembled by photolithographic approach on silicon dioxide (SiO2) for controlling the cell-free expression. ...
Figure 1. (A) Structure of SAMs on gold and chemical modifications to prepare template DNA-presenting biochips. The carboxylic acid- presenting monolayer was treated with 1-ethyl-3-(3-dimethylamino- propyl)carbodiimide hydrochloride (EDC) and N-aminoethyl male- imide, followed by thiolated template DNA. (B) Strategy for T7 RNAP activity assay on biochips and the sequences of oligonucleotides (T7 promoter sequences underlined) used in this study. In vitro synthesis of RNAs by bacteriophage T7 RNA poly- merase (T7 RNAP) has been applied extensively to the inves- tigation of RNA serving as a biologically active molecule. T7 RNAP is a single subunit enzyme with a molecular weight of 98 kDa that is capable of catalyzing transcription without any accessory proteins.
... He and Taussig [8] and He et al. [9] also used GFP to demonstrate the principle of PISA and DAPA for protein microarray development. In addition, Angenendt et al. [15] developed a platform that combined nanowell chip technology and cell-free protein expression using GFP and β-galactosidase as their proteins of choice. The recombinant B. pseudomallei OmpA protein was successfully expressed in E. coli by Hara et al. [16] and shown to be highly suitable as a serodiagnostic antigen for the tropical disease melioidosis using different diagnostic formats [17]. ...
Background
Protein microarrays have enormous
potential as in vitro diagnostic tools stemming from the ability to miniaturize whilst generating maximum evaluation of diagnostically relevant information from minute amounts of sample. In this report, we present a method known as repeatable arrays of proteins using immobilized DNA microplates (RAPID-M) for high-throughput in situ protein microarray fabrication. The RAPID-M technology comprises of cell-free expression using immobilized DNA templates and in situ protein purification onto standard microarray slides.
Results
To demonstrate proof-of-concept, the repeatable protein arrays developed using our RAPID-M technology utilized green fluorescent protein (GFP) and a bacterial outer membrane protein (OmpA) as the proteins of interest for microarray fabrication. Cell-free expression of OmpA and GFP proteins using beads-immobilized DNA yielded protein bands with the expected molecular sizes of 27 and 30 kDa, respectively. We demonstrate that the beads-immobilized DNA remained stable for at least four cycles of cell-free expression. The OmpA and GFP proteins were still functional after in situ purification on the Ni–NTA microarray slide.
Conclusion
The RAPID-M platform for protein microarray fabrication of two different representative proteins was successfully developed.
... The array was fabricated in polycarbonate by cold embossing (16 ). Similar arrays were recently reported for performing cellfree protein expression and functional assays of large sets of protein libraries (17 ). In vitro transcription and translation analysis before expression, as well as enzyme inhibition assays, were demonstrated. ...
Many cellular events occur at the nanoscale, and the desire to understand the mechanisms that control these systems is a core issue in life science research. Thomas Laurell, Johan Nilsson, and György Marko-Varga at Lund University (Sweden) explore the combination of dense arrays of nanovials with microfluidic platforms as a way to perform single-component analysis in biological fluids.
... More recently, the Felgner group has published a series of articles describing fabrication of protein microarrays in a variety of bacteria by directly spotting in vitro translated protein mixtures to glass (Crompton et al., 2010;Liang et al., 2011). However, although these systems can significantly decrease the reaction volume required for generation of recombinant proteins (Angenendt et al., 2004), the impurity of the translated proteins limits their applications. ...
The protein microarray technology provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput manner. It is viewed as a new tool that overcomes the limitation of DNA microarrays. On the basis of its application, protein microarrays fall into two major classes: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be directly spotted on a slide to form the so-called "reverse-phase" protein microarray. In the last decade, applications of functional protein microarrays in particular have flourished in studying protein function and construction of networks and pathways. In this chapter, we will review the recent advancements in the protein microarray technology, followed by presenting a series of examples to illustrate the power and versatility of protein microarrays in both basic and clinical research. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade.
... The types of a substrate for microarrays can be categorized according to the dimensionality of the surface (2-D or 3-D), and whether the binding of a ligand to the surface is covalent or occurred by means of a nonspecific adsorption. Nowadays, the two-dimensional (2-D) platforms are based on glass slides typically treated with organosilanes of different functionality (epoxy, amino or aldehyde) [2,3], thin films of non-porous synthetic polymers [4] or metals [5,6]. However, the functional properties of biosubstances used as complementary ligands strongly depend on their conformation and structure. ...
Macroporous monoliths with different surface functionalization (reactive groups) were utilized as platforms for DNA analysis in microarray format. The slides based on a copolymer glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) have been chosen as well known and thoroughly studied standard. In particular, this material has been used at optimization of DNA microanalytical procedure. The concentration and pH of spotting solution, immobilization temperature and time, blocking agent and coupling reaction duration were selected as varied parameters. The efficiency of analysis performed on 3-D monolithic platforms was compared to that established for commercially available glass slides. As a practical example, a diagnostic test for detection of CFTR gene mutation was carried out. Additionally, the part of presented work was devoted to preparation of aptamer-based test-system that allowed successful and highly sensitive detection both of DNA and protein.
... Thus, a 'nanotiter plate' has been developed containing 96 wells with a volume range from 100 to 1000 nL addressable by a piezo-nozzle. This microarray can be used in the same way as any 96-well plate in various cell assays, such as phenotypic characterization [5]. Another approach consists of printing cells onto a glass slide covered with cell-repulsive triethoxyaminopropylsilane to inhibit their adhesion to the slide, while a matrix of circular spots is left without a repulsive agent, allowing cells to specifically adhere in the spots and grow [6]. ...
To analyze the phenotypic consequences of perturbing mammalian cells with drugs, there is an increasing need for systematic cell-based assays in an HTS format. Cell microarrays provide an attractive solution as they offer more than a simple miniaturization and mechanization of conventional microtiter plates. While standard monolayer two-dimensional culture conditions are poor mimics of the cellular environment in situ, microfabricated systems enable three-dimensional organotypic cell cultures and have the potential to provide biological insight not achievable before. This article compares different cell microarray formats and evaluates their potential use in the drug discovery process.
... The IC 50 and Hill slope, especially after adjustment for molecular weight (SI Appendix, Fig. S9), were found to be in good agreement with the values obtained in microplate (a mean IC 50 of 2.04 versus 2.87 μM; a mean Hill slope of 1.07 versus 0.967; Fig. 2 B and C and SI Appendix, Table S2) and a published IC 50 (3.10 μM) (23). The precision of the IC 50 value was, however, found to be much higher in the microfluidic system than in a conventional eight-point microplate assay: For a single injection the 95% confidence interval was, on average, AE2.40% versus AE62.6% in microplate. ...
A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC(50) values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC(50) of 27 ± 0.83 μM.
... To achieve high-throughput protein expression and reduce reagent volumes and cost, CFPS is often performed in miniaturized or microfluidic devices (Angenendt et al., 2004;Agresti et al., 2010;Biyani et al., 2013;Fallah-Araghi et al., 2012;Hahn et al., 2007;Khnouf et al., 2010;Khnouf et al., 2009;Mei et al., 2007;Okano et al., 2012;Osaki et al., 2011;Park et al., 2009;Teh et al., 2011;Wu et al., 2009;Wu et al., 2009). However, in the CECF format, miniaturization is generally restricted due to the necessity for a porous membrane that allows passive chemical diffusion between reaction and feeding solutions. ...
Cell-free protein synthesis (CFPS), which entails synthesizing proteins outside of intact cells, is conducted in several formats with the continuous-exchange cell-free (CECF) format generally having the greatest protein expression yields. With this format, continuous chemical exchange occurs through a dialysis membrane separating a reaction solution from a feeding solution containing supplemental nutrient/energy molecules. Here, we describe the optimization of the miniaturized fluid array device (μFAD) by studying the effects of structural and experimental parameters responsible for the heightened chemical exchange across the dialysis membranes and enhanced protein expression capabilities of the high-throughput device. The interface area and number between the reaction and feeding solutions have a direct impact on protein expression, with a 1.6% enhancement in protein expression yield with each square millimeter increase in area and a 20% decrease with each additional interface. For nutrient/energy availability, an increasing solution volume ratio and height difference increase protein expression yield until the expression yield plateaus at a volume ratio of 20 to 1 (feeding to reaction solution) and a solution height difference of 2mm. This yield can be further increased by 7% every 30min with feeding solution replacement. Of the studied experimental factors (feeding solution stirring, device shaking, and temperature increase), feeding solution stirring has a significant effect on protein expression in this device. In the optimized system, green fluorescent protein (GFP), ß-glucuronidase (GUS), ß-galactosidase (LacZ), luciferase, and tissue plasminogen activator (tPA) expression increased 77.8-, 212-, 3.66-, 463-, and 5.43-fold, respectively, compared to the conventional batch format in a standard microplate. These results highlight the significance of structural/experimental conditions on the productive expression of proteins in the CECF format.
... The most popular cell-free systems are based in E. coli, wheat germ and rabbit reticulocytes extracts [4,5,27]. This platform has been applied in a number of proteomics-based studies including structural and functional proteomics [28,29,30], protein evolution [31], unnatural amino acids and protein labeling [32,33], protein interaction [34], diagnostics and therapeutics [35,36] and protein microarrays [15,37,38,39]. The protein microarray platform, exploiting antigen-specific antibodies present in plasma or sera from exposed or immunized animals or humans, has been of particular interest to our laboratory to identify potential target antigens for malaria vaccine development [10,11,40]. ...
Vaccine development against malaria and other complex diseases remains a challenge for the scientific community. The recent elucidation of the genome, proteome and transcriptome of many of these complex pathogens provides the basis for rational vaccine design by identifying, on a proteome-wide scale, novel target antigens that are recognized by T cells and antibodies from exposed individuals. However, there is currently no algorithm to effectively identify important target antigens from genome sequence data; this is especially challenging for T cell targets. Furthermore, for some of these pathogens, such as Plasmodium, protein expression using conventional platforms has been problematic but cell-free in vitro transcription translation (IVTT) strategies have recently proved successful. Herein, we report a novel approach for proteome-wide scale identification of the antigenic targets of T cell responses using IVTT products.
We conducted a series of in vitro and in vivo experiments using IVTT proteins either unpurified, absorbed to carboxylated polybeads, or affinity purified through nickel resin or magnetic beads. In vitro studies in humans using CMV, EBV, and Influenza A virus proteins showed antigen-specific cytokine production in ELIspot and Cytometric Bead Array assays with cells stimulated with purified or unpurified IVTT antigens. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost showed antigen-specific cytokine production using purified IVTT antigens only. Overall, the nickel resin method of IVTT antigen purification proved optimal in both human and murine systems.
This work provides proof of concept for the potential of high-throughput approaches to identify T cell targets of complex parasitic, viral or bacterial pathogens from genomic sequence data, for rational vaccine development against emerging and re-emerging diseases that pose a threat to public health.
... Likewise, high yield production (≈2 mg/ml concentration) and purification of a single therapeutic dose of a model protein (superfolder GFP) can be achieved on portable microfluidic bioreactors in which nanoporous membrane were adopted for swift and continuous exchange of small molecules (i.e., nucleotides, inorganic phosphates) and waste products for on-demand, point-of-care applications (Timm, Shankles, Foster, Doktycz, & Retterer, 2016). Similarly, to envision a high-throughput instrument similar to that of protein microarrays, microfluidic devices coupled with CFPS can be adopted for characterization of the expression, immobilization, display, and screening of massive protein libraries in an agile and cost-effective manner (Angenendt et al., 2004). Moreover, "cell mimics" capable of controlling the flux of a natural cell can be replicated by coupling cell-free protein production systems inside nanoporous picolitre volume containers. ...
Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab‐on‐chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell‐free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell‐cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell‐free protein synthesis formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed. This article is protected by copyright. All rights reserved.
... [27][28][29] In a structured PDMS flow cell containing the DNA microarray in cavities, spatial resolution and smallest spot size would be limited only by the capability of the IVTT mix to express proteins in very small volumes (nl to pl) and the technical limits of the spotting procedure. [30] This kind of approach would also lead to clear spot edges and minimize crosscontamination in case of the Halo-tag (as this enzyme is inactive at low temperatures and would not bind upon opening in cooled environment). In addition, a standard Nanowell NAPPA performed in such a PDMS cavity chip sealed with a HaloBind SCORE slide during expression could provide both a NAPPA array and a PMaC microarray copy in a single step. ...
Protein microarrays are essential to understand complex protein interaction networks. Their production, however, is a challenge rendering this technology unattractive for many laboratories. Recent developments in cell‐free protein microarray generation offer new opportunities, but are still expensive and cumbersome in practice. Here we describe a cost‐effective and user‐friendly method for the cell‐free production of protein microarrays. From a PDMS flow cell containing an expressible DNA microarray, proteins of interest are synthesised via cell‐free expression and then immobilized on a capture surface. The resulting protein microarray can be regarded as a “copy” of the DNA microarray. 2x6His‐ and Halo‐tagged fluorescent reference proteins were used to demonstrate the functionality of Ni‐NTA and Halo‐bind surfaces in this “copy” system. The described process can be repeated several times using the same DNA microarray. The identity and functionality of the proteins was proven, during the copy process by their fluorescence and on the surface via a fluorescent immune assay. Also, single color reflectometry (SCORE) was applied to show that on such copied arrays real‐time binding kinetic measurements are possible.
... It has been used for microelectromechanical systems (MEMS)-fabrication, [1][2][3] microfluidic-device design [4,5] bioscaffold creation, [6] or high-throughput screening efforts. [7][8][9][10][11] It is usually accomplished by applying liquids via a highly controlled method such as contact or noncontact printing, [12][13][14][15][16] prestructured surfaces, [17][18][19] micropatterning through thin liquid film instabilities, [20,21] or wettability patterning such as superhydrophobic-hydrophilic patterns. [22] The latter example enables the formation of patterns of aqueous solutions using the effect of discontinuous dewetting process. ...
Here a method is demonstrated to pattern liquids of varying surface tension and composition into droplets by utilizing slippery liquid‐infused surfaces prepared on chemically‐patterned substrates. The capability of different liquids to displace the lubricant from higher surface energy regions is studied and it is shown that both high and low surface tension liquids can imbibe the polymer, thereby forming droplets sharply following underlying surface energy patterns. For all liquids tested, droplet arrays of arbitrary shapes of each liquid are formed with precision down to 50 µm. By changing the chemical patterning from fluorinated to aliphatic groups, patterns of mineral and silicone oils are created. Finally, formation of 2D micropatterns of three‐phase liquid systems—fluorinated, organic, and aqueous phases—is demonstrated.
... Mostly, cell-free reactions are performed in batch, fed-batch or continuous exchange mode [4], whereas coupled systems combining translation and transcription yield highest protein amounts, but require the addition of RNA polymerases encoded by bacteriophages [5]. Since the first description of cellfree protein synthesis by Nirenberg and Matthaei [1] various applications have evolved, including the synthesis of therapeutics and pharmaceutical proteins, membrane proteins and virus-like particles [2,[5][6][7][8][9]. By now, cell-free protein synthesis has been optimized to allow manufacturing scale protein production [2] as well as high throughput production of protein libraries in the nanoliter scale [10]. ...
Cell-free systems exploit the transcription and translation machinery of cells from different origins to produce proteins in a defined chemical environment. Due to its open nature, cell-free protein production is a versatile tool to introduce specific labels such as heavy isotopes, non-natural amino acids and tags into the protein while avoiding cell toxicity. In particular, radiolabelled peptides and proteins are valuable tools for the functional characterization of protein-protein interactions and for studying binding kinetics. In this study we evaluated cell-free protein production for the generation of radiolabelled ligands for G protein-coupled receptors (GPCRs). These receptors are seven-transmembrane-domain receptors activated by a plethora of extracellular stimuli including peptide ligands. Many GPCR peptide ligands contain disulphide bonds and are thus inherently difficult to produce in bacterial expression hosts or in Escherichia coli-based cell-free systems. Here, we established an adapted E. coli-based cell-free translation system for the production of disulphide bond-containing GPCR peptide ligands and specifically introduce tritium labels for detection. The bacterial oxidoreductase DsbA is used as a chaperone to favour the formation of disulphide bonds and to enhance the yield of correctly folded proteins and peptides. We demonstrate the correct folding and formation of disulphide bonds and show high-affinity ligand binding of the produced radio peptide ligands to the respective receptors. Thus, our system allows the fast, cost-effective and reliable synthesis of custom GPCR peptide ligands for functional and structural studies.
... To address this issue, Angenendt et al. printed cDNA and expressed the proteins in nanowells using piezoelectric dispensers [31]. Takulapalli et al. demonstrated the fabrication of high-density cell-free protein arrays by combining photolithographicallyetched silicon nanowells (n=8,000/slide), NAPPA, and a piezo-inkjet printer [21]. ...
Rationale: Cell-free protein microarrays display naturally-folded proteins based on just-in-time in situ synthesis, and have made important contributions to basic and translational research. However, the risk of spot-to-spot cross-talk from protein diffusion during expression has limited the feature density of these arrays.
Methods: In this work, we developed the Multiplexed Nucleic Acid Programmable Protein Array (M-NAPPA), which significantly increases the number of displayed proteins by multiplexing as many as five different gene plasmids within a printed spot.
Results: Even when proteins of different sizes were displayed within the same feature, they were readily detected using protein-specific antibodies. Protein-protein interactions and serological antibody assays using human viral proteome microarrays demonstrated that comparable hits were detected by M-NAPPA and non-multiplexed NAPPA arrays. An ultra-high density proteome microarray displaying > 16k proteins on a single microscope slide was produced by combining M-NAPPA with a photolithography-based silicon nano-well platform. Finally, four new tuberculosis-related antigens in guinea pigs vaccinated with Bacillus Calmette-Guerin (BCG) were identified with M-NAPPA and validated with ELISA.
Conclusion: All data demonstrate that multiplexing features on a protein microarray offer a cost-effective fabrication approach and have the potential to facilitate high throughput translational research.
... Furthermore, applications of microdevices also include working with cell cultures and organs-on-a-chip, reducing the need for animal models and in vivo studies. Due to the diverse abilities of microchip technology, the capability of integrating conventional Polystyrene microchip as support to culture medium [174] Protein expression Transcription and translation of proteins inside nanowells, in a cell-free process 96-well glass microplates as support [175] Protein separation and analysis Isolation and enrichment of proteins on a 2-D capillary electrophoresis microchip PMMA multi-dimensional CE microchip [146] Pharmacokinetics and pharmacodynamics Evaluation of pharmacokinetic parameters of fluorescently labeled human mAb in serum Caliper LabChip GX commercial system [122] analytical and proteomic tools in a single system (gene isolation, DNA amplification, cell culture, protein expression, protein separation and analysis, pharmacokinetics, and pharmacodynamics e Table 5) meets the requirements for a quality-control system for recombinant drugs. Various applications brought by microchip technology are already being applied by various research groups; however, for recombinant drugs-on-chips to become a reality, it is necessary that the features mentioned herein be applied in a single device, thus creating one powerful and complete analytical tool for the recombinant drug industry. ...
We present here a critical review covering conventional analytical tools of recombinant drug analysis and discuss their evolution towards miniaturized systems foreseeing a possible unique recombinant drug-on-a-chip device. Recombinant protein drugs and/or pro-drug analysis require sensitive and reproducible analytical techniques for quality control to ensure safety and efficacy of drugs according to regulatory agencies. The versatility of miniaturized systems combined with their low-cost could become a major trend in recombinant drugs and bioprocess analysis. Miniaturized systems are capable of performing conventional analytical and proteomic tasks, allowing for interfaces with other powerful techniques, such as mass spectrometry. Microdevices can be applied during the different stages of recombinant drug processing, such as gene isolation, DNA amplification, cell culture, protein expression, protein separation, and analysis. In addition, organs-on-chips have appeared as a viable alternative to testing biodrug pharmacokinetics and pharmacodynamics, demonstrating the capabilities of the miniaturized systems. The integration of individual established microfluidic operations and analytical tools in a single device is a challenge to be overcome to achieve a unique recombinant drug-on-a-chip device.
This chapter starts with general considerations on wound healing; the main cells and their structures and functions; and methods of in vitro and in vivo evaluation. Implants with thin calcium phosphate (CaP) coatings can enhance or inhibit wound healing mechanisms on various levels of gene expression and protein production. Therefore, some techniques to elucidate the material and tissue interactions are mentioned. Bone development, the various types of bone, and bone fracture healing are presented. Thin CaP coatings of implant material can be investigated in in vitro test systems using primary cells or cell lines in culture. More reliable results can be expected from in vivo investigations with animal models. Important results of such investigations are presented. The prediction of the clinical performance of thin CaP coatings on the basis of in vitro and in vivo models is limited. Therefore, retrieval studies of surgically explanted or postmortem uncovered implants should be obligatory.
The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.
Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide.
Spot on! Cell-free protein expression on surfaces can be implemented in biosensors and in microfluidic devices like that shown in the picture. Here, proteins are generated and immobilized successively on separated spots in a microfluidic reactor. This approach opens up novel opportunities for basic and applied biomedical research.
Capturing and detecting viruses require specialized platforms and analysis techniques. The atomic force microscope (AFM) offers
not only tremendously high spatial and topographic resolution, but also allows localizing single molecules and determining
their interaction on a molecular level. AFM makes possible (a) generating nanopatterned surfaces, (b) detecting protein and
viral pathogen captured in these specific regions over time, and (c) gathering information about binding parameters. A section
of this chapter describes, for instance, the measurement of kinetic parameters governing the first step of viral infection
– the attachment of a human rhinovirus to a cell – at the single-molecule level. We review the principles of AFM and its applications
in the analysis of nanopatterned surfaces and protein arrays, in the detection of proteins and viruses, and in the characterization
of the interactions of these viruses with their cognate receptors under physiological conditions.
Natural genetic promoters are regulated by multiple cis and trans regulatory factors. For quantitative studies of these promoters, the concentration of only a single factor is typically varied to obtain dose response or transfer function of the promoters with respect to the factor. Such design of experiments has limited our ability to understand quantitative, combinatorial interactions between multiple regulatory factors at promoters. The limitation is primarily due to the intractable number of experimental combinations that arise from multifactorial design of experiments. To overcome this major limitation, we integrate impact printing and cell-free systems to enable multi-dimensional studies of genetic promoters. We first present a gradient printing system which comprises parallel piezoelectric cantilever beams as a scalable actuator array to generate droplets with tunable volumes in the range of 100pL – 10nL, which facilitates highly accurate direct dilutions in the range of 1 – 10,000 fold in a 1µL drop. Next, we apply this technology to study interactions between three regulatory factors at a synthetic genetic promoter. Finally, a mathematical model of gene regulatory modules is established using the multi-parametric and multi-dimensional data. Our work creates a new frontier in the use of cell-free systems and droplet printing for multi-dimensional studies of synthetic genetic constructs.
The ability of detecting multiple analyses simultaneously is an important advantage of protein sensors. But the denaturation of proteins on surfaces is still a challenge. In this article, a protein biosensor providing high selectivity is introduced. This approach is based on DNA-directed immobilization(DDI) method, in which protein-DNA conjugates are immobilized on surfaces by DNA hybridization. Nonspecific binding is reduced by applying fluorescence resonance energy transfer and scanning potential hairpin denaturizing technologies. Excellent discrimination on different recognition patterns is obtained.
The human genome contains ∼30,000 genes, but it is proposed that these genes could encode up to a million different proteins. Alternative splicing of genes results in the same gene encoding for multiple proteins that can then undergo further transformation via various posttranslational modifications – it is a combination of these two processes that could lead to diversity in the proteins produced. Characterization of the interaction of proteins with each other, DNA and ligands remains an enormous challenge, particularly for traditional techniques that typically enable resolution of the interactions of a single protein. It is not surprising that the technologies that facilitate the direct and concurrent probing of all (or a significant subset of) the proteins of an organism have generated considerable interest from researchers and pharmaceutical companies alike. This review highlights new technologies available for the study of protein function, with a particular focus on the applications of recombinant protein arrays.
Protein microarrays is a technology with great promise for high-throughput proteomics. Designing high-performance protein microarrays for global proteome analysis has, however, turned out to be challenging. To this end, major efforts are under way to design novel array formats capable of harboring the tremendous range of probes required to target complex proteomes composed of more than 10000 analytes. By adopting nanotechnology, the first generation of miniaturized nanoarrays has recently emerged, which opens up new avenues for global proteome analysis and disease proteomics. This review describes the progress and key issues in designing miniaturized protein arrays.
Cell-based protein production has low throughput and restricted flexibility in process design. For the development of a rapid platform for translational analysis of genetic sequences by bypassing time- and labor-consuming steps of cell-based gene expression methods, we attempted in this study to combine the techniques of gel electrophoresis and cell-free protein synthesis in a streamlined manner. After an electrophoretic separation of the DNAs of varying sizes, DNAs in the gel matrices were directly incubated in a reaction mixture for cell-free protein synthesis, which led to successful expression of functional proteins.
IntroductionChallenges for Extending Batch Duration and ProductivityScale-up of Reactions not Requiring Oxygen in Batch ModeScale-up of Reactions Requiring OxygenConclusions and ProjectionsReferences
Proteomics continues to grow in relevance and technical complexity as its importance in the investigation and understanding of protein expression in disease and drug development is recognized. Advances in proteomics have been supported by the continued expansion and availability of pharmacologically relevant genome information and driven by numerous and diverse technological and bioinformatic developments that underscore the complexity of the proteome. This chapter addresses some of the various technologies included in proteomic approaches, and provides salient examples where these technologies have been applied to pharmacology and/or the process of drug development.
Novel approaches in cell based and cell free micro reactors promise a new generation of high quality, high purity and on top personalized synthesis of drugs like antibiotics and functional proteins. These concepts are - however-relying on very specific conditions, under which the cells need to work and therefore require advanced sensing for e.g. ionic content, pH value and temperature. Also, the creation of product itself needs to be monitored to extract the valuable drug from the reactor, before side reactions start to deteriorate quality and yield. Microfluidics has come a long way since, resulting in advanced Point of Care devices nowadays, which can run complex protocols/1/. However, these reactions are time based and the process conditions are fixed and not monitored at all. To leverage the capability to drug production, monitoring sensors need to be integrated into the fluidic system, creating a complex electronic-optical microfluidic device. Exemplary techniques for the integration of sensors are provided in this paper, technological approaches and experimental results are given.
The sections in this article are
Drug discovery is a complex, multistep process, in which many challenges need to be overcome at each stage, from the discovery of a biomolecular target to the ensuring of the efficacy and safety of a compound in humans. Today's analytical methods allow tens of thousands of drug candidates to be screened for their ability to inhibit specific enzymes and the miniaturization of these approaches is highly desirable, accelerating the drug discovery process and reducing the associated costs. Herein, it is reviewed the miniaturized techniques currently used to evaluate enzymatic activity and inhibition giving special attention to microplate formats, microarrays, nanoarrays, and microfluidic technologies. It is, also, highlighted some of the characteristics and their abilities for potential uses are compared and discussed. In addition, the challenges of their applications in diagnosis, analysis, and therapy, which should help to improve the quality of healthcare globally are also pointed out.
We present a technology for the production of target proteins using novel cell-free systems derived from cultured human K562 cells and Chinese hamster ovary (CHO) cells. The protocol includes the cultivation of cells, the preparation of translationally active lysates, and the cell-free synthesis of desired proteins. An efficient expression vector based on the internal ribosome entry site (IRES) from the intergenic region (IGR) of the cricket paralysis virus (CrPV) was constructed for both systems. The coupled batch-based platforms enable the synthesis of a broad range of target proteins such as cytosolic proteins, secreted proteins, membrane proteins embedded into endogenous microsomes, and glycoproteins. The glycosylation of erythropoietin demonstrates the successful performance of posttranslational modifications in the novel cell-free systems. Protein yields of approximately 20 μg/ml (K562-based cell-free system) and 50 μg/ml (CHO-based cell-free system) of active firefly luciferase are obtained in the coupled transcription-translation systems within 3 h. As a result, both cell-free protein synthesis systems serve as powerful tools for high-throughput proteomics.
Because of strong demands for high throughput or high content cell-based assay, significant efforts have focused on the assay miniaturization by fabricating cell microarray using a variety of cell patterning techniques such as spotting, photolithography or soft lithography and by integrating cell microarray into microfluidic devices. Response of cells cultured on microarray can be monitored by using either electrochemical or optical detection methods. Impedancebased detection and potential-based detection have been widely used for electrochemical detection, while optical detection relies mostly on the fluorescence and bioluminescence-based techniques. Resultant cell microarray-based biosensor can be applied for high throughput/content drug screening and detection of pathogens, pollutants and warfare agents. For the successful application of cell-based biosensors to various areas, multi-phenotypic cell microarray should be developed and cells on microarray must be cultured in 3-dimensional environment as they do in real tissue to obtain accurate response of cells against target analytes.
We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.
We have developed a method for detecting toxins that inhibit protein synthesis. Biological synthesis of a protein starts from a gene to a messenger RNA and to a protein. This process can be implemented in a cell-free medium in a microfluidic array device. The device is comprised of reaction chambers for protein expression and feeding chambers as nutrient reservoirs. The device also incorporates dialysis membranes and microfluidic channels to connect chambers for supplying nutrients continuously and removing the reaction byproducts. To detect a toxin, a group of proteins are simultaneously synthesized in the array. The production yields of these proteins are inhibited differentially by the toxin. The toxin can thus be identified based on the unique response pattern (or signature) of the array. Three proteins have been synthesized in the device for the feasibility demonstration. The limit of detection for ricin, a bioterrorism agent, is determined at 10 pM.
The first goal of this thesis was to investigate the potential of two cationic polysaccharides (PQ-4 and PQ-10) for DNA delivery. We have shown that, compared to PEI based polyplexes, they were less efficient in transfecting cells. However, as they had very low toxicity, further tailoring of the nature and extent of cationic side chains on cationic hydroxyethylcellulose may be a promising avenue to further enhance their DNA delivery properties. As a second goal we investigated the applications of digitally encoded microcarriers for cell based assays. We succeeded to show that encoded microcarriers were suitable to grow cells on. Neither the coating at the surface of the beads (which facilitates the growth of the cells), nor the cells themselves hampered the decoding of the beads, even when the cells covering the microcarriers exhibited green or red fluorescence due to the expression of GFP and RFP respectively. We were able (a) to immobilize DNA, siRNA or adenoviral particles on the surfaces of the encoded microcarriers by the use of polyelectrolytes and, subsequently, (b) to grow cells on top of the nucleic acids/adenoviral particles. The DNA and siRNA immobilized on the surface of the microcarrier were not able to transfect cells. However, we showed that the cells growing on the polyelectrolyte layer could indeed become transduced with adenoviral particles hosted by the polyelectrolyte layer. In conclusion, a proof of principal to use photophysically encoded microcarriers as transfected microarray has been shown. As a third goal we investigated the use of digitally encoded microcarriers as tool to combat counterfeiting of tablets. We showed that the codes in the Memobeads in tables produced by granulation did not deform during tabletting and that the code in the beads remained readable. We also found evidence that, after oral intake, the encoded microparticles are highly unlikely toxic to humans.
A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed
robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes.
Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled
detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression
measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.
In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition.
Thousands of genes are being discovered for the first time by sequencing the genomes of model organisms, an exhilarating reminder that much of the natural world remains to be explored at the molecular level. DNA microarrays provide a natural vehicle for this exploration. The model organisms are the first for which comprehensive genome-wide surveys of gene expression patterns or function are possible. The results can be viewed as maps that reflect the order and logic of the genetic program, rather than the physical order of genes on chromosomes. Exploration of the genome using DNA microarrays and other genome-scale technologies should narrow the gap in our knowledge of gene function and molecular biology between the currently-favoured model organisms and other species.
Many new gene products are being discovered by large-scale genomics and proteomics strategies, the challenge is now to develop high throughput approaches to systematically analyse these proteins and to assign a biological function to them. Having access to these gene products as recombinantly expressed proteins, would allow them to be robotically arrayed to generate protein chips. Other applications include using these proteins for the generation of specific antibodies, which can also be arrayed to produce antibody chips. The availability of such protein and antibody arrays would facilitate the simultaneous analysis of thousands of interactions within a single experiment. This chapter will focus on current strategies used to generate protein and antibody arrays and their current applications in biological research, medicine and diagnostics. The shortcomings of these approaches, the developments required, as well as the potential applications of protein and antibody arrays will be discussed.
Rapid Translation System (RTS) is a cell-free protein production system employing an enhanced Escherichia coli lysate to perform coupled in vitro transcription-translation reactions. A continuous supply of energy substrates, nucleotides and amino acids combined with the removal of by-products guarantees a high yield of protein production. The gene to express is either cloned into a plasmid vector or introduced as a PCR product amenable to automation. The main property of this alternative system to cellular expression systems is its open design allowing direct manipulation of the reaction conditions and applications that are impossible or difficult in cell-based systems. RTS offers new promising possibilities in the postgenomic era.
In an earlier report (Litborn, E., Emmer, Å., Roeraade, J., Anal. Chim. Acta 1999, 401, 11—19), we described a technique for performing chemistry in chip-based vials. A major problem, solvent evaporation, was partially remedied by using a closed humidity chamber. In this paper we report an improved technique for performing parallel reactions in open, 15 nL volume, chip-based vials. The evaporation of solvent from the reaction fluid was continuously compensated by addition of solvent via an array of microcapillaries. The suitability of the method was demonstrated by performing eight separate peptide maps of myoglobin in parallel, using the three enzymes trypsin, α-chymotrypsin and endoproteinase Glu-C. The total amount of myoglobin utilized to perform the eight digests was less than 100 pmol. The corresponding amount of enzymes was ca. 0.1 pmol per reaction. In order to evaluate the operating limits of the technique, a study of the evaporation of solvents from a series of vials with proportionally smaller volumes operated at different temperatures was performed. The results showed that the concept for continuous compensation of solvent evaporation should be applicable to reaction volumes down to 30 pL.
The transition from slow, manual, low-throughput screening to industrialized robotic ultra-high throughput screening (uHTS) in the past few years has made it possible to screen hundreds of thousands of chemical entities against a biological target in a short time-frame. The need to minimize the cost of screening has been addressed primarily by reducing the volume of sample to be screened. This, in turn, has resulted in the miniaturization of HTS technology as a whole. Miniaturization requires new technologies and strategies for compound handling, assay development, assay adaptation, liquid handling and automation in addition to refinement of the technologies used for detection systems and data management. This review summarizes current trends in the field of uHTS and illustrates the technological developments that are necessary to enable the routine application of miniaturized uHTS systems within an industrial environment.
The current regulatory position of the Food and Drug Administration is discussed with regard to the approval of racemates and pure stereoisomers. Circumstances in which stereochemically sensitive analytical methods are necessary to ensure the safety and efficacy of a drug are described. Regulatory guidelines are interpreted for applications for the approval of a pure enantiomer in which the racemate is marketed, for the approval of either a racemate or a pure enantiomer in which neither is marketed, and for clinical investigations to compare the safety and efficacy of a racemate and its enantiomers. Examples of the basis for such regulation are drawn from historical situations (thalidomide, benoxaprofen) as well as currently marketed drugs (arylpropionic acids, disopyramide, indacrinone).
A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer [including amino acids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP)] through the reaction mixture and a continuous removal of a polypeptide product. Both prokaryotic (Escherichia coli) and eukaryotic (wheat embryos, Triticum sp.) versions of the system have been tested. In both cases the system has proven active for long times, synthesizing polypeptides at a high constant rate for tens of hours. With the use of MS2 phage RNA or brome mosaic virus RNA 4 as templates, 100 copies of viral coat proteins per RNA were synthesized for 20 hours in the prokaryotic or eukaryotic system, respectively. With synthetic calcitonin messenger RNA, 150 to 300 copies of calcitonin polypeptide were produced per messenger RNA in both types of continuous translation systems for 40 hours.
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.
Next Section
Partial or even complete cancer regression can be achieved in some patients with current cancer treatments. However, such initial responses are almost always followed by relapse, with the recurrent cancer being resistant to further treatments. The discovery of therapeutic approaches that counteract relapse is, therefore, essential for advancing cancer medicine. Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug sensitivity, and their potential to metastasize and cause relapse. Indeed, hypermalignant cancer cells, termed cancer stem cells or stemness-high cancer cells, that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types. Moreover, such stemness-high cancer cells are resistant to conventional chemotherapy and radiation. Here we show that BBI608, a small molecule identified by its ability to inhibit gene transcription driven by Stat3 and cancer stemness properties, can inhibit stemness gene expression and block spherogenesis of or kill stemness-high cancer cells isolated from a variety of cancer types. Moreover, cancer relapse and metastasis were effectively blocked by BBI608 in mice. These data demonstrate targeting cancer stemness as a novel approach to develop the next generation of cancer therapeutics to suppress cancer relapse and metastasis.
An automated enzyme assay was performed within a microfabricated channel network. Precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. Reagent dilution and mixing were controlled by regulating the applied potential at the terminus of each channel, using voltages derived from an equivalent circuit model of the microchip. The enzyme beta-galactosidase (beta-Gal) was assayed using resorufin beta-D-galactopyranoside (RBG), a substrate that is hydrolyzed to resorufin, a fluorescent product. Reaction kinetics were obtained by varying the concentration of substrate on-chip and monitoring the production of resorufin using laser-induced fluorescence. Derived Michaelis--Menten constants compared well between an on-chip and a conventional enzyme assay. Bias in the derived K(m) and kcat was primarily due to the limited solubility of RBG and the associated lack of measurements at substrate concentrations exceeding the K(m). A Ki of 8 microM for the inhibitor phenylethyl beta-D-thiogalactoside (PETG) was determined from plots of initial rate versus substrate concentration obtained at three concentrations of PETG. The relative inhibition of beta-Gal by lactose, p-hydroxymercuribenzoic acid, and PETG was determined by varying the inhibitor concentration with constant enzyme and substrate concentration. An enzyme assay performed on the microchip within a 20-min period required only 120 pg of enzyme and 7.5 ng of substrate, reducing the amount of reagent consumed by 4 orders of magnitude over a conventional assay.
Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.
At any time in vitro or in vivo expressed unlabeled proteins have to be quantified it is difficult to find a reliable method, especially with nonpurified samples. Quantification via protein activity can result in too low levels if the proteins analyzed tend to aggregate into inactive forms. Here, wild-type green fluorescent protein (GFPwt) was expressed in high amounts in vitro using the Rapid Translation System 500 based on Escherichia coli lysates. Fluorescent activity was determined in dependence of oxygen and compared to total protein levels. In the presence of low amounts of oxygen only 16% of the whole GFPwt amounts were detectable via determination of fluorescence activity. A reliable method to easily quantify whole protein levels even without specific antibodies and without purification steps by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining is described.
With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.
The system-wide study of proteins presents an exciting challenge in this information-rich age of whole-genome biology. Although traditional investigations have yielded abundant information about individual proteins, they have been less successful at providing us with an integrated understanding of biological systems. The promise of proteomics is that, by studying many components simultaneously, we will learn how proteins interact with each other, as well as with non-proteinaceous molecules, to control complex processes in cells, tissues and even whole organisms. Here, I discuss the role of microarray technology in this burgeoning area.
Historically, biotechnology has missed up to 99% of existing microbial resources by using traditional screening techniques. Strategies of directly cloning 'environmental DNA' comprising the genetic blueprints of entire microbial consortia (the so-called 'metagenome') provide molecular sequence space that along with ingenious in vitro evolution technologies will act synergistically to bring a maximum of available sequence-space into biocatalytic application.
Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.
The enantioselectivity of lipase from Burkhorderia cepacia KWI-56 has been inverted using a novel in vitro technique for construction and screening of a protein library by single-molecule DNA amplification by PCR followed by in vitro coupled transcription/translation system termed single-molecule-PCR-linked in vitro expression (SIMPLEX). Four amino acid residues (L17, F119, L167, and L266) in the hydrophobic substrate-binding pocket of the lipase were selected for mutation based on a structural model of a substrate-enzyme complex, and a combinatorial mutation library was constructed by SIMPLEX and screened for (R) and (S)-configurations of p-nitrophenyl 3-phenylbutyrate. Some combinations of amino acid substitutions in the four positions of the lipase were found as effective for changing the enantiopreference from the (S)-form substrate to the (R)-form. Two variants were expressed in the original host cells and purified to homogeneity, showing completely reversed enantioselectivity for the (R)-form of ethyl 3-phenylbutyrate (selectivity factor E(R)=38 or 33), whereas the wild-type lipase was (S)-selective (selectivity factor E(S)=33). Thus the semi-rational and semi-random combinatorial design of a mutant library followed by a high-throughput screening based on their enzymatic activity should be a powerful tool to engineer the enantioselectivity of enzymes.
Following the age of genomics having sequenced the human genome, interest is shifted towards the function of genes. This new age of proteomics brings about a change of methods to study the properties of gene products on a large scale. Protein separation technologies are now applied to allow high-throughput purification and characterisation of proteins. Two-dimensional-gel electrophoresis (2DE) and mass spectrometry (MS) have become widely used tools in the field of proteomics. At the same time, protein and antibody microarrays have been developed as successor of DNA microarrays to soon allow the proteome-wide screening of protein function in parallel. This review is aimed to introduce this new technology and to highlight its current prospects and limitations.
The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240,000 molecules) of analyte.