Article

The past, present and future of cell-free protein synthesis.

Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA.
Trends in Biotechnology (Impact Factor: 9.66). 04/2005; 23(3):150-6. DOI: 10.1016/j.tibtech.2005.01.003
Source: PubMed

ABSTRACT Recent technical advances have revitalized cell-free expression systems to meet the increasing demands for protein synthesis. Cell-free systems offer several advantages over traditional cell-based expression methods, including the easy modification of reaction conditions to favor protein folding, decreased sensitivity to product toxicity and suitability for high-throughput strategies because of reduced reaction volumes and process time. Moreover, improvements in translation efficiency have resulted in yields that exceed a milligram of protein per milliliter of reaction mix. We review the advances on this expanding technology and highlight the growing list of associated applications.

0 Bookmarks
 · 
106 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The human liver and lymph node sinusoidal endothelial cell C-type lectin (hLSECtin), a type II integral membrane protein, containing a Ca(2+)-dependent carbohydrate recognition domain (CRD), has a well-established biological activity, yet its three-dimensional structure is unknown due to low expression yields and aggregation into inclusion bodies. Previous study has demonstrated that the HIV-1 virus-encoded Tat peptide ('YGRKKRRQRRR') can increase the yields and the solubility of heterologous proteins. However, whether the Tat peptide could promote the high-yield and soluble expression of membrane proteins in Escherichia coli is not known. Therefore, the prokaryotic expression vector pET28b-Tat-hLSECtin-CRD (using pET28b and pET28b-hLSECtin-CRD as controls) was constructed, and transformed into E. coli BL21 (DE3) cells and induced with isopropyl-β-d-thiogalactoside (IPTG) followed with identifying by SDS-PAGE and Western blot. Subsequently, the bacterial subcellular structure, in which overexpressed the heterologous proteins Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD, was analyzed by transmission electron microscope (TEM) respectively, and the mannose-binding activity of Tat-hLSECtin-CRD was also determined. Expectedly, the solubility of Tat-LSECtin-CRD significantly increased compared to Tat-free LSECtin-CRD (**p < 0.01) with prolonged time, and the Tat-LSECtin-CRD had a significant mannose-binding activity. The subcellular structure analysis indicated that the bacterial cells overexpressed Tat-hLSECtin-CRD exhibited denser region compared with controls, while dot denser region aggregated in the two ends of bacterial cells overexpressed Tat-free hLSECtin-CRD. This study provided a novel method for improving the soluble expression of membrane proteins in prokaryotic systems by fusion with the Tat peptide, which may be potentially expanded to the expression of other membrane proteins.
    PLoS ONE 01/2013; 8(12):e83579. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The human dopamine D2L receptor has significant implications in neurological and neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Detailed structural knowledge of this receptor is limited due to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell-free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell-free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli (E.coli) lysate-based system and the wheat germ lysate-based system. The bacterial cell-free method used pET 100/ D-TOPO vector to synthesize hexa-histidine tagged D2 receptor using a dialysis bag system; the resulting protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity resin. The wheat germ system used pEU-glutathione-S-transferase (GST) vector to synthesize GST tagged D2L receptor using a bi-layer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays respectively. Additionally, in-gel protein sequencing via Nano LC-MS/MS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
    Biotechnology Progress 02/2013; · 1.85 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The lack of high-throughput approaches for expression and screening of large enzyme libraries remains a major bottleneck for current enzyme engineering efforts. To address this need, we have developed a high-throughput, fluorescence-based approach for rapid one-pot, microscale expression, and screening of industrial enzymes. In this chapter, we present the protocol for integration of cell-free protein expression with activity screening of enzymes in two formats: (1) a 96-well plate format and (2) a microscale-array format. Our one-pot method is ideally suited for rapid, first pass screening of enzymes and can also be used to perform detailed mechanistic analysis such as measurement of kinetics, determination of optimum temperature, and to study enzyme inhibition.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1118:55-69. · 1.29 Impact Factor

Full-text

View
4 Downloads
Available from