Tina Fink’s research while affiliated with National Institute of Chemistry and other places

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Publications (12)


Coiled-Coil Interaction Toolbox for Engineering Mammalian Cells
  • Article

March 2024

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22 Reads

Methods in molecular biology (Clifton, N.J.)

Erik Rihtar

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Tina Fink

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Protein interactions play a crucial role in a variety of biological processes. Therefore, regulation of these interactions has received considerable attention in terms of synthetic biology tool development. Of those, a toolbox of small peptides known as coiled coils (CCs) represents a unique effective tool for mediating protein–protein interactions because their binding specificity and affinity can be designed and controlled. CC peptides have been used as a building module for designing synthetic regulatory circuits in mammalian cells, construction of fast response to a signal, amplification of the response, and localization and regulation of function of diverse proteins. In this chapter, we describe a designed set of CCs used for mammalian cell engineering and provide a protocol for the construction of CC-mediated logic circuits in mammalian cells. Ultimately, these tools could be used for diverse biotechnological and therapeutic applications.


Fig. 2 (See legend on next page.)
Fig. 3 Construction of INSRTR protein logic gates based on intramolecular fusion with interacting and protease target segments. a Scheme of the implementation of INSRTR logic gates with protease inputs and allosterically activated protein function. b Schematic presentation of an AND logic gate. c Experimental testing of all nontrivial two-input Boolean logic functions by INSRTR in HEK293T cells, as regulated by a combination of PPV and SbMV protease inputs. The values in (c) are the means of four cell cultures ± SD and are representative of at least two independent experiments. Significance was tested with one-way ANOVA with Tukey's comparison between indicated ON and OFF states. P values 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), < 0.0001 (****) (significance, confidence intervals, degrees of freedom, F and P values are listed in Supplementary Table S2).
Fig. 4 (See legend on next page.)
Fig. 5 INSRTR regulation of CAR T cell activation. a Scheme of INSRTR-regulated CAR T receptor. P7 CC segment was inserted into a heavy chain of scFv at a loop neighboring CDR loops. b Antibody binding domain scFv CD19 N195 P7 in a CAR T receptor on cells activated by CD19-expressing Raji B cells. c Improved design of INSRTR CAR T receptor. An additional CC segment was fused to the N-terminus of the N195 P7 scFv(CD19) and a fusion peptide N5-P8A was used as a regulator. d Cells expressing N5_scFv(CD19)_N195_P7 were stimulated with Raji cells expressing CD19 antigen. e Antibody binding domain scFv Her2 S199 P7 in a CAR T receptor on cells activated by ErbB2-expressing SK-BR-3 cells. Statistical significance was tested with one-way ANOVA with Dunnett's multiple comparisons test between the INSRTR variant and the addition of a REG peptide; P values 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), < 0.0001 (****) (significance, confidence intervals, degrees of freedom, F and P values are listed in Supplementary Table S2).
Designed allosteric protein logic
  • Article
  • Full-text available

January 2024

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136 Reads

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9 Citations

Cell Discovery

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Estera Merljak

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Tina Fink

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[...]

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The regulation of protein function by external or internal signals is one of the key features of living organisms. The ability to directly control the function of a selected protein would represent a valuable tool for regulating biological processes. Here, we present a generally applicable regulation of proteins called INSRTR, based on inserting a peptide into a loop of a target protein that retains its function. We demonstrate the versatility and robustness of coiled-coil-mediated regulation, which enables designs for either inactivation or activation of selected protein functions, and implementation of two-input logic functions with rapid response in mammalian cells. The selection of insertion positions in tested proteins was facilitated by using a predictive machine learning model. We showcase the robustness of the INSRTR strategy on proteins with diverse folds and biological functions, including enzymes, signaling mediators, DNA binders, transcriptional regulators, reporters, and antibody domains implemented as chimeric antigen receptors in T cells. Our findings highlight the potential of INSRTR as a powerful tool for precise control of protein function, advancing our understanding of biological processes and developing biotechnological and therapeutic interventions.

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Designed allosteric protein logic

June 2022

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51 Reads

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2 Citations

Regulation of the activity of proteins enables control of complex cellular processes. Allosteric regulation has been introduced individually into few natural proteins. Here, we present a generally applicable regulation of diverse proteins called INSRTR (inserted peptide structure regulator), based on inserting a short unstructured peptide into a solvent-accessible loop that retains protein function. Function of the target protein can be inactivated by the addition of a peptide that forms a rigid coiled-coil dimer. This platform enables the construction of ON/OFF protein switches, their regulation by small molecules, and Boolean logic functions with a rapid response in mammalian cells. INSRTR can be used to regulate a wide range of proteins, as demonstrated on ten members of protein families with diverse biological activities including enzymes, signaling mediators, DNA binders/transcriptional regulators, fluorescent protein, and antibodies regulating chimeric antigen receptor. INSRTR platform presents an extraordinary potential for regulating biological systems and applications. One sentence summary Authors have designed a widely applicable system to activate or inactivate function of diverse proteins or form Boolean logic gates based on formation of a coiled-coil dimer within protein loops and demonstrated its implementation on a range of 10 diverse proteins.


Designed protease-based signaling networks

June 2022

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42 Reads

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18 Citations

Current Opinion in Chemical Biology

Regulated proteolysis is a pivotal regulatory mechanism in all living organisms from bacteria to mammalian cells and viruses. The ability to design proteases to sense, transmit, or trigger a signal opens up the possibility of construction of sophisticated proteolysis-regulated signaling networks. Cleavage of the polypeptide chain can either activate or inactivate the selected protein or process, often with a fast response. Most designs are based on sequence-selective proteases that can be implemented for transcriptional, translational, and ultimately post-translational control, aiming to engineer complex circuits that can dynamically control cellular functions and enable novel biotechnological and biomedical applications.


Fig. 5 Modular combinations and two-input signal-processing capacity of protein secretion systems. a A two-input Boolean logic processing system -SPOC, previously described by Fink et al., was used to control the reconstitution of a split TEVp and regulate secretion through the removal of KKYL. The input proteases SbMVp and PPVp produced the output according to A nimply B logical function and (b), AND logical function. For the KDEL system, SEAP was retained inside the ER by the addition of a C-terminal KDEL sequence, as well as the retention of the construct through the interaction of the P3/P4 coiled coils, where P3 also featured SbMVs and a KDEL sequence. Secretion was induced by the addition of erTEVp and erSbMVp. c To establish an AND function for the lumER system, P3-TEVs-KDEL was co-transfected and localized inside the ER with SEAP-P4-SbMVs-KDEL. Apart from the KDEL sequence (orange) present on the same protein, SEAP was retained inside of the ER through the P3/P4 coiled-coil interaction. To facilitate the secretion of SEAP, both erSbMVp and erTEVp have to be present, in order to remove the two retention signals. d Implementation of a protein secretion OFF switch. Rapamycin is used to reconstitute a split TEVp, which further drives secretion of the membER secretion construct. By inserting a PPVp cleavage site (PPVs) between the dimerization domain and protease fragment, the reconstitution of TEVp can be inhibited with an ABA-inducible split PPVp. Cells were stimulated with rapamycin to induce secretion. After 3 h ABA was added and cell media were harvested continuously for 6 h. Values are the mean of three (d) or four cell (a, b, c) cultures ± s.d. and are representative of two independent experiments. Significance was tested by one-way analysis of variance (ANOVA) with Dunnett's comparison between indicated ON and OFF states (values of confidence intervals, degrees of freedom, F and P are indicated). Source data are provided as a Source Data file.
Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

March 2022

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135 Reads

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53 Citations

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Response of protein secretion based on transcriptional control is rather slow, as it requires transcription, translation and transport from the endoplasmic reticulum (ER) to the plasma membrane via the conventional protein secretion (CPS) pathway. An alternative regulation to provide faster response would be valuable. Here we present two genetically encoded orthogonal regulatory secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by a cytosolic protease) or inside the ER lumen (lumER, released by an ER-luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables the combination of signals into logic functions. Its application was demonstrated on a chemically regulated therapeutic protein secretion and regulated membrane translocation of a chimeric antigen receptor (CAR) targeting cancer antigen. Regulation of the ER escape represents a platform for the design of fast-responsive and tightly-controlled modular and scalable protein secretion system for mammalian cells. Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Here the authors present two genetically encoded orthogonal regulatory secretion systems that enables inducible protein release and construction of logic gates.


Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

October 2021

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102 Reads

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Protein secretion based on transcriptional control is rather slow, as proteins requires transcription, translation, followed by the transport from the endoplasmic reticulum (ER) through the conventional protein secretion (CPS) pathway towards the plasma membrane. An alternative faster bypass would be valuable. Here we present two genetically encoded orthogonal secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by cytosolic protease) or inside the ER lumen (lumER, released by ER luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables precise combination of signals into logic functions and was demonstrated on a chemically regulated insulin secretion. Regulation of ER escape represents a platform for the design of fast responsive and tightly-controlled modular and scalable protein secretion system. Abstract Figure Abstract figure: MembER and lumER system By equipping a protein of interest (POI) with an N-terminal signaling sequence, which initiates the transport of proteins into the endoplasmic reticulum (ER), and a C-terminal KDEL ER retention sequence for luminal proteins or a KKXX sequence for transmembrane proteins, we can retain those proteins inside the ER and cis-Golgi apparatus (GA) through retrograde transport. Insertion of a protease cleavage site adjacent to the retention signal allows for the regulated fast secretion through proteolytic cleavage. The membrane bound, ER membrane (membER) and ER-luminal (lumER) systems allow for the controlled secretion of pre-synthesized protein, stored inside the ER. This platform enables release of target proteins several hours faster than systems relying transcription and translation.


Figure 1. Scaffolded RBDs induce high titers of antibody production in vivo. Molecular models of RBD domains are shown in violet and scaffold cores in blue. The number of RBD domains per particle, length of the scaffolding domain, and fraction of amino acid residues of RBD in the assembly are listed below each model (a). Mice were immunized with plasmid DNA encoding RBD, scaffolded RBD variants, or scaffolds alone, according to the indicated immunization schedule (b). End point titer (EPT) values of total IgG against RBD (c-e) and against whole length spike protein (f-h) are shown. Graphs represent the mean EPTs of groups of mice (n = 5 per group). Each dot represents an individual animal. * p < 0.05; ** p < 0.01. All p values are from the Mann-Whitney test compared to RBD group.
A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

April 2021

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389 Reads

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24 Citations

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.


Figure 1: Molecular models of designed scaffolded RBD domains. RBD domains are shown in violet and scaffold core in blue. Number of RBD domains per particle, length of the scaffolding domain and fraction of amino acid residues the RBD in the assembly are written below each model.
Figure 2: Secretion of RDB protein domains fused to different scaffolding proteins produced in plasmid-transfected mammalian cells and size analysis of the isolated RBD-bann protein. Supernatant of HEK293 cells transfected with indicated construct was harvested 3 days post transfection and the presence of differently scaffolded RBD domain variants was detected with anti RBD antibodies (A). Size analysis of the purified RBD-bann by DLS confirms the presence of particles around 500 nm (B).
Figure 3: Titer of total IgG antibodies against the RBD and Spike protein for immunization with plasmids for different scaffolded RBDs and scaffold alone. Mice were immunized with different combination of RBD plasmid DNA, complexed with jetPEI-in vivo transfection reagent, according to immunization protocol (A). End point titer (EPT) for total IgG against RBD (B-D) and against Spike protein (E-G). Graphs represent mean of EPT of group of mice (n=5 per group). Each dot represents an individual animal. *P< 0.05; **P < 0.01. All P values are from Mann-Whitney test.
Figure 4: Analysis of different classes of antibodies against RBD for different scaffolded RBDs and immunization by the scaffold. Mice were immunized with different combination of RBD plasmid DNA. End point titers 6 weeks after the first immunization of IgA (A), IgM (B), IgG1 (C), IgG2b (D) and IgG3 (E) against RBD protein were determined by ELISA. Graphs represent mean of EPT of group of mice (n=5 per group). Each dot represents an individual animal.
Figure 7: Spike protein-specific cytotoxic killing by lymphocytes from immunized animals. Cytotoxic T cell killing from immunized mice against cells expressing viral S protein. Mice were immunized with different combinations of RBD plasmid DNA and at the end of the experiment spleens
Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein

August 2020

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1,972 Reads

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7 Citations

Effective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and β-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue β-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.


Fig. 5 A coiled-coil dimer-based scaffold linked to mevalonate biosynthesis enzymes improves mevalonate accumulation in S. cerevisiae. a Scheme for the designed coiled-coil-based polypeptide scaffold. Extracellular mevalonate production (b) and total carotenoid accumulation (c) in carotenoid-producing yeast cells. The wild type is strain CAR-0002. CAR-0002 transformants express non-fused mevalonate pathway enzymes (Mev: ERG10, HMGS, or tHMGR) or coiled-coil scaffolded proteins (P3:GCN:P4-Mev). The data represent the average and standard deviation for mevalonate or the total carotenoid measurements for three independent transformants grown in duplicate
Schematic illustration of resveratrol biosynthesis and different strategies for the protein-based clustering of metabolic enzymes. a The enzymes 4-coumarate CoA ligase (4CL) and stilbene synthases (STS) are involved in resveratrol production from p-coumaric acid. b Native enzymes were used as a reference for resveratrol quantification. c, d Two strategies were based on the covalent fusion of the enzymes 4CL and STS: direct genetically encoded fusion (c) and intein-mediated fusion (d). e, f The interactions of designed coiled-coil-forming domains bring both enzymes into proximity. Coiled-coil dimers can have either a parallel (e) or an antiparallel (f) orientation
Comparison of the efficiency of covalently fused enzymes for resveratrol production. Resveratrol accumulation in E. coli strains expressing a the directly fused enzymes (4CL::STS) and c the enzymes covalently coupled via split intein fusion (4CL:STS). Expression of the directly fused (b) and intein-fused (d) enzymes was determined by WB. Native enzymes (4CL + STS) were used for reference. The data represent the average and standard deviation of the representative fermentations from three independent experiments with similar results
NiNTA-isolated coiled-coil-forming domains fused with enzymes interact and form clusters in vitro. a The SDS PAGE of the NiNTA-isolated proteins demonstrates the interaction between P3S and P4S from fusion proteins His4CL:P3S (*1) and AUSTS:P4S (*2). b Native PAGE analysis revealed that in vitro clusters of 4CL and STS are formed at two different temperatures only on the fusion of enzymes with the coiled-coil-forming segments P3S and P4S
The clustering of biosynthetic enzymes via coiled-coil-forming domains improves resveratrol production in E. coli. a Enhancement of resveratrol biosynthesis in E. coli after 8 h, depending on the type of coiled-coil-forming domain fused to the enzymes. Simultaneously expressed native enzymes (4CL + STS) served as a reference. b The expression of enzymes coupled via coiled-coil-forming domain fusion was determined by WB. The same protein expression in three representative fermentations is shown in Additional file 1: Fig. S2
Metabolic enzyme clustering by coiled coils improves the biosynthesis of resveratrol and mevalonate

May 2020

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172 Reads

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12 Citations

AMB Express

The clustering of biosynthetic enzymes is used in nature to channel reaction products and increase the yield of compounds produced by multiple reaction steps. The coupling of multiple enzymes has been shown to increase the biosynthetic product yield. Different clustering strategies have particular advantages as the spatial organization of multiple enzymes creates biocatalytic cascades with a higher efficiency of biochemical reaction. However, there are also some drawbacks, such as misfolding and the variable stability of interaction domains, which may differ between particular biosynthetic reactions and the host organism. Here, we compared different protein-based clustering strategies, including direct fusion, fusion mediated by intein, and noncovalent interactions mediated through small coiled-coil dimer-forming domains. The clustering of enzymes through orthogonally designed coiled-coil interaction domains increased the production of resveratrol in Escherichia coli more than the intein-mediated fusion of biosynthetic enzymes. The improvement of resveratrol production correlated with the stability of the coiled-coil dimers. The coiled-coil fusion-based approach also increased mevalonate production in Saccharomyces cerevisiae, thus demonstrating the wider applicability of this strategy.


Figure 1 Schematic illustration of resveratrol biosynthesis and different strategies for the proteinbased clustering of metabolic enzymes. (a) The enzymes 4-coumarate CoA ligase (4CL) and stilbene synthases (STS) are involved in resveratrol production from p-coumaric acid. (b) Native enzymes were used as a reference for resveratrol quantification. (c, d) Two strategies were based on the covalent fusion of the enzymes 4CL and STS: direct genetically encoded fusion (c) and intein-mediated fusion (d). (e, f) The interactions of designed coiledcoil-forming domains bring both enzymes into proximity. Coiled-coil dimers can have either a parallel (e) or an antiparallel (f) orientation.
Figure 2 Comparison of the efficiency of direct fusion and intein-fused enzymes for resveratrol production. Resveratrol accumulation in E. coli strains expressing: (a) the directly fused enzymes (4CL::STS) and (c) the enzymes covalently coupled via split intein fusion (4CL:STS). Expression of the directly fused (b) and intein-fused (d) enzymes. Native enzymes (4CL+STS) were used for reference. The data represent the average and standard deviation of the representative fermentations from three independent experiments with similar results.
Figure 3 NiNTA-isolated coiled-coil-forming domains fused with enzymes interact and form clusters in vitro. (a) The SDS PAGE of the NiNTA-isolated proteins demonstrates the interaction between P3S and P4S from fusion proteins His4CL:P3S (*1) and AUSTS:P4S (*2). (b) Native PAGE analysis revealed that in vitro clusters of 4CL and STS are formed at two different temperatures only on the fusion of enzymes with the coiled-coil-forming segments P3S and P4S.
Figure 4 The clustering of biosynthetic enzymes via coiled-coil-forming domains improves resveratrol production in E. coli. (a) Enhancement of resveratrol biosynthesis in E. coli, depending on the type of coiled-coil-forming domain fused to the enzymes. Simultaneously expressed native enzymes (4CL+STS) served as a reference. (b) The expression of enzymes coupled via coiled-coil-forming domain fusion determined by WB. The protein expression in three representative fermentations is shown in Fig. S2b.
Figure 5 A coiled-coil dimer-based scaffold linked to mevalonate biosynthesis enzymes improves mevalonate accumulation in S. cerevisiae. (a) Scheme for the designed coiled-coil-based polypeptide scaffold. Extracellular mevalonate production (b) and total carotenoid accumulation (c) in carotenoid-producing yeast cells. The wild type is strain CAR-0002. CAR-0002 transformants express non-fused mevalonate pathway enzymes (Mev: ERG10, HMGS, or tHMGR) or coiled-coil scaffolded proteins (P3:GCN:P4-Mev). The data represent the average and standard deviation for mevalonate or the total carotenoid measurements for three independent transformants grown in duplicate.
Metabolic enzyme clustering by coiled coils improves the biosynthesis of resveratrol and mevalonate

April 2020

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180 Reads

The clustering of biosynthetic enzymes is used in nature to channel reaction products and increase the yield of compounds produced by multiple reaction steps. The coupling of multiple enzymes has been shown to increase the biosynthetic product yield. Different clustering strategies have particular advantages as the spatial organization of multiple enzymes creates biocatalytic cascades with a higher efficiency of biochemical reaction. However, there are also some drawbacks, such as misfolding and the variable stability of interaction domains, which may differ between particular biosynthetic reactions and the host organism. Here, we compared different protein-based clustering strategies, including direct fusion, fusion mediated by intein, and noncovalent interactions mediated through small coiled-coil dimer-forming domains. The clustering of enzymes through orthogonally designed coiled-coil interaction domains increased the production of resveratrol in Escherichia coli more than the intein-mediated fusion of biosynthetic enzymes. The improvement of resveratrol production correlated with the stability of the coiled-coil dimers. The coiled-coil fusion-based approach also increased mevalonate production in Saccharomyces cerevisiae , thus demonstrating the wider applicability of this strategy.


Citations (8)


... For example, the integration of synthetic biology techniques, such as those developed at NIC, into the design of next-generation CAR T cells should facilitate the translation of these novel immunotherapies, such as those based on γδT cells, into clinical applications. The NIC has recently developed versatile approaches to increase the efficacy and control of CAR T-cell therapies by modifying CAR receptors to regulate their activity and by introducing versatile humanised switches to modulate therapeutic T-cell activity and precise recognition of cancer markers to reduce side effects and improve safety [29][30][31][32]. By increasing the efficacy of CAR T-cells and improving persistence and penetrance, CTGCT also aims to address the limiting factors of solid tumor therapies. ...

Reference:

CTGCT, Centre of Excellence for the Technologies of Gene and Cell Therapy: Collaborative Translation of Scientific Discoveries into Advanced Treatments for Neurological Rare Genetic Diseases and Cancer
Designed allosteric protein logic

Cell Discovery

... We designed an orthologous set of weakly interacting CC pairs, with adjustable affinities suitable for LLPS formation (sequences in Supplementary Table 1). The design was based on the same principles as used previously for the design of strongly interacting orthogonal pairs [50][51][52][53] . Canonical parallel CC dimers are composed of heptad repeats, with positions labeled abcdefg. ...

Designed allosteric protein logic
  • Citing Preprint
  • June 2022

... Virus-encoded proteases are the smallest proteases involved in the life cycle of both plant and animal viruses, including potato virus Y (PVY), human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or hepatitis C virus (HCV) [1][2][3]. A mechanism of action of these vertebrate viruses involves the proteolytic cleavage of host proteins, thereby inhibiting their activity [4], e.g., in innate immune system evasion [5,6]. ...

Designed protease-based signaling networks
  • Citing Article
  • June 2022

Current Opinion in Chemical Biology

... The eukaryotic protein secretory pathway is responsible for the delivery of up to one-third of the proteome to their proper destination (Gomez-Navarro and Miller 2016;Praznik et al. 2022). Newly synthesised proteins are first translocated into the endoplasmic reticulum (ER), where they undergo folding and preliminary modifications such as core glycosylation with the aid of various chaperone proteins, foldases and oligosaccharyltransferases (Benham 2012;Delic et al. 2013). ...

Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

... 68 In contrast, viruses and VLPs can bind to B cells with high affinity and avidity by crosslinking BCRs with repetitive antigens displayed on their surfaces, thereby enhancing B cell responses and retention in the lymph nodes. 100, 105 Cohen et al., as well as others groups, have demonstrated that immunization with heterotypic VLPs, which simultaneously display RBDs from eight different SARS-like beta coronaviruses (sarbecovirus), promote crosslinking interactions with high avidity to subdominant, conserved regions of the adjacent RBD antigens. [106][107][108][109][110] This leads to the induction of broadly crossreactive and neutralizing responses compared to homotypic VLPs that display only one type of RBD antigen. ...

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

... The titer of RBDspecific antibodies, which also recognize spiking proteins, neutralizes the interaction between the spiking proteins and ACE2 receptors and offers protection in alternative infection assays (RBD domains are shown in violet and scaffold core in blue). This is significantly increased when RBDs are fused to the scaffolding structural domains [82]. ...

Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein

... Reporting product titres in g/L is standard practice in academia [10,11,21,22]. However, these values provide little relevant context when considering scaling up a process, where time and amount of substrate are both important factors in considering scaling costs. ...

Metabolic enzyme clustering by coiled coils improves the biosynthesis of resveratrol and mevalonate

AMB Express

... In particular, compared to the more conventional transcriptional control, protein-level controls hold advantages for potential clinical use, such as fast operation, compact single-transcript delivery and context-independent performance 12 . Notably, orthogonal viral proteases have emerged as useful post-translational tools due to their ability to control the activity, degradation and localization of target proteins with high substrate specificity [13][14][15][16] . These orthogonal proteases are highly versatile and can be combined to implement robust sense-and-response behaviors in mammalian cells. ...

Design of fast proteolysis-based signaling and logic circuits in mammalian cells

Nature Chemical Biology