James R Swartz

Stanford University, Palo Alto, California, United States

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Publications (86)443.55 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous L-tyrosine. Using selectively-labeled tyrosine substrates, the cyanides were isotopically-labeled via a recently developed in vitro maturation procedure allowing. advanced electron par-amagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic 13C hyperfine interactions for the two CN- ligands-a reporter of spin density on their respective coordinating iron ions-collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [15N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine.
    Journal of the American Chemical Society 08/2014; · 10.68 Impact Factor
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    ABSTRACT: Three iron-sulfur proteins--HydE, HydF, and HydG--play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN(-) ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN(-) ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the (13)C, (15)N, and (57)Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe](H) component of the H-cluster.
    Science 01/2014; 343(6169):424-7. · 31.20 Impact Factor
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    ABSTRACT: Many organometallic cofactors are highly complex and require multiple accessory proteins for both their assembly and transfer to a target protein. A cell-free system in which the biosynthetic pathway for a prosthetic group has been fully or even partially reconstructed enables investigations of the reaction sequence as well as the cofactor itself. As a model for the in vitro assembly of protein-bound metal centers, we describe a procedure for the cell-free synthesis of the H-cluster in the context of producing purified and active [FeFe] hydrogenase samples for spectroscopic studies. In general terms, this in vitro system is a combination of non-purified accessory proteins, exogenous substrates, and purified hydrogenase apoprotein. We also describe methods for making the required components used in the cell-free system. Specifically, these procedures include anaerobic expression of heterologous metalloproteins in Escherichia coli, anaerobic cell lysate production, and anaerobic metalloprotein purification using Strep-Tactin(®) chromatography.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1122:49-72. · 1.29 Impact Factor
  • Yuan Lu, John P Welsh, James R Swartz
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    ABSTRACT: The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.
    Proceedings of the National Academy of Sciences 12/2013; · 9.81 Impact Factor
  • Cem Albayrak, James R Swartz
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    ABSTRACT: We report the synthesis of active polymers of super-folder green fluorescent protein (sfGFP) in one step using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p¬-azido-L-phenylalanine (pAzF) or p-propargyloxy-L-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were coupled by copper-catalyzed azide-alkyne cycloaddition to synthesize linear or branched protein polymers, respectively. The protein polymers retained ≥63% of their specific activity (i.e. fluorescence) after coupling. Polymerization of a concentrated solution of triply-substituted sfGFP resulted in fluorescent macromolecular particles. Our method can be generalized to synthesize polymers of a protein or co-polymers of any two or more proteins, and the conjugation sites can be determined exactly by standard genetic manipulation. Polymers of proteins and small molecules can also be created with this technology to make a new class of scaffolds or biomaterials.
    ACS Synthetic Biology 11/2013; · 3.95 Impact Factor
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    ABSTRACT: The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN(-) for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of (2)H, (13)C, and (15)N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the Cα-Cβ bond forms a transient 4-oxidobenzyl (4OB(•)) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB(•) radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN(-), a key intermediate in the assembly of the 2Fe subunit of the H cluster.
    Science 10/2013; 342(6157):472-5. · 31.20 Impact Factor
  • Cem Albayrak, K. C. Jones, James R. Swartz
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    ABSTRACT: Cell-free protein synthesis (CFPS) has emerged as a practical method for producing a broad variety of proteins. In addition, the direct accessibility to the reaction environment makes CFPS particularly suitable as a learning vehicle for fundamental biological concepts. Here, we describe its implementation as a teaching tool for a high school laboratory course. Ninety students in a biotechnology class used CFPS to study the effects of the concentrations of amino acids, cell extract, DNA, and the energy source on accumulation of active super-folder green fluorescent protein. Students estimated product concentrations simply by comparing solution colors to a printed green color gradient. This simple and inexpensive method allows for immediate measurements, and 26 of the 30 groups observed measurable product concentrations within 60 min. These student-generated data were then discussed to illustrate concepts of data analysis such as outliers and standard deviation. We also combined the laboratory experience with a visit to a university campus that included a laboratory tour and a college-style lecture. Our overall objective was to excite the students about the scientific enterprise and to instill a sense of personal relevance and attainability so that these students could realistically consider technical careers.
    Journal of Science Education and Technology 07/2013; · 0.94 Impact Factor
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    Cem Albayrak, James R Swartz
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    ABSTRACT: We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.
    Nucleic Acids Research 04/2013; · 8.81 Impact Factor
  • Yuan Lu, John P Welsh, Wei Chan, James R Swartz
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    ABSTRACT: Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this paper, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/ml and the product had high affinity for the TLR5 receptor (EC50 = 2.4 ± 1.4 pM). The flagellin coding sequence was first optimized for cell-free expression. We then found that the D0 domain at the C-terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease-deficient cell extracts or deletion of the flagellin D0 domain. A human Toll-Like Receptor 5 (hTLR5)-specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non-natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus-like particle (VLP) using bioorthogonal azide-alkyne cycloaddition reaction. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10-fold. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 03/2013; · 4.16 Impact Factor
  • Cem Albayrak, William C Yang, James R Swartz
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    ABSTRACT: HIGHLIGHTS: ► Both R9Sox2 and Sox2 bind heparin with comparable affinity. ► Both R9Sox2 and Sox2 bind to fibroblasts, but only R9Sox2 is internalized. ► Internalization efficiency of R9Sox2 is 0.3% of the administered protein. ► Heparan sulfate adsorption may be part of a mechanism for managing cell death. ► Abstract. ► The binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD. However, only the R9Sox2 fusion protein is internalized by the cells. These results collectively show that binding to heparan sulfate is not sufficient for cellular uptake, thereby supporting a recent hypothesis that other proteins play a role in cell internalization of PTD-conjugated proteins.
    Biochemical and Biophysical Research Communications 01/2013; · 2.28 Impact Factor
  • Cem Albayrak, James R Swartz
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    ABSTRACT: Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in E. coli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e. proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated k(cat) for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4×10(-5) s(-1). Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported cell-free accumulation of two different modified proteins (450 μg/ml CAT109pAzF and 428 ± 2 μg/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP cell-free recipe.
    Biochemical and Biophysical Research Communications 01/2013; · 2.28 Impact Factor
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    ABSTRACT: The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four [FeS] clusters that facilitate electron transfer to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H(2). Here, we have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe-4S] ferredoxin as well as a model complex resembling the [2Fe](H) catalytic domain of the H-cluster. In order to enrich the hydrogenase with (57)Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with (56)Fe-labeled [FeS] clusters was activated in vitro using (57)Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe](H) subcluster with (57)Fe, which NRVS confirms by detecting (57)Fe-CO and (57)Fe-CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second [(57)FeS] cluster. EPR spectroscopy indicates that this (57)Fe-enriched metal center is not the [4Fe-4S](H) subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an (56)Fe-enriched [4Fe-4S](H) cluster during in vitro maturation, providing unambiguous evidence for stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.
    Biochemistry 12/2012; · 3.38 Impact Factor
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    ABSTRACT: Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the αCD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4(+) helper T cells. Consequently, the αCD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.
    Proceedings of the National Academy of Sciences 08/2012; 109(36):14526-31. · 9.81 Impact Factor
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    ABSTRACT: In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the "head" domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C-terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 06/2012; · 4.16 Impact Factor
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    ABSTRACT: [FeFe] hydrogenases catalyze H(2) production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO), cyanide (CN(-)), and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN(-) ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN(-) ligands that is a precursor to the H-cluster [2Fe](H) subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe](H) subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG.
    PLoS ONE 01/2012; 7(9):e45850. · 3.53 Impact Factor
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    ABSTRACT: Biological technologies for the renewable conversion of biomass to hydrogen, an important chemical feedstock and potential fuel, could reduce greenhouse gas emissions by displacing the current source of virtually all hydrogen: fossil fuels. However, current fermentative methodologies suffer from low productivities and conversion yields. Previous work has shown that purified enzymes from the pentose phosphate pathway can be used to efficiently transfer the reducing equivalents in glucose to NADPH; this approach used a [NiFe] hydrogenase to directly produce hydrogen from NADPH. However, [FeFe] hydrogenases offer much higher hydrogen production activities. We demonstrate a new enzymatic method for the conversion of NADPH reducing equivalents to hydrogen by first using ferredoxin-NADPH-reductase to transfer electrons from NADPH to ferredoxin. The reduced ferredoxin then delivers the electrons to a [FeFe] hydrogenase for hydrogen production. This alternative in vitro pathway enables utilization of the fastest known hydrogenases, [FeFe] hydrogeneases, and activates electron delivery by the native electron donor for these hydrogenases, ferredoxin. We report proof-of-principle data for this synthetic enzyme pathway, showing high volumetric production rates and hydrogenase turnover numbers relative to previous results utilizing [NiFe] hydrogenases.
    Fuel and Energy Abstracts 01/2012;
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    ABSTRACT: Escherichia coli cell-free protein synthesis (CFPS) uses E. coli extracts to make active proteins in vitro. The basic CFPS reaction mixture is comprised of four main reagent components: (1) energy source and CFPS chemicals, (2) DNA encoding the protein of interest, (3) T7 RNA Polymerase (RNAP) for transcription, and (4) cell extract for translation. In this work, we have simplified and shortened the protocols for preparing the CFPS chemical mixture, cell extract, and T7 RNAP. First, we streamlined the workflow for preparing the CFPS chemical solutions by combining all the chemicals into a single reagent mixture, which we call Premix. We showed that productive cell extracts could be made from cells grown in simple shake flasks, and we also truncated the preparation protocol. Finally, we discovered that T7 RNAP purification was not necessary for CFPS. Crude lysate from cells over-expressing T7 RNAP could be used without deleteriously affecting protein production. Using chloramphenicol acetyltransferase (CAT) as a model protein, we showed that these streamlined protocols still support high-yielding CFPS. These simplified procedures save time and offer greater accessibility to our laboratory's CFPS technology.
    Biotechnology Progress 12/2011; 28(2):413-20. · 1.85 Impact Factor
  • James R. Swartz
    AIChE Journal 12/2011; 58(1):5 - 13. · 2.58 Impact Factor
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    ABSTRACT: Directed evolution is a powerful strategy for protein engineering; however, evolution of pharmaceutical proteins has been limited by the reliance of current screens on binding interactions. Here, we present a method that identifies protein mutants with improved overall cellular efficacy, an objective not feasible with previous approaches. Mutated protein libraries were produced in soluble, active form by means of cell-free protein synthesis. The efficacy of each individual protein was determined at a uniform dosage with a high-throughput protein product assay followed by a cell-based functional assay without requiring protein purification. We validated our platform by first screening mock libraries of epidermal growth factor (EGF) for stimulation of cell proliferation. We then demonstrated its effectiveness by identifying EGF mutants with significantly enhanced mitogenic activity at low concentrations compared to that of wild-type EGF. This is the first report of EGF mutants with improved biological efficacy despite much previous effort. Our platform can be extended to engineer a broad range of proteins, offering a general method to evolve proteins for improved biological efficacy.
    Journal of Molecular Biology 08/2011; 413(2):406-15. · 3.91 Impact Factor
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    ABSTRACT: Since the discovery that somatic cells could be reprogrammed back to a pluripotent state through the viral expression of a certain set of transcription factors, there has been great interest in reprogramming using a safer and more clinically relevant protein-based approach. However, the search for an efficient reprogramming approach utilizing the transcription factors in protein form requires a significant amount of protein material. Milligram quantities of transcription factors are challenging to obtain due to low yields and poor solubility. In this work, we describe enhanced production of the pluripotency transcription factors Oct4, Sox2, Klf4, Nanog, and Lin28 after fusing them to a solubility partner, IF2 Domain I (IF2D1). We expressed and purified milligram quantities of the fusion proteins. Though the transcription factor passenger proteins became insoluble after removal of the IF2D1, the un-cleaved Oct4, Sox2, Klf4, and Nanog fusion proteins exhibited specific binding to their consensus DNA sequences. However, when we administered the un-cleaved IF2D1-Oct4-R9 and IF2D1-Sox2-R9 to fibroblasts and measured their ability to influence transcriptional activity, we found that they were not fully bioactive; IF2D1-Oct4-R9 and IF2D1-Sox2-R9 influenced only a subset of their downstream gene targets. Thus, while the IF2D1 solubility partner enabled soluble production of the fusion protein at high levels, it did not yield fully bioactive transcription factors.
    Protein Expression and Purification 07/2011; 80(1):145-51. · 1.43 Impact Factor