Use of<Emphasis Type="SmallCaps">l</Emphasis>-buthionine sulfoximine for the efficient expression of disulfide-containing proteins in cell-free extracts of<Emphasis Type="Italic">Escherichia coli</Emphasis>

We have developed a technique to improve the formation of correct disulfide bonds within cell-free synthesized proteins. Via the use of a metabolic inhibitor of glutamate-cysteine ligase, the accumulation of glutathione was effectively prevented in cell-free extracts, thereby enabling the stable maintenance of redox potential for extended reaction periods. As a result, in a reaction in which a model protein contatining 9 disulfide bonds was synthesized under cell-free conditions, the final amount of active protein products was increased by 50%. The method presented in this study will provide a rapid and robust route to the high-throughput expression and screening of proteins which require multiple disulfide bonds for their activity.     

Expression of active murine granulocyte-macrophage colony-stimulating factor in an Escherichia coli cell-free system

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.     

Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli

In this report, we demonstrate that a complex mammalian protein containing multiple disulfide bonds is successfully expressed in an E.coli-based cell-free protein synthesis system. Initially, disulfide-reducing activities in the cell extract prevented the formation of disulfide bonds. However, a simple pretreatment of the cell extract with iodoacetamide abolished the reducing activity. This extract was still active for protein synthesis even under oxidizing conditions. The use of a glutathione redox buffer coupled with the DsbC disulfide isomerase and pH optimization produced 40 microg/mL of active urokinase protease in a simple batch reaction. This result not only demonstrates efficient production of complex proteins, it also emphasizes the control and flexibility offered by the cell-free approach.     

Enhancing multiple disulfide bonded protein folding in a cell-free system

A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli-based cell-free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30 degrees C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 microg/mL of bioactive PA in a simple 3-h batch reaction.     

Mapping superoxide dismutase 1 domains of non-native interaction: roles of intra- and intermolecular disulfide bonding in aggregation

Superoxide dismutase 1 (SOD1) proteins harboring mutations linked to familial amyotrophic lateral sclerosis (FALS) uniformly show heightened potential to form high molecular weight structures. Here, we examine the domains of SOD1 that are involved in forming these structures (aggregates) and study the role of intra- and intermolecular disulfide bonds. An analysis of disease mutations identified to date reveals a non-random distribution with predominant occurrence at residues within highly conserved beta-strands or at highly conserved residues in loop domains. Using a cell transfection assay for aggregation, we determined that no single domain in SOD1 is indispensable in the formation of sedimentable aggregates, suggesting multiple potential motifs in the protein mediate non-native interactions. By a cell-free aggregation assay, analysis of transgenic mouse tissues, and mutagenesis approaches, we found evidence that redox conditions may modulate SOD1 aggregation; reduction of the native intramolecular disulfide bonds may predispose SOD1 to unfolding and aggregation, whereas non-native intermolecular disulfide linkages may help stabilize aggregates in vivo. The results suggest a possible mechanism for diversity in the structures formed by different SOD1 mutants, and define a potential contribution of redox conditions to SOD1 aggregation.     

Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.     

Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.     

Cell-free production of aggregation-prone proteins in soluble and active forms

We have developed an efficient cell-free protein synthesis system for the production of soluble and active eukaryotic proteins that are predominantly produced as inclusion bodies in bacteria. S30 extracts (indicating the supernatant of cell homogenate when centrifuged at 30,000g) for cell-free protein synthesis were prepared from Escherichia coli that was modified to overexpress a set of chaperones (GroEL/ES or DnaK/J-GrpE) and disulfide isomerase (leader sequence-free mature DsbC expressed in the cytoplasm). The solubility and biological activity concentration (biological activity per unit volume of cell-free protein synthesis reaction mixture) of the protein synthesized by the new cell-free protein synthesis system showed a dramatic improvement. Solubility enhancement was most dramatic with the existence of DnaK/J-GrpE. It shows that the co-translational interaction with DnaK/J-GrpE prior to folding trial is important in maintenance of the aggregation-prone protein in a folding-competent soluble state. For maximizing the biological activity concentration of the expressed protein, the additional presence of GroEL/ES and DsbC was required. When human erythropoietin was expressed in the developed cell-free protein synthesis system including endogenously overexpressed chaperones and/or DsbC, the biological activity concentration of erythropoietin was enhanced by 700%. It implies that the post-translational folding and disulfide bond reshuffling as well as co-translational folding are important in acquiring functionally active protein from cell-free expression system. This is the first report of using S30 extracts including endogenously overexpressed chaperones and/or disulfide isomerase for the efficient production of soluble and active proteins in cell-free protein synthesis. This new cell-free protein synthesis system was capable of introducing much larger amounts of chaperones and disulfide isomerase compared to a conventional method that supplements them separately. The developed cell-free protein synthesis system supported efficient expression of the eukaryotic proteins in soluble and active forms without the need of any exogenous addition or coexpression of folding effectors.     

Acceleration of disulfide-coupled protein folding using glutathione derivatives

Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives - glutamyl-cysteinyl-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (K(RCG) = 3.69 × 10(-3) s(-1)) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.     

Thioredoxins and glutaredoxins as facilitators of protein folding

Thiol-disulfide oxidoreductase systems of bacterial cytoplasm and eukaryotic cytosol favor reducing conditions and protein thiol groups, while bacterial periplasm and eukaryotic endoplasmatic reticulum provide oxidizing conditions and a machinery for disulfide bond formation in the secretory pathway. Oxidoreductases of the thioredoxin fold superfamily catalyze steps in oxidative protein folding via protein-protein interactions and covalent catalysis to act as chaperones and isomerases of disulfides to generate a native fold. The active site dithiol/disulfide of thioredoxin fold proteins is CXXC where variations of the residues inside the disulfide ring are known to increase the redox potential like in protein disulfide isomerases. In the catalytic mechanism thioredoxin fold proteins bind to target proteins through conserved backbone-backbone hydrogen bonds and induce conformational changes of the target disulfide followed by nucleophilic attack by the N-terminally located low pK(a) Cys residue. This generates a mixed disulfide covalent bond which subsequently is resolved by attack from the C-terminally located Cys residue. This review will focus on two members of the thioredoxin superfamily of proteins known to be crucial for maintaining a reduced intracellular redox state, thioredoxin and glutaredoxin, and their potential functions as facilitators and regulators of protein folding and chaperone activity.     

Cell-free production of functional antibody fragments

Botulinum neurotoxin serotype B (BoNT/B)-specific Fab was expressed in a cell-free protein synthesis system derived from an E. coli extract. The cell-free synthesized antibody fragment was found to be effective in neutralizing the toxicity of BoNT/B in animal studies. Expression of functional Fab required an appropriately controlled and stably maintained redox potential. Under an optimized redox condition, the cell extract, whose disulfide reducing activity had been exhausted, could generate bio-functional Fab molecules. Use of a cell extract enriched with molecular chaperones (GroEL/ES) and disulfide bond isomerases were effective in obtaining larger quantities of functional Fab. Under the optimized reaction conditions, approximately 30 μg of functional Fab was obtained after purification from 1 mL reaction mixture.     

Production of disulfide-bonded proteins in Escherichia coli

Disulfide bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. A disulfide bond can serve structural, catalytic, and signaling roles. However, there is an inherent problem to the process of disulfide bond formation: mis-pairing of cysteines can cause misfolding, aggregation and ultimately result in low yields during protein production. Recent developments in the understanding of the mechanisms involved in the formation of disulfide bonds have allowed the research community to engineer and develop methods to produce multi-disulfide-bonded proteins to high yields. This review attempts to highlight the mechanisms responsible for disulfide bond formation in Escherichia coli, both in its native periplasmic compartment in wild-type strains and in the genetically modified cytoplasm of engineered strains. The purpose of this review is to familiarize the researcher with the biological principles involved in the formation of disulfide-bonded proteins with the hope of guiding the scientist in choosing the optimum expression system.     

High throughput screening of disulfide‐containing proteins in a complex mixture

The formation of disulfide bonds between cysteine residues is crucial for the stabilization of native protein structures and, thus, determination of disulfide linkages is an important facet of protein structural characterization. Nonetheless, the identification of disulfide bond linkages remains a significant analytical challenge, particularly in large proteins with complex disulfide patterns. Herein, we have developed a new LC/MS strategy for rapid screening of disulfides in an intact protein mixture after a straightforward reduction step with tris(2‐carboxyethyl)phosphine. LC/MS analysis of reduced and nonreduced protein mixtures quickly revealed disulfide‐containing proteins owing to a 2 Da mass increase per disulfide reduction and, subsequently, the total number of disulfide bonds in the intact proteins could be determined. We have demonstrated the effectiveness of this method in a protein mixture composed of both disulfide‐containing and disulfide‐free proteins. Our method is simple (no need for proteolytic digestion, alkylation, or the removal of reducing agents prior to MS analysis), high throughput (fast on‐line LC/MS analysis), and reliable (no S–S scrambling), underscoring its potential as a rapid disulfide screening method for proteomics applications.     

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG⁰) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.     

Development of cell-free protein synthesis platforms for disulfide bonded proteins

The use of cell-free protein synthesis (CFPS) for recombinant protein production is emerging as an important technology. For example, the openness of the cell-free system allows control of the reaction environment to promote folding of disulfide bonded proteins in a rapid and economically feasible format. These advantages make cell-free protein expression systems particularly well suited for producing patient specific therapeutic vaccines or antidotes in response to threats from natural and man-made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. In this work we assess the versatility of modern cell-free methods, optimize expression and folding parameters, and highlight the importance of rationally designed plasmid templates for producing mammalian secreted proteins, fusion proteins, and antibody fragments in our E. coli-based CFPS system. Two unique CFPS platforms were established by developing standardized extract preparation protocols and generic cell-free reaction conditions. Generic reaction conditions enabled all proteins to express well with the best therapeutic protein yield at 710 microg/mL, an antibody fragment at 230 microg/mL, and a vaccine fusion protein at 300 microg/mL; with the majority correctly folded. Better yields were obtained when cell-free reaction conditions were optimized for each protein. Establishing general CFPS platforms enhances the potential for cell-free protein synthesis to reliably produce complex protein products at low production and capital costs with very rapid process development timelines.     

Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.     

Impact of an easily reducible disulfide bond on the oxidative folding rate of multi-disulfide-containing proteins.

The burial of native disulfide bonds, formed within stable structure in the regeneration of multi-disulfide-containing proteins from their fully reduced states, is a key step in the folding process, as the burial greatly accelerates the oxidative folding rate of the protein by sequestering the native disulfide bonds from thiol-disulfide exchange reactions. Nevertheless, several proteins retain solvent-exposed disulfide bonds in their native structures. Here, we have examined the impact of an easily reducible native disulfide bond on the oxidative folding rate of a protein. Our studies reveal that the susceptibility of the (40-95) disulfide bond of Y92G bovine pancreatic ribonuclease A (RNase A) to reduction results in a reduced rate of oxidative regeneration, compared with wild-type RNase A. In the native state of RNase A, Tyr 92 lies atop its (40-95) disulfide bond, effectively shielding this bond from the reducing agent, thereby promoting protein oxidative regeneration. Our work sheds light on the unique contribution of a local structural element in promoting the oxidative folding of a multi-disulfide-containing protein.     

Providing an oxidizing environment for the cell-free expression of disulfide-containing proteins by exhausting the reducing activity of Escherichia coli S30 extract

We developed a novel method of producing proteins containing multiple disulfide bonds in a cell-free protein synthesis system. To provide an optimized redox potential during the synthesis of truncated plasminogen activator (rPA), we pretreated the E. coli S30 extract with an excess amount of oxidized glutathione based on the anticipation that the reducing potential of the S30 extract would be exhausted through the reduction of the oxidized glutathione molecules. As expected, it was found that the reducing activity of the S30 extract was remarkably decreased through the pretreatment, and active rPA was produced when the pretreated S30 extract was used after removing the residual glutathione molecules. In particular, compared to the method involving the iodoacetamide treatment of S30 extract, the present protocol was effective in producing active rPA during the batch reaction of cell-free protein synthesis.     

Efficient disulfide bond formation in virus-like particles

Virus-like particles (VLPs) consist of a virus's outer shell but without the genome. Similar to the virus, VLPs are monodisperse nano-capsules which have a known morphology, maintain a high degree of symmetry, and can be engineered to encapsidate the desired cargo. VLPs are of great interest for vaccination, drug/gene delivery, imaging, sensing, and material science applications. Here we demonstrate the ability to control the disulfide bond formation in VLPs by directly controlling the redox potential during or after production and assembly of VLPs. The open cell-free protein synthesis environment, which has been reported to produce VLPs at yields comparable or greater than traditional in vivo technologies, was employed. Optimal conditions for disulfide bond formation were found to be VLP dependent, and a cooperative effect in the formation of such bonds was observed.     

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

The significant contribution of disulfide bonds to the conformational stability of proteins is generally considered to result from an entropic destabilization of the unfolded state causing a faster escape of the molecules to the native state. However, the introduction of extra disulfide bonds into proteins as a general approach to protein stabilization yields rather inconsistent results. By modeling studies, we selected positions to introduce additional disulfide bonds into ribonuclease A at regions that had proven to be crucial for the initiation of the folding or unfolding process, respectively. However, only two out of the six variants proved to be more stable than unmodified ribonuclease A. The comparison of the thermodynamic and kinetic data disclosed a more pronounced effect on the unfolding reaction for all variants regardless of the position of the extra disulfide bond. Native-state proteolysis indicated a perturbation of the native state of the destabilized variants that obviously counterbalances the stability gain by the extra disulfide bond.