Incorrect Disulfide Bond Formation Research Articles

Harnessing the potential of bacterial oxidative folding to aid protein production.

Protein folding is central to both biological function and recombinant protein production. In bacterial expression systems, which are easy to use and offer high protein yields, production of the protein of interest in its native fold can be hampered by the limitations of endogenous posttranslational modification systems. Disulfide bond formation, entailing the covalent linkage of proximal cysteine amino acids, is a fundamental posttranslational modification reaction that often underpins protein stability, especially in extracytoplasmic environments. When these bonds are not formed correctly, the yield and activity of the resultant protein are dramatically decreased. Although the mechanism of oxidative protein folding is well understood, unwanted or incorrect disulfide bond formation often presents a stumbling block for the expression of cysteine-containing proteins in bacteria. It is therefore important to consider the biochemistry of prokaryotic disulfide bond formation systems in the context of protein production, in order to take advantage of the full potential of such pathways in biotechnology applications. Here, we provide a critical overview of the use of bacterial oxidative folding in protein production so far, and propose a practical decision-making workflow for exploiting disulfide bond formation for the expression of any given protein of interest.

Collateral fitness effects of mutations

The distribution of fitness effects of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutation's collateral fitness effects, which we define as effects that do not derive from changes in the protein's ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in the Escherichia coli TEM-1 β-lactamase antibiotic resistance gene using growth competition experiments in the absence of antibiotic. At least 42% of missense mutations in TEM-1 were deleterious, indicating that for some proteins collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper posttranslational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently in TEM-1 than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against deleterious effects on protein activity and protein abundance.

Rapid Identification of Disulfide Bonds and Cysteine-Related Variants in an IgG1 Knob-into-Hole Bispecific Antibody Enhanced by Machine Learning.

Bispecific antibodies are regarded as the next generation of therapeutic modalities as they can simultaneously bind multiple targets, increasing the efficacy of treatments for several diseases and opening up previously unattainable treatment designs. Linking two half antibodies to form the knob-into-hole bispecific antibody requires an additional in vitro assembly step, starting with reduction of the antibodies and then reoxidization. Analysis of the disulfide bonds (DSBs) is vital to ensuring the correct assembly, stability, and higher-order structures of these important biomolecules because incorrect disulfide bond formation and/or presence of cysteine-related post-translational modifications can cause a loss of biological activity or even elicit an immune response from the host. Despite advancements in analytical methods, characterization of cysteine forms remains technically challenging and time-consuming. Herein, we report the development of an improved nonreduced peptide map method coupled with machine learning to enable rapid identification of disulfide bonds and cysteine-related variants in an IgG1 knob-into-hole bispecific antibody. The enhanced method offers a fast, consistent, and accurate workflow in mapping-out expected disulfide bonds in both half antibodies and bispecific antibodies and identifying cysteine-related modifications. Comparisons between two versions of the bispecific antibody molecule and analysis of stressed samples were also accomplished, indicating this method can be utilized to identify alterations originating from bioprocess changes and to determine the impact of assembly and postassembly stress conditions to product quality.

Role of Disulfide Bonds on DNA Packaging Forces in Bull Sperm Chromatin

Short arginine-rich proteins called protamines mediate the near crystalline DNA packaging in most vertebrate sperm cells. Protamines are synthesized during spermiogenesis and condense the paternal genome into a transcriptionally inactive state in late-stage spermatids. Protamines from eutherian mammals, including bulls and humans, also contain multiple cysteine residues that form intra- and interprotamine sulfur-sulfur bonds during the final stages of sperm maturation. Although the cross-linked protamine network is known to stabilize the resulting nucleoprotamine structure, little is known about the role of disulfide bonds on DNA condensation in the mammalian sperm. Using small angle x-ray scattering, we show that isolated bull nuclei achieve slightly lower DNA packing densities compared to salmon nuclei despite salmon protamine lacking cysteine residues. Surprisingly, reduction of the intermolecular sulfur-sulfur bonds of bull protamine results in tighter DNA packing. Complete reduction of the intraprotamine disulfide bonds ultimately leads to decondensation, suggesting that disulfide-mediated secondary structure is also critical for proper protamine function. Lastly, comparison of multiple bull collections showed some to have aberrant x-ray scattering profiles consistent with incorrect disulfide bond formation. Together, these observations shed light on the biological functions of disulfide linkages for in vivo DNA packaging in sperm chromatin.

Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A

Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy.

Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm

Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together β-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond.

Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys2nd and Cys6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be applied in both health care and agricultural industries, and could lead to new methods for breeding fungus-resistant transgenic crops and antifungal transgenic silkworm strains.

Novel Cell Envelope Proteins Related to Copper Resistance in <i>Acidithiobacillus ferrooxidans</i>

The presence in At. ferrooxidans of canonical copper resistance determinants does not explain the extremely high copper concentrations this microorganism is able to tolerate. This suggests that At. ferrooxidans may have additional copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE and real time PCR (qRT-PCR). Results showed the up-regulation of RND-type Cus systems and different RND-type efflux pumps in At. ferrooxidans grown in the presence of copper, suggesting that these proteins may be implied in resistance to this metal. Furthermore, the up-regulation of putative periplasmatic disulfide isomerases was also seen in the presence of copper. These proteins are most likely involved in the formation and rearrangement of disulfide bonds in proteins in the periplasm. Copper ions catalyze the formation of incorrect disulfide bonds in proteins. However, the up-regulated disulfide isomerases found could restore native disufide bonds allowing cell survival. In conclusion, At. ferrooxidans may resist high copper concentrations by using additional copper resistance strategies in which cell envelope proteins are very important. This knowledge could be used to select the best fit members of the bioleaching community to attain more efficient industrial biomining processes.

Backbone 1H, 15N, 13C and side chain 13Cβ NMR chemical shift assignment of murine interleukin-10

Almost complete assignment of backbone (1)H, (13)C, (15)N and side chain (13)Cβ resonances for the immune-regulatory cytokine IL-10 is reported. The protein was overexpressed in Escherichia coli and was refolded from inclusion bodies. The point mutation C149Y was introduced to suppress incorrect disulfide bond formation and to improve protein refolding.

Hereditary pancreatitis caused by mutation-induced misfolding of human cationic trypsinogen: A novel disease mechanism

We investigated the biochemical properties and cellular expression of the c.346C>T (p.R116C) human cationic trypsinogen (PRSS1) mutant, which we identified in a German family with autosomal dominant hereditary pancreatitis. This mutation leads to an unpaired Cys residue with the potential to interfere with protein folding via incorrect disulfide bond formation. Recombinantly expressed p.R116C trypsinogen exhibited a tendency for misfolding in vitro. Biochemical analysis of the correctly folded, purified p.R116C mutant revealed unchanged activation and degradation characteristics compared to wild type trypsinogen. Secretion of mutant p.R116C from transfected 293T cells was reduced to approximately 20% of wild type. A similar secretion defect was observed with another rare PRSS1 variant, p.C139S, whereas mutants p.A16V, p.N29I, p.N29T, p.E79K, p.R122C, and p.R122H were secreted normally. All mutants were detected in cell extracts at comparable levels but a large portion of mutant p.R116C was present in an insoluble, protease-sensitive form. Consistent with intracellular retention of misfolded trypsinogen, the endoplasmic reticulum (ER) stress markers immunoglobulin-binding protein (BiP) and the spliced form of the X-box binding protein-1 (XBP1s) were elevated in cells expressing mutant p.R116C. The results indicate that mutation-induced misfolding and intracellular retention of human cationic trypsinogen causes hereditary pancreatitis in carriers of the p.R116C mutation. ER stress triggered by trypsinogen misfolding represents a new potential disease mechanism for chronic pancreatitis.

Preparative expression of secreted proteins in bacteria: status report and future prospects

The expression of heterologous secreted proteins in Escherichia coli is widely employed for laboratory and preparative purposes. Thanks to advances in expression technologies over the past 25 years, many mammalian proteins can now be produced routinely in secreted form with yields in the gram/litre scale. Nonetheless, ensuring efficient secretion across the inner membrane, and preventing proteolytic degradation, incorrect disulfide-bond formation and aggregation into periplasmic inclusion bodies, frequently presents significant challenges. Recent advances in the understanding of the periplasmic folding quality control system are leading to new strategies to facilitate the expression of heterologous secreted proteins. In parallel, protein design and directed evolution approaches are beginning to be exploited for engineering of the cellular protein folding machinery to achieve further improvements in protein expression.

Complete refolding of bovine β-lactoglobulin requires disulfide bond formation under strict conditions

β-Lactoglobulin (β-LG) denatured with 6 M guanidine hydrochloride (GdnHCl) containing a reducing agent and subsequently dialysed against phosphate-buffered saline (PBS) resulted in incomplete refolding of this protein despite the fact that the biological activity for retinol-binding was recovered to almost the same degree as that of the native molecule [Hattori, M., Ametani, A., Katakura, Y., Shimizu, M., Kaminogawa, S. J., Biol. Chem. 268 (1993) 22414–22419]. The enzyme probe method, evaluation of hydrophilicity values, in-gel mobility on SDS-PAGE, and evaluation of disulfide bonds with the Ellman method showed exposure of the hydrophobic region(s) and incorrect disulfide bond formation in such dialyzed β-LG molecules. We reveal in this present work that complete refolding could be attained by diluting denatured β-LG with PBS containing a reducing agent, before slow reoxidation of the sulfhydryl groups upon dialysis for gradient removal of the reducing agent in 6 steps. Complete renaturation was confirmed by analyzing the retinol-binding activity, CD spectra, intrinsic fluorescence, binding ability of monoclonal antibodies (mAbs), and SDS-PAGE. Step-by-step disulfide bond formation was considered to be critical for the complete refolding of denatured β-LG. Our method can contribute to establish a procedure for complete refolding of useful recombinant proteins in vitro without such biological aids as chaperones.

Positional expression effects of cysteine mutations in the Thermobifida fusca cellulase Cel6A and Cel6B catalytic domains

Ten cysteine mutations in the Thermobifida fusca cellulase Cel6A and Cel6B catalytic domains were created at different positions by site-directed mutagenesis for the purpose of testing their effects on thermostability and catalytic activity. The level of in vivo expression of these mutant proteins was significantly reduced by many of these mutations. There were positional effects of the mutations, which are probably due to the formation of incorrect disulfide bonds. These results increase our understanding of the role of cysteine residues in protein folding, and also help guide the choice of sites for other mutations in these enzymes.

A Free Cysteine Residue at the Dimer Interface Decreases Conformational Stability of Xenopus laevis Copper,Zinc Superoxide Dismutase

The two Cu,Zn superoxide dismutases from the amphibian Xenopus laevis (denoted XSODA and XSODB) display different heat sensitivities, XSODA being more thermolabile than XSODB. In this study, we have investigated the contribution of a free cysteine residue located close to the subunit interface of XSODA to its lower thermal stability. We have found that mutation of residue Cys 150 to Ala in XSODA makes the thermal stability of this enzyme comparable to that of the wild-type XSODB isoenzyme, while the introduction of a cysteine residue in the same position of XSODB renders this enzyme variant much more heat-sensitive. Differential scanning calorimetry experiments showed that XSODA has a melting temperature about 8.5°C lower than that of XSODB. On the contrary, the melting temperature of XSODACys150Ala is very close to that of XSODB, while the melting temperature of XSODBSer150Cys is even lower than that of wild-type XSODA. These data indicate that the free cysteine residue present in XSODA affects not only the reversibility of unfolding of the enzyme but also its conformational stability. We suggest that the large effect of the Cys 150 residue on XSODA stability might be due to incorrect disulfide bond formation or disulfide bond interchange during heat-induced unfolding rather than to alteration of the interaction between the enzyme subunits.

Folding, heterodimeric association and specific peptide recognition of a murine αβ T-cell receptor expressed in Escherichia coli

In a systematic study of the murine T-cell receptor UZ3-4, expressed and refolded from inclusion bodies in Escherichia coli, it was found that functional molecules can be obtained only under a very narrow set of conditions. The refolded T-cell receptor UZ3-4 specifically recognizes its cognate peptide (from mycobacterial Hsp60) in the context of H-2Db, but not another peptide bound to H-2Db, and the dissociation constant was determined by BIAcore as 10−4 M. Using T-cell receptor constructs comprising all extracellular domains (VαCα and VβCβ), found to be necessary for stability of the final product, significant amounts of native molecules were obtained only if the intermolecular Cα-Cβ disulfide bridge bond was deleted, even though the interaction between the complete α and β-chain was determined to be very weak and fully reversible (KD ≈ 10−7 to 10−6 M). Fusion of Jun and Fos to the constant domains also decreased the folding yield, because of premature association of intermediates leading to aggregation. Furthermore, only in a very narrow set of concentrations of oxidized and reduced glutathione, native disulfide bonds dominated. This shows that T-cell receptor domains are very prone to aggregation and misassociation during folding, compounded by incorrect disulfide bond formation. Once folded, however, the heterodimeric molecule is very stable and could be concentrated to millimolar concentration.

Origin of dimeric structure in the ribonuclease superfamily.

To enable application of postgenomic evolutionary approaches to understand the divergence of behavior and function in ribonucleases (RNases), the impact of divergent sequence on the divergence of tertiary and quaternary structure is analyzed in bovine pancreatic and seminal ribonucleases, which differ by 23 amino acids. In a crystal, seminal RNase is a homodimer joined by two "antiparallel" intersubunit disulfide bonds between Cys-31 from one subunit and Cys-32' from the other and having composite active sites arising from the "swap" of residues 1-20 from each subunit. Specialized Edman degradation techniques have completed the structural characterization of the dimer in solution, new cross-linking methods have been developed to assess the swap, and sequence determinants of quaternary structure have been explored by protein engineering using the reconstructed evolutionary history of the protein family as a guide. A single Cys at either position 32 (the first to be introduced during the divergent evolution of the family) or 31 converts monomeric RNase A into a dimer. Even with an additional Phe at position 31, another residue introduced early in the seminal lineage, swap is minimal. A hydrophobic contact formed by Leu-28, however, also introduced early in the seminal lineage, increases the amount of "antiparallel" connectivity of the two subunits and facilitates swapping of residues 1-20. Efficient swapping requires addition of a Pro at position 19, a residue also introduced early in the divergent evolution of the seminal RNase gene. Additional cysteines required for dimer formation are found to slow refolding of the protein through formation of incorrect disulfide bonds, suggesting a paradox in the biosynthesis of the protein. Further studies showed that the dimeric form of seminal RNase known in the crystal is not the only form in vivo, where a substantial amount of heterodimer is known. These data complete the acquisition of the background needed to understand the evolution of new structure, behavior, and function in the seminal RNase family of proteins.

Competition between DsbA-mediated oxidation and conformational folding of RTEM1 beta-lactamase.

Similar to other proteins of the periplasm of Escherichia coli, TEM 1 beta-lactamase contains only a single disulfide bond. It can fold to its native conformation in both the presence and the absence of this disulfide bond. The GdmC1-dependent equilibrium unfolding of beta-lactamase in vitro is well described by a N reversible I reversible U three-state model in which the native protein (N) first reacts to an intermediate of the molten globule type (I) and then to the unfolded state (U). We find that the disulfide bond of beta-lactamase stabilizes I relative to U, but does not change the stability of N relative to I. The I reversible U transition is an extremely rapid reaction for both reduced and oxidized beta-lactamase, but the N reversible I folding kinetics are slow and identical in the presence and the absence of the disulfide bond. This insensitivity of the N reversible I equilibrium and kinetics suggests that the region around the disulfide bond is already native-like folded and is presumably buried in the intermediate I, prior to the slow and rate-limiting events of folding. This was confirmed by measuring the stability of the disulfide bond, which, to a first approximation, is identical in N and I. In native, reduced beta-lactamase, the thiol groups are inaccessible for oxidation by DsbA, but at the stage of the molten globule intermediate I oxidation is still possible, because I is in fast exchange with the unfolded protein U. The introduction of the disulfide bond into beta-lactamase by DsbA competes with conformational folding at the stage of the final slow steps in the folding of the reduced protein. The major problem in the oxidation of proteins with one or two disulfide bonds (such as beta-lactamase) is not the formation of incorrect disulfide bonds, but the premature burial of the thiol groups by the rapid conformational folding of the reduced protein. DsbA, the major thiol/ disulfide isomerase of the bacterial periplasm, meets this problem. It is a very strong oxidant, and its reaction with cysteine residues in unfolded proteins is extremely fast.

Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants

The nucleotide sequence encoding the scorpion insectotoxin I 5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I 5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I 5A produced using the bacteria secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I 5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I 5A was unstable in bacterial cells. I 5A secreted from yeast using an α-factor signal sequence was shown to have an N-terminal (Glu-Ala) 2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I 5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.