Intrachain Disulfide Bonds Research Articles

Characterization and analysis of scFv-IgG bispecific antibody size variants

ABSTRACTBispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

Structural Changes and Aggregation Mechanisms of Two Different Dimers of an IgG2 Monoclonal Antibody

Protein therapeutics, monoclonal antibodies (mAbs) in particular, are large, structurally complex molecules that are prone to numerous modes of degradation during their production and long-term storage. Physical degradation via protein aggregation is a major concern when developing protein therapeutic candidates for clinical use. A dimer is perhaps the simplest element of protein aggregation, and thus, a better understanding of protein dimers in terms of their structures, intermolecular interactions, and chemical nature will help in the development of rational strategies for reducing aggregation propensity. In this study, two different mAb dimers were generated from an IgG2 monoclonal antibody solution, i.e., a native dimer generated under long-term storage and a thermal dimer from a thermal stress condition. Both IgG2 dimers were characterized in terms of their chemical and physical properties, bioactivity, and conformational dynamics. The native IgG2 dimer was formed mainly through noncovalent association. It displayed minimal differences in biophysical properties and higher-order structure compared to the monomer yet showed compromised in vitro potency, likely because of steric hindrance. In contrast, the thermal IgG2 dimer was mainly disulfide-linked, but even so, no new non-native disulfide bonds were detected by peptide mapping. Two regions within the Fc-CH2 domain of the thermal IgG2 dimer exhibited significantly increased flexibility as measured by hydrogen-deuterium exchange mass spectrometry, and notably, these regions are connected by an intrachain disulfide bond under natively folded conditions. These findings provide a better understanding of dimer formation under long-term storage and thermal stress conditions for this IgG2 mAb, and possible aggregation mechanisms are discussed.

An All-Atomistic Molecular Dynamics Study to Determine the Structural Importance of Disulfide Bonds in Immunoglobulin G and Bovine Serum Albumin

A fully-atomistic molecular dynamics study was performed to determine the importance of disulfide bonds on the stability of immunoglobulin G (IgG) and bovine serum albumin (BSA).The transferability of a previous prescreening methodology to assess contributions from individual disulfide bonds on conformational stability was tested on both proteins. In IgG, it was apparent that inter-chain and intra-chain disulfide bonds play different roles in maintaining structure, evidenced by clear separation of inter-chain cysteine residues upon cleavage of disulfide bonds. In BSA, a set of double disulfide bonds required both to be broken in order to observe significant structural changes, equivalently seen in a previous study of human serum albumin (HSA), a structurally similar protein. Structural analysis of IgG showed deviations in distances between domains, while analysis of BSA suggested more local structural changes. This work helps confirm the efficacy and reproducibility of the prescreening methodology on both a novel, larger protein such as IgG and a more homologous (to HSA), globular protein such as BSA. The results provide insight into the role of specific disulfide bonds in the stability of IgG and BSA. KEYWORDS: Molecular Dynamics; Atomistic Simulations; Immunoglobulin G; Bovine Serum Albumin; Disulfide Bonds

Abstract 65: RESPECT (REsidue-SPEcific Conjugation Technology): A platform technology utilizing native cysteine and lysine residues for the generation of homogeneous antibody-drug conjugates

Abstract The random conjugation of toxins, dyes, peptides, or other payloads to monoclonal antibodies often targets free thiol groups generated by partial reduction methods or lysine residues using succinimide- or isothiocyanate-based chemistry. There remains a need for conjugation technologies targeting specific amino acid residues as a way to produce a homogeneous antibody-drug conjugate (ADC) product with a defined drug-to-antibody ratio (DAR). Our REsidue-SPEcific Conjugation Technology (RESPECT) utilizes two methods by which payloads can be conjugated to specific residues in an antibody. Our cysteine-specific conjugation method exploits a unique intrachain disulfide bond in the light chain of rabbit antibodies between residues 80 and 171 of the variable and constant domains, respectively. Our humanization strategy allows retention of the cysteine at position 80 with a free thiol group that is both amenable for residue-specific conjugation and compatible with optimal antibody biophysical properties including antigen binding and structural stability. This platform has been optimized via antibody engineering strategy stems from in silico modeling, extensive mutagenesis, and crystallographic studies, which have allowed defining the contribution of neighboring residues to the retention of a reactive thiol group as well as the desired humanized antibody’s properties. Our C-terminal lysine-specific linkage method employs the transglutaminase enzyme that catalyzes the formation of a stable isopeptide bond between the γ-carboxyamide group (acyl donor) of a glutamine and the ϵ-amino group (acyl acceptor) of a lysine. While we found no acyl acceptor sites in recombinant wild-type IgG, all antibodies investigated lacked the C-terminal Lys447 due to cleavage by carboxypeptidase B in the antibody production cell line. Blocking the cleavage of Lys447 by addition of a C-terminal amino acid resulted in transamidation of Lys447 by a variety of acyl donor substrates in the presence of any non-acidic, non-proline amino acid residue at position 448. Antibody-drug conjugates (ADCs) prepared using our RESPECT technology targeting the tumor associated-mesothelin protein produced uniform drug-to-antibody ratios (DAR) and were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model. Citation Format: Earl Albone, Jared Spidel, Xin Cheng, Young Chul Park, Sara Jacob, Arielle Verdi, Andrew Milinichik, Ben Vaessen, J. Bradford Kline, Luigi Grasso. RESPECT (REsidue-SPEcific Conjugation Technology): A platform technology utilizing native cysteine and lysine residues for the generation of homogeneous antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 65. doi:10.1158/1538-7445.AM2017-65

Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi.

Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization. Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection. We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis. We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.

The SAMHD1 dNTP Triphosphohydrolase Is Controlled by a Redox Switch.

Proliferative signaling involves reversible posttranslational oxidation of proteins. However, relatively few molecular targets of these modifications have been identified. We investigate the role of protein oxidation in regulation of SAMHD1 catalysis. Here we report that SAMHD1 is a major target for redox regulation of nucleotide metabolism and cell cycle control. SAMHD1 is a triphosphate hydrolase, whose function involves regulation of deoxynucleotide triphosphate pools. We demonstrate that the redox state of SAMHD1 regulates its catalytic activity. We have identified three cysteine residues that constitute an intrachain disulfide bond "redox switch" that reversibly inhibits protein tetramerization and catalysis. We show that proliferative signals lead to SAMHD1 oxidation in cells and oxidized SAMHD1 is localized outside of the nucleus. Innovation and Conclusions: SAMHD1 catalytic activity is reversibly regulated by protein oxidation. These data identify a previously unknown mechanism for regulation of nucleotide metabolism by SAMHD1. Antioxid. Redox Signal. 27, 1317-1331.

Generation of therapeutic immunoconjugates via Residue-Specific Conjugation Technology (RESPECT) utilizing a native cysteine in the light chain framework of Oryctolagus cuniculus

ABSTRACTThe conjugation of toxins, dyes, peptides, or proteins to monoclonal antibodies is often performed via free thiol groups generated by either partial reduction methods or engineering free cysteine residues into the antibody sequence. Antibodies from the rabbit Oryctolagus cuniculus have an additional intrachain disulfide bond, whereby the light chain variable kappa domain is bridged to the constant kappa region between cysteine residues at positions 80 and 171, respectively. Chimerization of rabbit antibodies with human constant domains allows for the generation of a free thiol group at the light chain position 80 (C80) that can be used for site-specific conjugation. An efficient process for the purification and simultaneous removal of cysteinylation at the C80 site was developed. The unpaired C80 was shown to be efficiently conjugated using several different maleimido-based ligands. REsidue SPEcific Conjugation Technology (RESPECT) antibody-drug conjugates prepared using rabbit-human chimeric anti-human mesothelin rabbit antibodies and maleimido-PEG2-auristatin conjugated to C80 were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model.

Mass Spectrometry Characterization of a Novel Insulin Mimetic Peptide s597

Novel peptide-based drugs have recently gained high popularity, especially among athletes seeking ways to enhance their performance. Although the World Anti-Doping Agency (WADA) has banned the use of any nonapproved for human therapeutic use pharmacological substances in Sports, a huge variety of such peptides with potential performance-enhancing properties are available for sale in the black market and in illegal online websites. The difficulty of determination of these molecules in biological fluids depending on their low concentrations and their similarity to endogenous compounds has boosted their use not only among athletes, but also in the amateurs’ world. The goal of this study was to perform the mass spectrometry characterization of a novel s597 peptide drug purchased via an online store. The study was carried out using nanoscale liquid chromatography/quadrupole Orbitrap mass spectrometry with accurate mass determination and sequence analysis for both intact drug and after its trypsinolysis. The purchased drug was found to be a peptide with 31 amino acid residues, intra-chain disulfide bond and modified by C-terminal amide and N-terminal acetyl groups. The proposed sequence was consistent with s597 peptide, designed to be used as insulin receptor ligand mimetic.

Soluble Expression of Recombinant Nerve Growth Factor in Cytoplasm of Escherichia coli.

Pivotal roles of Nerve growth factor (NGF) in the development and survival of both neuronal and non-neuronal cells indicate its potential for the treatment of neurodegenerative diseases. However, investigation of NGF deficits in different diseases requires the availability of properly folded human β-NGF. In previous studies bacterial expression of hNGF demonstrated the feasibility of its overproduction. However, known limitations in the use of E. coli as an expression host for a protein with three intra-chain disulfide bonds were evident. Here an optimized system was developed to overexpress the soluble NGF in E. coli. The gene encoding the β subunit of mature hNGF was optimized based on E. coli codon preference and cloned into pET-32a expression vector providing His- and Trx- tags for detection and increasing the solubility of recombinant protein, respectively. The recombinant DNA was expressed in E. coli Origami (DE3), which enhances the correct formation of disulfide bonds in the cytoplasm of E. coli. Different culture conditions were evaluated to increase soluble expression of the target protein. The highest soluble expression level was achieved when E. coli Origami (DE3) cells expressing NGF were grown at 30ºC in TB medium with 0.2 mM IPTG induction at OD600nm = 1 for 4 h. Our results indicated that the recombinant NGF was successfully expressed as a soluble form.

The creation of synthetic crystalline bovine insulin.

The creation of synthetic crystalline bovine insulin.

Abstract 653: Antibody-drug conjugates: engineered N-terminal serine residues as a novel approach for site-specific conjugation

Abstract Emerging data from the growing number of antibody-drug conjugates (ADCs) in clinical studies reflects the critical contributions from each component of the ADC. The tumor-targeting antibody and the cytotoxic effector molecule are recognized as key elements of an ADC, but increasingly the linkers and linkage mechanisms are being seen as critical for optimizing the design of an ADC based on the specific properties of the target antigen, tumor types, and clinical indications. Traditionally, antibodies have been conjugated to their cytotoxic payloads either at their lysine residues or through cysteine residues accessed through reduction of intrachain disulfide bonds. Recently a number of site-specific conjugation methods have been reported. Here we describe the construction of antibodies and antibody fragments with engineered N-terminal serine residues to facilitate a novel approach for site-specific antibody conjugation. Serine residues can be engineered into at least four different N-terminal positions on an antibody. Serines are found at the N-termini of some naturally occurring human antibodies, but in most cases they must be introduced by either replacing the native N-terminal residue or by including an additional serine residue at the N-terminus. These modifications can be made on the light chain, heavy chain, or both to provide as many as 4 potential linkage sites per antibody. Modification at the N-terminus situates the linked cytotoxic molecule well outside of the antibody's target binding sites in the variable region complementarity determining regions (CDRs). Molecular modeling demonstrates that this is the case whether the linkage is on either the light or heavy chain N-termini. ELISA and flow cytometry binding data demonstrated that changing the N-terminal residue to serine itself had no impact on the binding properties of the parent antibody. Likewise, ADCs utilizing the N-terminal serine for conjugation had similar binding properties compared with lysine or engineered cysteine conjugates of the same antibody. Finally, in vitro cytotoxicity assays demonstrated that N-terminal serine conjugates were at least as potent as conventional ADCs against antigen-expressing tumor cells. The utilization of a variable region framework residue means that the same N-terminal serines used for antibody conjugation can be applied to antibody fragments. Due to the size and residue usage limitations of antibody fragments, conjugation of hydrophobic, cytotoxic agents to these molecules can be challenging. We have built and tested N-terminal serine conjugates with Fabs, scFvs, and sdAbs, establishing the utility of this approach across a wide range of antibody fragment formats. In conclusion, conjugation of engineered antibody N-terminal serine residues represents a promising approach to site-specific ADCs that is worthy of further development. Citation Format: Daniel Tavares, Lingyun Rui, Olga Ab, Luke Harris, Erin Maloney, Thomas Keating, Thomas Chittenden, Nathan Fishkin. Antibody-drug conjugates: engineered N-terminal serine residues as a novel approach for site-specific conjugation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2015-653

Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity toward human cancer cell lines

BackgroundThe objective of this study is to investigate the cytotoxic activity of three isolectins purified from the latex of Euphorbia trigona Miller. HypothesisAmong lectins are the ribosome-inactivating proteins (RIPs), which are potent inhibitors of protein synthesis in cells and in cell-free systems. ResultsThree isolectins, ETR1, ETR2 and ETR3, were purified by anion exchange chromatography. Both ETR1 and ETR3 yielded a single band on SDS–PAGE under reducing conditions, corresponding to a molecular weight of 32 g mol−1, while ETR2 yielded two bands corresponding to 31 and 33 g mol−1. When non-reducing conditions were used molecular weight decreased, indicating the presence of intrachain disulfide bonds. Size-exclusion chromatography revealed proteins of apparent molecular weight of 59–63 g mol−1, suggesting a dimeric nature, with subunits not being held together by disulfide linkage. ETR1, ETR2 and ETR3 hemagglutinated human, sheep and rat erythrocytes and this hemagglutination was specifically inhibited by galactose and its derivatives. The lectins studied were thermostable up to 60 °C and their observed activity was maintained across pH range 5–12. These lectins, from the latex of Euphorbia trigona, are potent inhibitors of eukaryotic protein synthesis in a cell-free system. Flow cytometry analysis revealed the antiproliferative activity of them toward A549, HeLa, H116, HL-60 cell lines. ConclusionEuphorbia trigona isolectins are RIPs with cytotoxic activity toward human cancer cell lines.

L-rhamnose-binding lectin from eggs of the Echinometra lucunter: Amino acid sequence and molecular modeling

An l-rhamnose-binding lectin named ELEL was isolated from eggs of the rock boring sea urchin Echinometra lucunter by affinity chromatography on lactosyl-agarose. ELEL is a homodimer linked by a disulfide bond with subunits of 11kDa each. The new lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as l-rhamnose, melibiose, galactose and lactose. The amino acid sequence of ELEL was determined by tandem mass spectrometry. The ELEL subunit has 103 amino acids, including nine cysteine residues involved in four conserved intrachain disulfide bonds and one interchain disulfide bond. The full sequence of ELEL presents conserved motifs commonly found in rhamnose-binding lectins, including YGR, DPC and KYL. A three-dimensional model of ELEL was created, and molecular docking revealed favorable binding energies for interactions between ELEL and rhamnose, melibiose and Gb3 (Galα1-4Galβ1-4Glcβ1-Cer). Furthermore, ELEL was able to agglutinate Gram-positive bacterial cells, suggesting its ability to recognize pathogens.

Thioredoxin superfamily and its effects on cardiac physiology and pathology.

A precise control of oxidation/reduction of protein thiols is essential for intact cardiac physiology. Irreversible oxidative modifications have been proposed to play a role in the pathogenesis of cardiovascular diseases. An imbalance of redox homeostasis with diminution of antioxidant capacities predisposes the heart to oxidant injury. There is growing interest in endoplasmic reticulum (ER) stress in the cardiovascular field, since perturbation of redox homeostasis in the ER is sufficient to cause ER stress. Because a number of human diseases are related to altered redox homeostasis and defects in protein folding, many research efforts have been devoted in recent years to understanding the structure and enzymatic properties of the thioredoxin superfamily. The thioredoxin superfamily has been well documented as thiol oxidoreductases to exert a role in various cell signaling pathways. The redox properties of the thioredoxin motif account for the different functions of several members of the thioredoxin superfamily. While thioredoxin and glutaredoxin primarily act as antioxidants by reducing protein disulfides and mixed disulfide, another member of the superfamily, protein disulfide isomerase (PDI), can act as an oxidant by forming intrachain disulfide bonds that contribute to proper protein folding. Increasing evidence suggests a pivotal role of PDI in the survival pathway that promotes cardiomyocyte survival and leads to more favorable cardiac remodeling. Thus, the thiol redox state is important for cellular redox signaling and survival pathway in the heart. This review summarizes the key features of major members of the thioredoxin superfamily directly involved in cardiac physiology and pathology.

Experimental and Computational Studies of Pulmonary Surfactant Protein SP-B Interacting with Lipid Bilayers

Pulmonary lung surfactant protein B (SP-B) is a 79 residue hydrophobic protein, from the Saposin superfamily. Saposin super family proteins share common features including 3 intra-chain disulfide bonds and 4-5 helical regions. SP-B appears to carry out its essential functions in respiration by binding to and modifying the structures of phospholipid bilayers and monolayers at the air-water interface. Due to difficulties arising from SP-B's extreme hydrophobicity, the 3D structure of full SP-B is not yet known. Thus we are using computational methods in combination with solid-state NMR to investigate the structure of SP-B. Our approach is to generate candidate structures via computational methods, predict the 15N spectra for the computed configurations and then compare them to the experimental NMR spectra.For the computational studies, we employ Mini-b, a construct of SP-B with known structure, and add the rest of residues to make a full length SP-B. We use several positions of SP-B in a POPC bilayer as initial structures. Calculations are carried out using GROMACS, with OPLS-aa, an all-atom force field, as well as with PACE, a hybrid model force field. Simulation methods include Molecular Dynamics (MD) and Replica Exchange Molecular Dynamics (REMD). For the experimental part, we use recombinantly expressed 15N labeled SP-B in mechanically oriented POPC bilayers to collect 1D 15N solid-state NMR spectra.

Clustering-based model of cysteine co-evolution improves disulfide bond connectivity prediction and reduces homologous sequence requirements.

Cysteine residues have particular structural and functional relevance in proteins because of their ability to form covalent disulfide bonds. Bioinformatics tools that can accurately predict cysteine bonding states are already available, whereas it remains challenging to infer the disulfide connectivity pattern of unknown protein sequences. Improving accuracy in this area is highly relevant for the structural and functional annotation of proteins. We predict the intra-chain disulfide bond connectivity patterns starting from known cysteine bonding states with an evolutionary-based unsupervised approach called Sephiroth that relies on high-quality alignments obtained with HHblits and is based on a coarse-grained cluster-based modelization of tandem cysteine mutations within a protein family. We compared our method with state-of-the-art unsupervised predictors and achieve a performance improvement of 25-27% while requiring an order of magnitude less of aligned homologous sequences (∼10(3) instead of ∼10(4)). The software described in this article and the datasets used are available at http://ibsquare.be/sephiroth. wvranken@vub.ac.be Supplementary material is available at Bioinformatics online.

A chromophore-containing agglutinin from Haliclona manglaris: Purification and biochemical characterization

A new chromophore-containing agglutinin (Haliclona manglaris agglutinin (HMA)) was isolated from the tropical sponge H. manglaris. HMA was purified by a combination of hydrophobic interaction chromatography and ion exchange chromatography. Native HMA is a heterotrimer formed by two β-chains (15kDa) and one α-chain (22kDa). HMA is a glycoprotein and possesses three intrachain disulfide bonds. Hemagglutinating activity of HMA was stable at neutral pH and temperatures up to 60°C. HMA was only inhibited by thyroglobulin. Mass spectrometry sequencing and Edman degradation revealed a unique amino acid sequence of about 30%. Moreover, HMA has an organic chromophore of 581Da, and this characteristic seems to be important to its antioxidant activity. Interestingly, while HMA showed no toxicity against Artemia nauplii and was unable to agglutinate bacterial cells, it did show a high capacity to protect β-carotene against oxidation. Thus, our findings suggest the putative involvement of HMA in the protection of the sponge against oxidation.

Intra-molecular reactions between cysteine sulfinyl radical and a disulfide bond within peptide ions

Cysteine sulfinyl radical (SO•Cys) is a reactive intermediate discovered in the inactivation of enzymes utilizing the glycyl/thiyl radical in their catalytic functions upon exposure to air. SO•Cys has been recently formed and investigated in the gas phase via mass spectrometry (MS), with the aim being to acquire direct experimental evidence of the radical’s intrinsic chemical reactivity. Ion/molecule reaction studies showed that SO•Cys was relatively chemically inert toward thiol (SH) and disulfide (SS) functional groups under the explored experimental conditions. Herein, we utilized intra-molecular reactions aided by collision-induced dissociation (CID) to overcome the limitations associated with the traditional bimolecular reactions and explore the reactivity of SO•Cys. Our results revealed a new reaction pathway in which the sulfinyl radical exchanged with an intrachain or interchain disulfide bond within a peptide ion, leading to the formation of a new disulfide bond and a sulfinyl radical. As a consequence, CID of peptide disulfide regio-isomers consisting of SO•Cys led to enhanced sequence information, however the disulfide bond linkage patterns could not be accurately assigned. This reaction pathway also has implications on disulfide bond scrambling in proteins initiated by a radical intermediate.

Expression, purification, and biological activity of the recombinant pramlintide precursor.

Pramlintide is an artificially designed protein which has the same function as amylin in human body. This protein is extremely difficult to synthesize through prokaryotic expression method because of its two essential active sites, intrachain disulfide bond and C-terminal amide group. Since α-amidating monooxygenase is widely distributed in human and animal, it is possible to use pramlintide precursor with an additional C-terminal glycine (PAG), which is the potential substrate of α-amidating monooxygenase, for in vivo applications. The recombinant PAG was expressed in Escherichia coli using the small ubiquitin-related modifier (SUMO) as the molecular chaperone, and the optimal fusion expression level reached to 36.3% of the total supernatant protein. Under optimal conditions in a 10-L fermentor, the recombinant PAG was obtained with a purity of greater than 95%, and the average expression level was reached to 20 mg/L. The authenticity and the intrachain disulfide bridge of PAG were confirmed by Western blotting and matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS) as well as N-terminal sequencing of protein. Based on an L6 myoblast cell model in vitro and an animal model of gastric emptying in vivo, the results of activity revealed that PAG showed a lower biological activity in vitro but has almost the same activity as the chemically synthesized pramlintide in vivo.

Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57

Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57VL85S and FV57VL84S. Furthermore, the disulfide bonds in the light chain of FV57, FV57VL85S and FV57VL84S were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.