Protein Chemical Methods Research Articles

Vaccine Based on Recombinant Fusion Protein Combining Hepatitis B Virus PreS with SARS-CoV-2 Wild-Type- and Omicron-Derived Receptor Binding Domain Strongly Induces Omicron-Neutralizing Antibodies in a Murine Model.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum-hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Protein Chemistry Combined with Mass Spectrometry for Protein Structure Determination.

The advent of soft-ionization mass spectrometry for biomolecules has opened up new possibilities for the structural analysis of proteins. Combining protein chemistry methods with modern mass spectrometry has led to the emergence of the distinct field of structural proteomics. Multiple protein chemistry approaches, such as surface modification, limited proteolysis, hydrogen-deuterium exchange, and cross-linking, provide diverse and often orthogonal structural information on the protein systems studied. Combining experimental data from these various structural proteomics techniques provides a more comprehensive examination of the protein structure and increases confidence in the ultimate findings. Here, we review various types of experimental data from structural proteomics approaches with an emphasis on the use of multiple complementary mass spectrometric approaches to provide experimental constraints for the solving of protein structures.

Use of PASEF for Accelerated Protein Sequence Confirmation and De Novo Sequencing with High Data Quality.

Biopharmaceutical sequences can be well confirmed by multiple protease digests-e.g., trypsin, elastase, and chymotrypsin-followed by LC-MS/MS data analysis. High quality data can be used for de novo sequencing as well. PASEF (Parallel Accumulation and Serial Fragmentation) on the timsTOF instrument has been used to accelerate proteome and protein sequence studies and increase sequence coverage concomitantly.Here we describe the protein chemical and LC-MS methods in detail to generate high quality samples for sequence characterization from only 3 digests. We applied PASEF to generate exhaustive protein sequence coverage maps by combination of results from the three enzyme digests using a short LC gradient. The data quality obtained was high and adequate for determining antibody sequences de novo.Nivolumab and dulaglutide were digested by 3 enzymes individually. For nivolumab, 94/94/90% sequence coverage and 86/84/85% fragment coverage were obtained from the individual digest analysis with trypsin/chymotrypsin/elastase, respectively. For dulaglutide, 96/100/90% sequence coverage and 92/90/83% fragment coverage were obtained. The merged peptide map from the 3 digests for nivolumab resulted in ∼550 peptides; enough to safely confirm the full sequences and to determine the nivolumab sequence de novo.

Gelation Methods to Assemble Fibrous Proteins.

Gelation is an efficient way to fabricate fibrous protein materials. Briefly, it is an aggregation process where protein molecules assembly from a random structure into an organized structure such as nanofibrillar networks. According to their mechanisms, the fibrous proteins gelation can be classified into physical gelation and chemical gelation. The physical gelation is formed by the conformational transformation of fibroin proteins, which can be triggered by temperature, concentration, pH, or shear force. On the other hand, the chemical gelation is to cross-link fibrous proteins through chemical and/or enzymatic reactions. In this chapter, we summarize the protocols for preparing fibrous protein hydrogels, including both physical and chemical methods. The mechanisms of these gelation methods are also highlighted.

Protein Chemical Cross-linking/Mass Spectrometry: From raw data to fully immersive visualizations

Proteins are the base component that make all living organisms function. They are made up of amino acids and are responsible for the structure, function, and regulation of organisms’ tissues and organs. They can exist alone or as part of a multi-unit structure. They interact with other proteins to form stable or transient complexes. Protein function, such as transporting signals or converting energy, is determined by its structure, therefore being critical that we understand protein complexes at the cellular level. Chemical cross-linking combined with mass spectrometry is an established method in protein chemistry to explain lowresolution 3D protein structures and interacting sequences in protein complexes. Chemical cross-linking of a protein enables scientists to understand how the protein folds, whereas intermolecular cross-linking between different proteins enables to determine which components interact and how and where they physically contact each other. Mass spectrometry is used to acquire distance information and distance constraints within a molecule. The challenge is to how to best take advantage of the information provided by these methods utilizing novel visualization analytics that can help explore much more detailed information on protein structure than traditional biomechanical methods, such as the Edman degradation which sequentially removes one residue at a time and the structures are observed through chromatographic procedures. Additionally, data resulting from chemical cross-linking in most cases come in a raw format that makes it hard for the user to absorb the data and get a bigger picture of the existing interactions as well as getting a close insight on smaller reactions. This has motivated the department of Bioinformatics at the University of Arkansas at Little Rock to develop an algorithm named “X-Linked Peptide Mapping” that allows the analysis of the data and identify interacting peptides (short chains of amino acids) in an easier, more accurate way avoiding the limitations mentioned before. Since the field of chemical cross-linking mass spectrometry of protein-protein interactions hasn’t been explored visually in a comprehensive way before, the results encouraged us at the Emerging Analytics Center at the University of Arkansas at Little Rock to develop an interactive web application that allows the user to visualize the results of the analysis including 2D information representations as well as 3D structural modeling representation of the interactions,. This paper presents preliminary research in this area, including the development of a prototype immersive environment to explore the molecular structures.

Evidence that ubiquitylated H2B corrals hDot1L on the nucleosomal surface to induce H3K79 methylation.

Ubiquitylation of histone H2B at lysine 120 (H2B-Ub), a post-translational modification first discovered in 1980, plays a critical role in diverse nuclear processes including the regulation of transcription and DNA damage repair. Herein, we use a suite of protein chemistry methods to explore how H2B-Ub stimulates hDot1L-mediated methylation of histone H3 on lysine 79 (H3K79me). By using semisynthetic ‘designer' chromatin containing H2B-Ub bearing a site-specifically installed photocrosslinker, here we report an interaction between a functional hotspot on ubiquitin and the N-terminus of histone H2A. Our biochemical studies indicate that this interaction is required for stimulation of hDot1L activity and leads to a repositioning of hDot1L on the nucleosomal surface, which likely places the active site of the enzyme proximal to H3K79. Collectively, our data converge on a possible mechanism for hDot1L stimulation in which H2B-Ub physically ‘corrals' the enzyme into a productive binding orientation.

The Rise of Mass Spectrometry and the Fall of Edman Degradation.

Featured Article: Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal Chem 1996;68:850–8.2 Many of today's key proteins in biology and biomedicine were originally identified by protein chemical methods. Frederick Sanger obtained his first Nobel Prize for determining the sequence of insulin in the 1950s, and the Edman degradation, in which one amino acid after another is cleaved off the end of a protein or peptide and identified by HPLC, reigned supreme into the 1990s. My background is in mass spectrometry (MS),3 in particular electrospray ionization (1), for which my advisor John Fenn won a Nobel Prize. On coming to the European Molecular Biology Laboratory (EMBL) in Heidelberg as a young group leader, I was determined to challenge what I saw as the old-fashioned chemical methods with high-tech and cool MS technology. Little did I know what I was up against, because this would require not only highly sensitive peptide sequencing by MS …

Application of the protein semisynthesis strategy to the generation of modified chromatin.

Histone proteins are subject to a host of posttranslational modifications (PTMs) that modulate chromatin structure and function. Such control is achieved by the direct alteration of the intrinsic physical properties of the chromatin fiber or by regulating the recruitment and activity of a host of trans-acting nuclear factors. The sheer number of histone PTMs presents a formidable barrier to understanding the molecular mechanisms at the heart of epigenetic regulation of eukaryotic genomes. One aspect of this multifarious problem, namely how to access homogeneously modified chromatin for biochemical studies, is well suited to the sensibilities of the organic chemist. Indeed, recent years have witnessed a critical role for synthetic protein chemistry methods in generating the raw materials needed for studying how histone PTMs regulate chromatin biochemistry. This review focuses on what is arguably the most powerful, and widely employed, of these chemical strategies, namely histone semisynthesis via the chemical ligation of peptide fragments.

Chemical Polysialylation of Recombinant Human Proteins.

Design of drug with prolonged therapeutic action is one of the rapid developing fields of modern medical science and required implementation of different methods of protein chemistry and molecular biology. There are several therapeutic proteins needing increasing of their stability, pharmacokinetic, and pharmacodynamics parameters. To make long-live DNA-encoded drug PEGylation was proposed. Alternatively polysialic (colominic) acid, extracted from the cell wall of E. coli, fractionated to the desired size by anion-exchange chromatography and chemically activated to the amine-reactive aldehyde form, may be chemically attached to the polypeptide chain. Conjugates of proteins and polysialic acid generally resemble properties of protein-PEG conjugates, but possess significant negative net charge and are thought to be fully degradable after endocytosis due to the presence of intracellular enzymes, hydrolyzing the polysialic acid. Complete biodegradation of the polysialic acid moiety makes this kind of conjugates preferable for creation of drugs, intended for chronic use. Here, we describe two different protocols of chemical polysialylation. First protocol was employed for the CHO-derived human butyrylcholinesterase with optimized for recovery of specific enzyme activity. Polysialic acid moieties are attached at various lysine residues. Another protocol was developed for high-yield conjugation of human insulin; major conjugation point is the N-terminal residue of the insulin's light chain. These methods may allow to produce polysialylated conjugates of various proteins or polypeptides with reasonable yield and without significant loss of functional activity.

Laying the groundwork for proteomics: Mass spectrometry from 1958 to 1988

The development of mass spectrometric methods in peptide and protein chemistry in the author's laboratory is reviewed, from the first determination of the amino acid sequence of small peptides in the late 1950s to its use for the determination of the primary structure of large proteins by a combination of mass spectrometry and DNA sequencing in the late 1980s. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

Features of actomyosin superprecipitation reaction in different environment conditions

The influence of divalent cations (Cd 2+ , Zn 2+ , Sr 2+ ) and staphylococcus protein A on the superprecipitation reaction of cardiac muscle actomyosin was investigated using methods of preparative protein chemistry, optical spectroscopy and mechanokinetic analysis. It was shown that the metal ions in the range of concentrations 0.1–5 mM inhibit the Mg 2+ -dependent SPP reaction of actomyosin cardiac muscle and alter the kinetic parameters of this process. It was revealed that protein A modulates superprecipitation dynamics decreasing the maximal value of optical density and time of its half-maximum achievement as well as starting and normalized superprecipitation rates. Thus, investigated factors are able to influence the actin myosin interaction changing their functional parameters of proteins from cardiomyocyte contractile complex. The kinetic characteristics of actomyosin superprecipitation are sensitive to the influence of physicochemical and pharmacological factors and can be used to study their influence on the molecular mechanisms of muscle contraction. Keywords: actomyosin, superprecipitation, kinetic parameters, metal ions, S. aureus protein A, cardiac muscle.

Streptavidin–biotin technology: improvements and innovations in chemical and biological applications

Streptavidin and its homologs (together referred to as streptavidin) are widely used in molecular science owing to their highly selective and stable interaction with biotin. Other factors also contribute to the popularity of the streptavidin-biotin system, including the stability of the protein and various chemical and enzymatic biotinylation methods available for use with different experimental designs. The technology has enjoyed a renaissance of a sort in recent years, as new streptavidin variants are engineered to complement native proteins and novel methods of introducing selective biotinylation are developed for in vitro and in vivo applications. There have been notable developments in the areas of catalysis, cell biology, and proteomics in addition to continued applications in the more established areas of detection, labeling and drug delivery. This review summarizes recent advances in streptavidin engineering and new applications based on the streptavidin-biotin interaction.

Open questions: two challenges in chemical biology - chemical engineering and the science of diet

A respected scientist was once asked what research project he thought he would be working on in five years. He responded that if he knew the answer, he would be working on it now. As the great New York Yankee baseball catcher Yogi Berra said, “It’s difficult to make predictions, especially about the future.” It is thus with considerable trepidation that I offer a short discussion about two research topics at the chemistry/biology interface that I believe may offer opportunities for major advances: protein chemical engineering and chemoprevention. Understanding protein dynamics, relating structure and function, and deciphering the specific contributions of post-translational modifications has attracted intense effort but has yielded slow progress. Part of the challenge has been the limitation of the genetic code and the natural 20 amino acids. While making recombinant proteins has become relatively routine, the inability to introduce biophysical probes, isotopic labels, unnatural amino acids, and post-translational modifications site-specifically into proteins has been a major challenge historically. Recently developed methods for the chemical manipulation of proteins using semisynthetic strategies and unnatural amino acid mutagenesis promise to be increasingly powerful in the analysis of protein structure and function. Exploitation of inteins, sortase, and chemoselective modification reactions have already been valuable in modifying enzymes and histone proteins for the introduction of a wide range of post-translational modifications and fluorescent probes. Unnatural amino acid mutagenesis by the development of evolved aminoacyl tRNA synthases and nonsense suppression approaches have produced a range of chemically altered proteins. As synthetic biology develops organisms with artificial genomes lacking particular nonsense codons, it should be possible to create cells and animals with a wide range of chemical modifications. Such techniques are likely to be of special utility for membrane proteins, which are often very complicated to study. As these protein chemical methods are adapted to cellular and in vivo experiments, they should be transformative in illuminating the contributions such proteins make to cell signaling and gene regulation. Another emerging area for chemistry/biology interface research is in disease prevention using natural products and their analogs. The search for, and application of, chemical substances in dietary plants has garnered increasing attention among clinical and basic science investigators in recent years. Whereas herbal products have had a rich history as a source of pharmaceuticals in Chinese and Western medicine, the focus on prevention rather than treatment is gaining currency with researchers and the general public. As health care costs rise inexorably, massive late stage expenditures in the last year of life to stave off advanced illness are unsustainable. It is generally recognized that identifying a health problem at an early phase offers the best chance for successful and cost-effective therapeutic intervention. In this light, chemoprevention through dietary adjustment is an attractive public health strategy. However, there has been a justified skepticism surrounding research in this area, which has sometimes lacked rigor and molecular detail. To move this field from the phenomenological to the mechanistic, defining key molecular pathways that are affected by chemoprevention compounds is critical. Such has been elegantly demonstrated with the broccoli compound sulforaphane and its interactions with the Keap1/Nrf2 anti-oxidant response pathway. Dozens of clinical trials are underway for myriad conditions to determine if broccoli extracts rich in sulforaphane can protect against cancer, inflammatory diseases, and photo-irradiation-induced skin damage. Going forward, there are likely to be many molecular mechanisms yet to be discovered that provide opportunities for chemoprevention. There is also much to still be learned in nutritional science and metabolomics that could have a major impact on public health.

Rhodocetin-αβ–induced Neuropilin-1–cMet Association Triggers Restructuring of Matrix Contacts in Endothelial Cells

The snake venom component rhodocetin-αβ (RCαβ) stimulates endothelial cell motility in an α2β1 integrin-independent manner. We aimed to elucidate its cellular and molecular mechanisms. We identified neuropilin-1 (Nrp1) as a novel target of RCαβ by protein-chemical methods. RCαβ and vascular endothelial growth factor (VEGF)-A avidly bind to Nrp1. Instead of acting as VEGF receptor 2 coreceptor, Nrp1 associates upon RCαβ treatment with cMet. Furthermore, cell-based ELISAs and kinase inhibitor studies showed that RCαβ induces phosphorylation of tyrosines 1234/1235 [corrected] and thus activation of cMet. Consequently, paxillin is phosphorylated at Y31, which is redistributed from streak-like focal adhesions to spot-like focal contacts at the cell perimeter, along with α2β1 integrin, thereby regulating cell-matrix interactions. Cortactin is abundant in the cell perimeter, where it is involved in the branching of the cortical actin network of lamellipodia, whereas tensile force-bearing actin stress fibers radiating from focal adhesions disappear together with zyxin, a focal adhesion marker, on RCαβ treatment. Our data demonstrate that (1) Nrp1 is a novel target for venom components, such as RCαβ; (2) Nrp1 coupled to cMet regulates the type of cell-matrix interactions in a manner involving paxillin phosphorylation; and (3) altered cell-matrix interactions determine endothelial cell migration and cellular force management.

New actinoporins from sea anemone Heteractis crispa: Cloning and functional expression

A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10-27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins.

Newborn Screening for Hemoglobin Disorders

The epidemiology of hemoglobinopathies in the United States reflects recent changes in the demographic make-up of the country’s population. The incidence of thalassemias, in particular, has risen in parallel with immigration of high-risk populations. With an increase in Asian immigration by 2000% in the past three decades, hemoglobinopathies such as Hb H (β4) and Hb E-β-thalassemia (β-thal) have acquired proportionally greater clinical significance in several states, including California. Newborn screening (NBS) for hemoglobinopathies allows for the identification of affected individuals so that prophylactic treatment and comprehensive care may be provided prior to the development of clinical complications. Although the more common hemoglobin (Hb) variants, such as Hb S or Hb C, are readily identified by NBS programs, other clinically significant Hb disorders often remain unresolved or are not reported by NBS programs.The state of California mandates NBS and follow-up for thalassemic disorders as well as sickle cell disease. A centralized laboratory for diagnostic testing and rapid reporting of NBS results is a necessary component of a comprehensive NBS program. Confirmatory diagnostic testing for the California NBS program is provided by the Hemoglobinopathy Reference Laboratory at Children’s Hospital & Research Center Oakland (CHRCO), Oakland, CA, USA. Using electrophoresis, high performance liquid chromatography (HPLC) and DNA-based methods, the laboratory annually performs diagnostic testing for sickle cell disease, β-thal and Hb H disease on more than 1,000 samples. While most screening results are straightforward, 5% of newborns identified with a presumptive hemoglobinopathy by primary screening are modified after confirmatory testing. The availability of rapid and reliable genotyping assays to complement traditional protein chemistry methods for hemoglobinopathy screening has facilitated the diagnosis of thalassemias and sickling disorders in newborns with ambiguous screening results. Together with coordinated follow-up systems in place, the Hemoglobinopathy Reference Laboratory has contributed to the success of the NBS program in the rapid identification and delivery of care to newborns with clinically significant Hb disorders.

Hairpin conformation of an 11-mer peptide

An 11-mer peptide taken from a subsequence of the human protein ubiquitin was synthesized. The peptide has been fully characterized by NMR spectroscopy using chemical shift analysis and by NOE measurements. The conformation was calculated using state of the art MD methods of protein chemistry. A hairpin conformation was found which is to a large part identical with the structure of this peptide fragment within the human ubiquitin. The surprising result that already an 11-mer peptide adopts a hairpin conformation in aqueous solution is discussed in terms of initials sites for protein folding.

RhDNase I is a Well Characterized Protein

rhDNase I (Pulmozyme, Dornase Alpha) is a glycoprotein composed of 260 amino acids with two disulphide bridges and two sites for N-linked glycosylation. The purity and potency of the enzyme was demonstrated by a variety of methods. Techniques available today for the characterization of proteins provide a wealth of information. The techniques that proved most useful in the characterization of rhDNase I include peptide mapping/mass spectrometry, cation-exchange chromatography to measure deamidation of asparagine-74, potency by hydrolysis of a methyl green: DNA complex, high pH anion-exchange chromatography to profile released oligosaccharides and isoelectric focusing to compare charge heterogeneity. As clinical trials progressed it was necessary to modify the production of the protein to accommodate increased clinical demands. During the course of the phase III trials the fermentation vessel used in the production process was increased from 1000 L to the current market scale of 12000 L. Additional modifications of the purification steps to utilize larger fluid volumes were also necessary. Utilizing the above analytical techniques we were able to demonstrate the comparability of rhDNase I produced at the two scales without additional double-blind placebo-controlled clinical trials. The ability to use state-of-the-art protein chemistry methods to assess the comparability of rhDNase I made at two process scales resulted in rapid, worldwide approval of rhDNase I for the treatment of cystic fibrosis.

Myeloperoxidase-catalyzed oxidative inactivation of human kininogens: the impairment of kinin-precursor and prekallikrein-binding functions

Bradykinin-related vasoactive peptides (kinins) are important mediators of local and systemic inflammatory reactions. However, at local inflammatory foci, the production of kinins from proteinaceous precursors (kininogens) can be affected by reactive oxygen species released by phagocyte cells. One of the predominant oxidants at these places is hypochlorous acid which is formed from hydrogen peroxide and chloride ions by neutrophil myeloperoxidase. In this study, inactivation of human kininogens after oxidation with the myeloperoxidase-H₂O₂-chloride system was observed and analyzed by protein chemistry methods. The kinin release from oxidized kininogens by major kinin-producing enzymes, plasma and tissue kallikreins, proceed with a very low rate. This effect was assigned to apparent inability of kallikreins to process the kinin N-terminus owing to the conversion of the adjacent Met-361 residue to methionine sulfoxide. Additionally, the oxidized high-molecular mass kininogen lost its natural ability to bind plasma prekallikrein. This effect was assigned to the oxidation of Trp-569 residue within the prekallikrein-binding region which is subsequently destructed owing to cleavage of the peptide bond after that residue. One possible pathophysiological consequence of the described effects on kininogens could be the impairment of the normal assembly and triggering of the kinin-forming system on defense cell surfaces.

Design of an in vivo Cleavable Disulfide Linker in Recombinant Fusion Proteins

In order to achieve optimal biological activity and desired pharmacokinetic profiles, a dithiocyclopeptide linker was designed for in vivo release of protein domains from a recombinant fusion protein. This novel in vivo cleavable disulfide linker, based on a dithiocyclopeptide containing a thrombin-sensitive sequence and an intramolecular disulfide bond, was inserted between transferrin and granulocyte colony-stimulating factor (G-CSF) recombinant fusion protein domains. After expression of the fusion protein, G-C-T, from HEK293 cells, thrombin treatment in vitro generated a fusion protein linked via a reversible disulfide bond that was quickly cleaved in vivo, separating the protein domains. After release from the fusion protein, free G-CSF exhibited an improved biological activity in a cell proliferation assay. Although reversible disulfide bonds are commonly used in protein chemical conjugation methods, to our knowledge this report is the first example of the construction of a recombinant fusion protein with a disulfide linkage for the release of the functional domain. This linker design can be adapted to diverse recombinant fusion proteins in which in vivo separation of protein domains is required to achieve an improved therapeutic effect and a desirable pharmacokinetic profile and biodistribution of the functional domain.