title: Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.
abstract: Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (VH+CH1) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli.
authors.affiliation: Department of Systems Immunology, Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon 200-701, South Korea.
authors.forename: Hyeon-Ju
authors.initials: HJ
authors.lastname: Kang
authors.affiliation: Department of Systems Immunology, Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon 200-701, South Korea.
authors.forename: Hye-Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: AprilBio Co., Ltd, Kangwon National University, Rm. A202, Biomedical Science Building, Chuncheon, Gangwon 200-701, South Korea.
authors.forename: Mun-Sik
authors.initials: MS
authors.lastname: Jung
authors.affiliation: AprilBio Co., Ltd, Kangwon National University, Rm. A202, Biomedical Science Building, Chuncheon, Gangwon 200-701, South Korea.
authors.forename: Jae-Kyu
authors.initials: JK
authors.lastname: Han
authors.affiliation: Department of Systems Immunology, Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon 200-701, South Korea; AprilBio Co., Ltd, Kangwon National University, Rm. A202, Biomedical Science Building, Chuncheon, Gangwon 200-701, South Korea; Institute of Biological Technology, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon 200-701, South Korea. Electronic address: chash@kangwon.ac.kr.
authors.forename: Sang-Hoon
authors.initials: SH
authors.lastname: Cha
mesh_terms: D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D017384:Sequence Deletion; D012995:Solubility
chemical_list: D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; D003545:Cysteine
keywords: Anti-serum albumin Fab; Human growth hormone; Interchain disulfide bond; Periplasmic expression