title: Expression and biochemical characterization of recombinant human epididymis protein 4.
abstract: Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.
authors.affiliation: Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Huazhong Agricultural University, 1 Shi-zi-shan Street, Wuhan 430070, Hubei, PR China; Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Ling
authors.initials: L
authors.lastname: Hua
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Yunhui
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Zhen
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Deyou
authors.initials: D
authors.lastname: Wan
authors.affiliation: Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Huazhong Agricultural University, 1 Shi-zi-shan Street, Wuhan 430070, Hubei, PR China. Electronic address: caojiyue@mail.hzau.edu.cn.
authors.forename: Jiyue
authors.initials: J
authors.lastname: Cao
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China. Electronic address: gaoxinamms@163.com.
authors.forename: Xin
authors.initials: X
authors.lastname: Gao
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology; D001424:Bacterial Infections / Q000188:drug therapy / Q000201:enzymology; D002240:Carbohydrate Sequence; D015870:Gene Expression; D006031:Glycosylation; D057809:HEK293 Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D000081102:WAP Four-Disulfide Core Domain Protein 2
chemical_list: D000900:Anti-Bacterial Agents; D011480:Protease Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; D000081102:WAP Four-Disulfide Core Domain Protein 2; C569698:WFDC2 protein, human
keywords: Antimicrobial activity; Antiprotease activity; N-glycosylation; Recombinant human epididymis protein 4