Expression and biochemical characterization of recombinant human epididymis protein 4
| Autor: | Wan Deyou, Shuai Zhen, Xin Gao, Yunhui Liu, Ling Hua, Jiyue Cao |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Glycosylation
Molecular Sequence Data Gene Expression Pichia Pichia pastoris law.invention chemistry.chemical_compound WAP Four-Disulfide Core Domain Protein 2 N-linked glycosylation law Protein A/G Humans Protease Inhibitors biology Bacteria Proteins Bacterial Infections biology.organism_classification Recombinant Proteins Anti-Bacterial Agents Secretory protein HEK293 Cells Biochemistry chemistry Carbohydrate Sequence biology.protein Recombinant DNA Protein G Whey Acidic Protein Biotechnology |
| Zdroj: | Protein expression and purification. 102 |
| ISSN: | 1096-0279 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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