title: Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgGκ signal peptide in optimized HEK293F transient transfection.
abstract: VSTM1-v2 is a secretory glycoprotein identified by our laboratory. Our previous study revealed that VSTM1-v2 could promote differentiation and activation of Th17 cells. To explore the role of VSTM1-v2 in the immune system further, a source of abundant high-quality recombinant protein is warranted. However, high-level expression of bioactive VSTM1-v2 is difficult due to its weak secretion capacity. To obtain sufficient recombinant VSTM1-v2, we developed an improved expression and purification system by replacing the native signal peptide with a mouse IgGκ signal peptide that did not alter the protein cleavage site. We also optimized parameters for a transient gene expression system in HEK293F cells suspended in serum-free media with polyethyleneimine. Finally, 3.6 mg/L recombinant VSTM1-v2 protein with N-glycosylation and no less than 95% purity was obtained through one-step purification with Ni affinity chromatography. The final yield after purification was increased by more than 7-fold compared to the yield from our previously reported HEK293T system (from 0.5 mg/L to 3.6 mg/L). More importantly, VSTM1-v2 protein exhibited excellent bioactivity. In conclusion, the improved system is not only a dependable source of abundant bioactive VSTM1-v2 for functional studies but also demonstrates a highly efficient approach for enhancing the production of proteins in a short time period, especially for secretory proteins with poor yields.
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Medical Immunology, Ministry of Health, 38 Xueyuan Road, Beijing 100191, China; Peking University Center for Human Disease Genomics, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China.
authors.forename: Huihui
authors.initials: H
authors.lastname: Liu
authors.affiliation: Proteomics Laboratory, Medical and Healthy Analytical Center, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China.
authors.forename: Xiajuan
authors.initials: X
authors.lastname: Zou
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Medical Immunology, Ministry of Health, 38 Xueyuan Road, Beijing 100191, China; Peking University Center for Human Disease Genomics, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Medical Immunology, Ministry of Health, 38 Xueyuan Road, Beijing 100191, China; Peking University Center for Human Disease Genomics, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Medical Immunology, Ministry of Health, 38 Xueyuan Road, Beijing 100191, China; Peking University Center for Human Disease Genomics, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China.
authors.forename: Wanqiong
authors.initials: W
authors.lastname: Yuan
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Medical Immunology, Ministry of Health, 38 Xueyuan Road, Beijing 100191, China; Peking University Center for Human Disease Genomics, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China.
authors.forename: Yingyu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Medical Immunology, Ministry of Health, 38 Xueyuan Road, Beijing 100191, China; Peking University Center for Human Disease Genomics, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100191, China. Electronic address: hanwl@bjmu.edu.cn.
authors.forename: Wenling
authors.initials: W
authors.lastname: Han
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D016895:Culture Media, Serum-Free; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection
chemical_list: D016895:Culture Media, Serum-Free; D006023:Glycoproteins; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D021382:Protein Sorting Signals; D011994:Recombinant Proteins
keywords: Cytokine; Secretory glycoprotein; Signal peptide; TMPP-Ac derivatization; Transient gene expression; VSTM1-v2