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Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgGκ signal peptide in optimized HEK293F transient transfection

Paper ID Volume ID Publish Year Pages File Format Full-Text
20010 43153 2016 7 PDF Available
Title
Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgGκ signal peptide in optimized HEK293F transient transfection
Abstract

•Mouse IgGκ signal peptide greatly enhanced secretion capacity of VSTM1-v2.•The yield of VSTM1-v2 in the optimized suspension HEK293F transient expression system was increased by 7 folds.•VSTM1-v2 was synthesized and secreted as a hyper-glycosylated protein with complex oligosaccharides.•The purified VSTM1-v2 exhibited excellent bioactivity.•The study provides a high-efficient way to rapidly enhance production of proteins with weak secretion capacity.

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.

Keywords
Cytokine; VSTM1-v2; Secretory glycoprotein; Signal peptide; Transient gene expression; TMPP-Ac derivatization
First Page Preview
Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgGκ signal peptide in optimized HEK293F transient transfection
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 121, Issue 2, February 2016, Pages 133–139
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering