Open AccessRecombinant human B7‐H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL‐2 secretion in vitro 1
Author(s) -
MAO Yixiang,
CHEN YongJing,
GE Yan,
MA Hongbing,
YU Jianfeng,
WU Hongya,
HU Yumin,
WANG Qin,
SHI Qin,
ZHANG Xueguang
Publication year - 2006
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2006.00338.x
Subject(s) - fusion protein , recombinant dna , microbiology and biotechnology , lac operon , escherichia coli , complementary dna , extracellular , biology , expression vector , gene expression , biochemistry , chemistry , gene
Aim: To explore the biofunctions of human B7‐H4 generated from prokaryotic system. Methods : The gene of human B7‐H4 extracellular region (IgV‐like and IgC‐like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX‐5X‐3 expressing glutathione s‐transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21‐RIL(DE3). A 47 kDa fusion protein (GST/hB7‐H4) was induced by isopropyl‐beta‐ D ‐thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7‐H4 on proliferation of T cells was observed in vitro by CD3mAb activated T‐cell culturing system and [ 3 H]‐thymidine incorporation assay. The concentrations of interleukin‐2 and iterferon‐g in the supernatants of T cells were determined by ELISA. Results : We successfully constructed the method for high‐level expression and purification of the hB7‐H4 extracellular domain as GST fusion protein from E coli. The GST/hB7‐H4 fusion protein produced in bacteria had obvious biological activity to inhibit T‐lymphocyte proliferation and IL‐2 secretion. Conclusion : The prokaryote expression system could be used to generate hB7‐H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.