Cloning and expression of the human augmenter of liver regeneration at low temperature in Escherichia coli.
Acute and chronic hepatic failure is a devastating illness of varied causes with considerable mortality. Human augmenter of liver regeneration (hALR) is a hepatotrophic protein and the unique cytokine which can specially stimulate hepatic origin cells to grow regardless of genus. It has been proven that ALR can promote regeneration and avoid all kinds of injury in rat and canine models. In this study, the recombinant protein hALR was expressed successfully with recombinant prokaryotic expression vector pET28a(+) in Escherichia coli BL21 (DE3). We constructed the recombinant expression vector pET28a(+)/hALR with a full-length cDNA encoding hALR protein from normal human liver tissue by one-step reverse transcription-polymerase chain reaction and his-tag recognition sequence encoding polyhistidine (6 x His). Under IPTG (isopropyl-beta-d-thiogalactopyranoside) induction for 2 h at 37 degrees C, recombinant protein hALR was expressed. The expression of recombinant polyhistidine-tagged hALR was increased under low temperature and was confirmed that the temperature of 23 degrees C was the most suitable IPTG induction condition. Under low temperature induction of IPTG, recombinant protein can be expressed as a soluble protein. Recombinant protein hALR was also purified with His Bind Kits and characterized with SDS-PAGE and Western blotting. The results showed that recombinant hALR could be expressed as a soluble protein under low temperature induction of IPTG. The successful expression of ALR in E. coli makes it possible to further study its biological function and purified recombinant hALR could be developed into a new anti-hepatic damage product.
Sheng
Jifang
J
Department of Infectious Disease, the First Affiliated Hospital, College of Medicine, Zhejiang University, Key Lab. of Infectious Disease of Ministry of Health, Qingchun Rd. 79, Hangzhou, 310003, China. shengjf@mail.hz.zj.cn
Yu
Haiying
H
Li
Jun
J
Sheng
Guoping
G
Zhou
Linfu
L
Lu
Yingyan
Y
D001483:Base Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D003579:Cytochrome Reductases / Q000096:biosynthesis* / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007544:Isopropyl Thiogalactoside; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012995:Solubility
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D003579:Cytochrome Reductases; C500897:GFER protein, human; D050862:Oxidoreductases Acting on Sulfur Group Donors