Genetic recombinant expression and characterization of human augmenter of liver regeneration.
OBJECTIVE
To establish a highly effective prokaryotic recombinant expression system for human augmenter of liver regeneration (hALR) and to characterize the recombinant hALR both in vitro and in vivo.


METHODS
ALR cDNA was synthesized and inserted into expression vector pET28a+, the recombinant plasmid was transformed into BL21, and expression of hALR was induced by IPTG. Recombinant hALR (rhALR) was purified by sequential detergent wash, enterokinase (EK) digestion, gel-filtration, and chelating chromatography. The rhALR was identified by SDS-PAGE, immunoblotting, MALDI-TOF-MS, and N-terminal sequencer. Cell proliferative effect of rhALR on human hepatocytes was analyzed by MTT. The protective effect of rhALR on liver function was observed on CCl(4)-induced intoxicated mice.


RESULTS
Recombinant expression plasmid of ALR [pET28(a+)-hALR] was confirmed by restriction enzyme digestion and DNA sequencing. The expressed rhALR constituted 30% of total bacterial protein. Molecular weight was 15,029 for monomer and 30,136 for dimer by mass determination. N-terminal was M-R-T-Q-Q, exactly the same as anticipated for hALR. The purified protein migrating at about 15 KD showed excellent antigenicity in immunoblotting. The rhALR also showed a strong stimulative effect on hepatocyte proliferation. ALT and AST levels, liver histological structure, as well as the survival rate of CCl(4)-intoxicated mice were significantly improved when rALR was administrated at 40 microg/kg or 200 microg/kg.


CONCLUSIONS
The rhALR is successfully expressed highly effectively with anticipated MW, N-terminal, and antigenicity. It could play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and improving liver function in CCl(4)-intoxicated mice.
Gao
Chun-Fang
CF
Department of Laboratory Medicine, Eastern Hepatobiliary Hospital, Second Military Medical University, 225 Changhai Road, Shanghai, 200438, China. gaocf1115@yahoo.com
Zhou
Fei Guo
FG
Wang
Hao
H
Huang
Ying-Feng
YF
Ji
Qiang
Q
Chen
Jie
J
D000818:Animals; D002252:Carbon Tetrachloride Poisoning; D002460:Cell Line; D049109:Cell Proliferation; D003579:Cytochrome Reductases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use; D004926:Escherichia coli / Q000378:metabolism; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000378:metabolism; D022781:Hepatocytes / Q000502:physiology; D006801:Humans; D017114:Liver Failure, Acute / Q000139:chemically induced / Q000188:drug therapy*; D008297:Male; D051379:Mice; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000627:therapeutic use
D011994:Recombinant Proteins; D003579:Cytochrome Reductases; C500897:GFER protein, human; D050862:Oxidoreductases Acting on Sulfur Group Donors