Genetic Recombinant Expression and Characterization of Human Augmenter of Liver Regeneration
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Aims 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 CCl4-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 CCl4-intoxicated mice were significantly improved when rALR was administrated at 40 μg/kg or 200 μg/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 CCl4-intoxicated mice.
KeywordsGene Augmenter of liver regeneration (ALR) Recombinant Hepatocytes Liver function
Augmenter of liver regeneration
Plasmid for expression by T7 RNA polymerase
We are especially grateful for the technical support of Dr. Tong-Hai Dou from Fu-Dan University regarding protein purification.
- 8.Polimeno L, Margiotta M, Marangi L, Lisowsky T, Azzarone A, Ierardi E et al (2000) Molecular mechanisms of augmenter of liver regeneration as immunoregulator: its effect on interferon-gamma expression in rat liver. Dig Liver Dis 32(3):217–225. doi: 10.1016/S1590-8658(00)80824-5 PubMedCrossRefGoogle Scholar
- 10.Hagiya M, Francavilla A, Polimeno L, Ihara I, Sakai H, Seki T et al (1994) Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution. Proc Natl Acad Sci USA 91(17):8142–8146. doi: 10.1073/pnas.91.17.8142 PubMedCrossRefGoogle Scholar
- 18.Francavilla A, Vujanovic NL, Polimeno L, Azzarone A, Iacobellis A, Deleo A et al (1997) The in vivo effect of hepatotrophic factors augmenter of liver regeneration, hepatocyte growth factor, and insulin-like growth factor-II on liver natural killer cell functions. Hepatology 25(2):411–415PubMedGoogle Scholar
- 19.Polimeno L, Capuano F, Marangi LC, Margiotta M, Lisowsky T, Ierardi E et al (2000) The augmenter of liver regeneration induces mitochondrial gene expression in rat liver and enhances oxidative phosphorylation capacity of liver mitochondria. Dig Liver Dis 32(6):510–517. doi: 10.1016/S1590-8658(00)80009-2 PubMedCrossRefGoogle Scholar