Yeast Secretory Expression, Purification And Bioactive Characterization Of Recombinant Human Hepassocin

Hepassocin (HPS) is a liver-specific hepatocyte proliferation factor and its expression is tissue-specific. HPS is highly expressed in adult liver, moderately in fetal liver and low in pancreas. The expression of HPS in other tissues has not been detected. Partial hepatectomy can induce HPS expression, suggesting that it may participate in the process of liver regeneration. HPS is regulated by liver-specific transcription factor HNF-1. Human IL-6 can also induce HPS expression through STAT pathway. Early experiment suggested that HPS stimulated only hepatic parenchymal cell proliferation in vitro, no corresponding activity on non-liver cells or liver cancer cells. Recent studies in our laboratory show that the HPS may also stimulate liver cell proliferation in vivo with a clear role in the anti-hepatic injury. Exogenous injection of HPS increased cell proliferation in mouse liver after most PH., effectively reduced CCl4, and D-gal induced liver injury in the level of serum transaminase, improved liver damage repairing and the survival rate of acute liver failure in experimental rats. These studies indicate that HPS plays an important role not only in the liver development and maintenance of liver function, but also protection of liver and liver damage repairing. Research work on HPS is intended to reveal its biological activity and molecular mechanism. It may also provide an effective drug treatment for serious liver disease in the near future. (HPS has been listed in major scientific and technological innovation and specific drug candidates.)Human HPS is located on chromosome 8p22-p21.3. It has an ORF length of 936bp that encodes a 312 amino acid peptide with the signal peptide of N-terminal 22 amino acids. The monomer of HPS is a 34KDa protein which forms a 68KDa homodimer with mitogenic activitiy in hepatocytes.The aim of our research project is to explore mechanism of HPS in accelerating hepatocytes proliferation and protection against liver injury. Initially we amplified HPS cDNA eliminated the sequence coding signal peptide from the human liver cDNA library. The cDNA fragment was cloned into expression vector pPIC9K. Recombinant clones were subsequently transformed into Pichia pastoris strain GS115 by electroporation. Pichia transformants were screened for HIS4, G418 resistance and MutS phenotypes, verified with yeast colony rapid PCR amplification and inducing expression. Forty eight positive clones were obtained in which five are yeast strains with high expression level of rhHPS.Yeast secretory expression of rhHPS and fermentation process have been optimized in a 10-liter table top glass bioreactor. We have further explored efficient protein purification processes. Hollow fiber column was used for concentration, desalination and removal of pigment. CHT chromatography was subsequently performed to conduct a preliminary purification of concentrated product and to remove pigment residues. Using strong anion exchange resin to remove majority of protein impurities, desalting and freeze-dry, we obtained rhHPS protein with a purity of greater than 90%. The purified rhHPS was identified by western blot and MALDI-TOF-MS.Purified recombinant human HPS enhanced L02 cells proliferation stimulation in vitro, also stimulated the proliferation of hepatocytes in rats with 70% PHx and protected against the liver injury with D-gal and CCl4 treatment.We hope our study and research results may shed some lights to exploration of therapeutic prospect of HPS in hepatic failure and add some value to pharmaceutical production of HPS in the future.