[Expression of rat augmenter of liver regeneration in pichia pastoris and evaluation of its bioactivity in vitro].
OBJECTIVE
To construct yeast expression plasmid of rat augmenter of liver regeneration (rALR), express it in GS115, and examine its bioactivity in vitro.


METHODS
With PCR and genetic recombination techniques, the gene fragment of rALR was amplified from recombinant plasmid pcDNA3.1-rALR, and the recombinant plasmid pPIC9K-rALR was constructed. The recombinant plasmid pPIC9K-rALR was transducted into GS115 by electroporation after identified with endonuclease digestion and PCR amplification. The target protein was expressed by GS115 under the induction of 0.5% methanol. The recombinant rALR (rrALR) was purified with ultrafiltration after demonstrated by 15% SDS-PAGE and western blot. The effects of rrALR on the proliferation of QGY, HepG2 cells and primary rat hepatocytes in vitro were evaluated by 3H-TdR intake method.


RESULTS
Recombinant plasmid pPIC9K-rALR was identified by restriction digestion and PCR methods. The rrALR as a secretive protein was successfully expressed in GS115. Its molecular weight, 1.5x10(4), was in correspondence with theoretic value. The rrALR could bind with the polyclonal antibody against human ALR in western blot, which demonstrated that there was cross-antigenicity between human and rat ALR. The high qualitative rrALR was obtained through ultrafiltration. Among the experimental concentrations, the rrALR could stimulate the proliferations of QGY and HepG2 cells in a dose-dependent manner in vitro, but had little effect on the proliferation of primary rat hepatocytes in vitro.


CONCLUSIONS
The rrALR as a secretive protein is expressed in GS115 efficiently. The rrALR can stimulate the proliferations of QGY and HepG2 cells in a dose-dependent manner in vitro, but had no effect on primary rat hepatocytes, which demonstrates that there exists different receptors on the normal hepatocytes and hepatocarcinoma cells. There are cross-bioactivity and cross-antigenicity between human and rat ALR.
Yu
Hui-feng
HF
Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China.
Liu
Qi
Q
D000818:Animals; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D050862:Oxidoreductases Acting on Sulfur Group Donors
D011506:Proteins; D011994:Recombinant Proteins; C088825:GFER protein, rat; D050862:Oxidoreductases Acting on Sulfur Group Donors