Expression, purification and bioactivity of human augmenter of liver regeneration.
OBJECTIVE
To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration (hALR) and to study their biological activity.


METHODS
hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase (GST) sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation.


RESULTS
The product of PCR from plasmid pGEM-T-hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups (P < 0.01).


CONCLUSIONS
Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.
Zhang
Yang-De
YD
National Hepatobiliary and Enteric Surgery Research Center, Ministry of Health, 141 Xiangya Road, Changsha 410008, Hunan Province, China. zyd@2118.cn
Zhou
Jian
J
Zhao
Jin-Feng
JF
Peng
Jian
J
Liu
Xiao-Dong
XD
Liu
Xin-Sheng
XS
Jia
Ze-Ming
ZM
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003579:Cytochrome Reductases / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D022781:Hepatocytes / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D008297:Male; D050862:Oxidoreductases Acting on Sulfur Group Donors; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003579:Cytochrome Reductases; C500897:GFER protein, human; D050862:Oxidoreductases Acting on Sulfur Group Donors; D005982:Glutathione Transferase