Cloning and expression of the gene of augmenter of liver regeneration in yeast cells.
OBJECTIVE
To study the function of augmenter of liver regeneration (ALR) as a regulatory factor that specifically stimulates hepatic cell regeneration, we constructed yeast expressive vector of ALR and expressed it in yeast cells.


METHODS
Total RNA was extracted from HepG2 cells, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the coding region of ALR. The products were cloned into PGEM-T vector and sequenced, then cloned into PGBK T7 vector. The recombinant plasmid PGBK T7-ALR was transformed into yeast AH109. The yeast protein was extracted and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization technique.


RESULTS
DNA sequencing results confirmed that the coding region of ALR was correctly inserted into the yeast expression vector, and Western blotting assay showed that recombinant ALR was successfully expressed in yeast. Its molecular weight was identical to the theoretical value of 15 000 Da; the protein was found inside the yeast cells.


CONCLUSIONS
The successful expression of ALR in yeast cells makes it possible to study further on its biological function.
Cheng
Jun
J
Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of PLA, Beijing 100039, China. cj@genetherapy.com.cn
Wang
Lin
L
Li
Ke
K
Lu
Yin-Ying
YY
Liu
Yan
Y
Duan
Hui-Juan
HJ
Hong
Yuan
Y
Wang
Gang
G
Li
Li
L
Zhang
Ling-Xia
LX
D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D008113:Liver Neoplasms; D008115:Liver Regeneration / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping; D015003:Yeasts; D050862:Oxidoreductases Acting on Sulfur Group Donors
D018076:DNA, Complementary; D011506:Proteins; D011994:Recombinant Proteins; C088825:GFER protein, rat; D050862:Oxidoreductases Acting on Sulfur Group Donors