[Expression of human brain myelin basic protein gene cDNA in Escherichia coli].
A 612bp full-length cDNA sequence coding for 21.5 kd myelin basic protein (MBP) was amplified from human brain cDNA library by polymerase chain reaction, cloned into vector pGEM-3Zf(+) with blunt-end. The 561bp cloned fragment was recovered from recombinant pGEMP after BamHI digestion, and then inserted into the BamHI site downstream of the glutathione-S-transferase gene of the expression vector pGEX-5T. The recombinant vector p5TBP was transformed into E. coli JM 109 and positive colonies were incubated in LB medium containing 1 mmol/L IPTG for six hours at 37 degrees C. A new polypeptide band with an apparent molecular weight 49 kd was detected in transformed cell lysates by SDS-PAGE. Western blotting analysis confirmed that this fusion protein reacted specifically with rabbit antibodies to human MBP. The expression level of MBP in this system was about 50 mg/L of bacterial culture estimate by immuno-dot blot, ELISA and absorbance scanning of SDS-PAGE.
Cheng
H
H
Recombinant DNA Laboratory, School of Basic Medical Sciences, Chengdu.
Chen
J
J
Wang
R
R
Li
C
C
D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D004676:Myelin Basic Protein / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D018076:DNA, Complementary; D004676:Myelin Basic Protein; D011993:Recombinant Fusion Proteins