[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.