CLONING AND EFFICIENT EXPRESSION OF CYTOKINE HUMAN MK IN E. coli
HUANG Jian LIN Wan Min LUO Zu Yu XIE Yi ( Department of Physiology and Biophysics, Fudan University, Shanghai 200433) ( State key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433)
For cloning the cytokine human Midkine (MK) gene, we designed by PCgene program and synthesized a pair of PCR specific primers according to the reported human MK cDNA sequence. Total cellular RNA was extracted from a human hepatoblastoma cell line HepG2, and then the target DNA fragment was obtained by RT-PCR and subcloned into plasmid pUC118. Checked with radioisotope sequencing and ABI 377A sequencer, the nu-cleotide sequence of the cloned MK cDNA was identical with the reported one. A prokaryotic expression vector, named pBV220, was used to express the MK protein effi- ciently in E. coli strain TG1 and a predicted band of 16. 5 kD in Mr by 15% SDS-PAGE was found. The expressed recombinant protein was found in insoluble aggregated form and accounted for about 31.21% of the total cellular proteins. The first 15 N-terminal amino acid sequence analysis of this protein by Edman degradation method showed that it was accordant with that predicted from the cDNA sequence. The activity of neurite outgrowth-promoting of the MK crude samples was tested with brain cells isolated from 18-day embryos of SD rat.
【CateGory Index】： Q78