[Cloning and efficient expression of cytokine human MK in E. coli].
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 nucleotide 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 efficiently 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.
Huang
J
J
Department of Physiology and Biophysics, Fudan University, Shanghai 200433.
Lin
W M
WM
Luo
Z Y
ZY
Xie
Y
Y
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016207:Cytokines; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D006801:Humans; D000078792:Midkine; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002352:Carrier Proteins; D016207:Cytokines; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000078792:Midkine