[Two incompatible plasmids coexpress the human endostatin and predigested human plasminogen kringle 5 in E. coli].
OBJECTIVE
To construct the recombinant plasmid pET28a/hES and coexpress the human endostatin (hES) and predigested human plasminogen kringle 5 (predhPK-5) in E. coli.


METHODS
The mRNA was extracted from human liver tissue, and the endostatin gene was amplified by RT-PCR, which then was cloned into pET-28a(+). Under screening pressure by ampicillin and kanamycin simultaneously, E. coli BL21 (DE3) was cotransformed with pET28a/hES and pGEX-1lambdaT/predhPK-5 and induced with IPTG to express the recombinant proteins. The stability of cotransformants existing in E. coli was measured through two aspects in the serial culture time and passage number of bacterium.


RESULTS
The two incompatible plasmids could be coexisted under the pressure of two antibiotics (ampicillin and kanamycin). After induced with IPTG, both human endostation and predhPK-5 gene were coexpressed, and the recombinant proteins comprised about 20% and 21% of total cell proteins, respectively. The two incompatible plasmids could still be maintained in over 75% E. coli cells for at least 16 hours or after 120 passages under the pressure of two antibiotics.


CONCLUSIONS
The two incompatible plasmids pET28a/hES and pGEX-1lambda T/predhPK-5 may coexpress the recombinant proteins in E. coli. A new method for coexpression of proteins in E. coli containing two incompatible plasmids is proved to be feasible.
Sun
Jian
J
Department of Biochemistry and Molecular Biology, West China School of Preclinicial and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Wang
Jian-Qi
JQ
Zhai
Zhao-Yang
ZY
D043169:Endostatins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D010958:Plasminogen / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction
D043169:Endostatins; D010446:Peptide Fragments; C491499:plasminogen kringle 5; D010958:Plasminogen