TY - JOUR
T1 - A new method for protein coexpression in Escherichia coli using two incompatible plasmids
AU - Yang, Wei
AU - Zhang, Lan
AU - Lu, Zhigang
AU - Tao, Wei
AU - Zhai, Zhonghe
N1 - Funding Information:
We gratefully thank Dr. Xiaodong Wang from U.T. Southwestern Medical Center at Dallas (Dallas, TX) for the mouse polyclonal antibody against DFF40 and Dr. Joe C. Wu from IDUN Pharmaceuticals Inc. (La Jolla, California) for purified recombinant caspase-3. This work is supported by Grant G1999053905 from the Special Funds for Major State Basic Research of China.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2001
Y1 - 2001
N2 - It is commonly believed that incompatible plasmids carrying the same replicon cannot coexist stably in one Escherichia coli cell. However, we found that two incompatible plasmids carrying different antibiotic resistance genes, if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding regions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors - pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli BL21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Induced by isopropyl β-D-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression product contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhibited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately.
AB - It is commonly believed that incompatible plasmids carrying the same replicon cannot coexist stably in one Escherichia coli cell. However, we found that two incompatible plasmids carrying different antibiotic resistance genes, if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding regions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors - pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli BL21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Induced by isopropyl β-D-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression product contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhibited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately.
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U2 - 10.1006/prep.2001.1453
DO - 10.1006/prep.2001.1453
M3 - Article
C2 - 11483011
AN - SCOPUS:0034887510
SN - 1046-5928
VL - 22
SP - 472
EP - 478
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 3
ER -