High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli.
Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.
Su
Zhijian
Z
State Key Laboratory for Biocontrol, Zhongshan University College of Life Science, Guangzhou 510235, P R China.
Huang
Yadong
Y
Zhou
Quannan
Q
Wu
Zhiling
Z
Wu
Xiaoping
X
Zheng
Qing
Q
Ding
Changcai
C
Li
Xiaokun
X
D000818:Animals; D041702:BALB 3T3 Cells; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D051379:Mice; D008938:Mitosis / Q000187:drug effects; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D004815:Epidermal Growth Factor