High-level expression and purification of heparin-binding epidermal growth factor (HB-EGF) with SUMO fusion.
Heparin-binding epidermal growth factor (HB-EGF) can stimulate the division of various cell types and has potential clinical applications that stimulate growth and differentiation. HB-EGF has an EGF-like domain typical of all members of the EGF family. The high expression of active HB-EGF in Escherichia coli has not been successful as the protein contains three intra-molecular disulfide bonds, the same as other members of the EGF super family that are difficult to form correctly in the bacterial intracellular environment. This work fused the non-glycosylated HB-EGF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fusion gene SUMO-HBEGF was highly expressed in BL21(DE3) that the soluble SUMO-HBEGF was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to obtain the native HB-EGF, which was further purified by Ni-NTA affinity chromatography. MTT assays indicated the purified HB-EGF, as well as SUMO-HBEGF, had mitogenic activity in a dose-dependent manner.
Lu
Wuguang
W
Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, Nanjing, Jiangsu, China.
Cao
Peng
P
Lei
Huangzong
H
Zhang
Shuangquan
S
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004396:Coloring Agents / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008938:Mitosis / Q000187:drug effects; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
D004396:Coloring Agents; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue