High-level expression and purification of heparin-binding epidermal growth factor (HB-EGF) with SUMO fusion

Mol Biotechnol. 2010 Mar;44(3):198-203. doi: 10.1007/s12033-009-9226-0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Cloning, Molecular
  • Coloring Agents / metabolism
  • Dose-Response Relationship, Drug
  • Escherichia coli / genetics
  • Gene Expression
  • Genetic Vectors
  • Heparin-binding EGF-like Growth Factor
  • Humans
  • Intercellular Signaling Peptides and Proteins / genetics
  • Intercellular Signaling Peptides and Proteins / isolation & purification*
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • Intercellular Signaling Peptides and Proteins / pharmacology
  • Mice
  • Mitosis / drug effects
  • Molecular Sequence Data
  • Plasmids / genetics
  • Protein Binding
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / pharmacology
  • Small Ubiquitin-Related Modifier Proteins / genetics
  • Small Ubiquitin-Related Modifier Proteins / metabolism*
  • Solubility
  • Tetrazolium Salts / metabolism
  • Thiazoles / metabolism

Substances

  • Coloring Agents
  • HBEGF protein, human
  • Hbegf protein, mouse
  • Heparin-binding EGF-like Growth Factor
  • Intercellular Signaling Peptides and Proteins
  • Recombinant Fusion Proteins
  • Small Ubiquitin-Related Modifier Proteins
  • Tetrazolium Salts
  • Thiazoles
  • thiazolyl blue