01 Production of a C-terminal fragment of CTGF in Escherichia coli that mimics activity of the intact CTGF molecule

D.K. Ball, D.R. Brigstock. Department of Surgery, Children’s Hospital, Columbus, Ohio, USA

The primary translational product of human connective tissue growth factor (CTGF) is predicted to comprise 349 residues, which, after cleavage of the signal peptide, is expected to produce a protein of 323 residues with a M_r of 38 000. Lower mass forms of CTGF comprising modules 3 and 4 or module 4 alone appear to exhibit some of the activities of the full-length CTGF protein, suggesting that functional domains are present in the C-terminal region of CTGF. To facilitate studies of the lowest mass form of CTGF identified to date, we have used an E coli expression system to produce a 10 kDa CTGF protein that comprises essentially module 4 and corresponds to residues 247–349 of human CTGF.

The cDNA encoding 10 kDa CTGF (commencing at Glu247) was cloned into the pMAL-c2 vector (New England Biolabs) and the construct was transformed into E coli strain BL21. Escherichia coli carrying the cDNA for the maltose-binding protein–CTGF fusion protein were induced with isopropylthiogalactoside (IPTG) for one hour and then mechanically lysed using a French press. The cell extract was passed through an amylose resin and the bound fusion was eluted with 10 mM maltose. This protein was digested with factor Xa and the cleavage products further purified by heparin-affinity chromatography and reverse-phase high performance liquid chromatography. Recombinant 10 kDa CTGF demonstrated comparable immunoreactive and heparin-binding properties to native CTGF and promoted adhesion of several cell types including fibroblasts, endothelial cells, and epithelial cells. For each cell type tested, CTGF-mediated cell adhesion was heparin dependent and was ablated by prior treatment of the CTGF with reducing agents.

In conclusion, recombinant 10 kDa CTGF produced in E coli appears to mimic the biological activity and heparin-binding properties of native CTGF. The …