Establishment of a recombinant CTGF expression system in vitro that models CTGF processing in vivo: Structural and functional characterization of multiple mass CTGF proteins.

摘要

Connective tissue growth factor (CTGF) was expressed using recombinant DNA technology. A cDNA encoding full-length 38kDa human CTGF was cloned into the mammalian expression vector pcDNA3.1 and transfected into mutant Chinese hamster ovary (CHO) 745 cells. Stably transfected clones were isolated and screened for recombinant CTGF expression by Western blot. Clones were shown to produced not only 38kDa CTGF but also low mass (10–20kDa) CTGFs as well. The clone with the highest level of expression was selected for large scale production of the recombinant proteins. Conditioned media was collected from transfected cells and subjected to heparin-affinity FPLC. All mass forms of CTGF (38kDa, 16–20kDa, 10–12kDa) were heparin-binding and were subsequently separated from one another using a combination of cation-exchange FPLC, gel filtration HPLC and/or reverse-phase HPLC. Structural analysis of the purified proteins showed that they commenced at Ala181 (20kDa), Leu184 (18kDa), Ala197 (16kDa) and Gly253 (10kDa). A single preparative run of 4L of conditioned medium produced 1–2mg of 16–20kDa CTGFs, 100μg 38kDa CTGF and 50–75μg 10kDa CTGF. Additionally, it was shown that pig uterine luminal fluids were able to digest 38kDa CTGF to stable 16–20kDa proteins, a phenomenon blocked by antithrombin III. Thrombin treatment of 38kDa CTGF produced a 16kDa CTGF isoform that commenced at Asn198. These data implicate serine proteases as one class of enzyme that can cause limited proteolysis of CTGF. Gel filtration purifications indicated that all forms of CTGF had the ability to stimulate DNA synthesis in Balb/c 3T3 cells. Additionally, different mass forms of CTGF stimulated dose-dependent adhesion of Balb/c 3T3 cells at concentrations between 5 and 20μg/ml. These results show that cell adhesion motifs in CTGF are located in the C-terminal region of the CTGF molecule. An in vitro expression system has been developed that mimics physiological CTGF processing and is amenable for future studies of the individual biological and cell binding properties of CTGF isoforms.