The development of a non-lytic insect-cell expression system for the production of fusion proteins of insulin-like growth factor II and stromal-cell derived factor I

Insulin-like growth factors (IGFs) and stromal cell derived factor 1 (SDF-1) have been suggested as being potential therapeutic agents in HIV infected patients. The cDNA coding for a recombinant fusion protein of IGF 1I and SDF-1 was synthesized by PCR and inserted into a commercial non-lytic insect cell expression system. The successful production of the recombinant protein in the culture medium of insect cells transfected with the recombinant plasmid was monitored using HPLC. Purified protein was characterized by following 3H-thymidine incorporation into bovine fetal liver cells for the identification of IGF II and using Western blots and enzyme-linked immunosorbent assays for the identification of SDF-1.;A modified IGF II-SDF 1 chimera, which included a N-glycosylation site, was also successfully produced by insect cells. Mass spectrometry analysis indicated that the glycosylated and non-glycosylated proteins had molecular masses of 17.6 kDa and 14.5 kDa, respectively. The recovery of the glycosylated protein was higher than that obtained with the non-glycosylated form after purification by heparin affinity chromatography, a property that could be an advantage for the large scale production of the protein. Finally, three additional cDNAs were successfully generated and cloned into insect cell expression vectors. Two of them encoded the glycosylated-and non glycosylated forms of the chimera incorporating a Kozak sequence to improve translation efficiency. The third cDNA encoded a chimera of IGF II and a soluble form of the HIV-binding protein CD4, a potential inhibitor of HIV infection.