Recently a lot of in vitro and in vivo studies demonstrated that CTLA4Ig, a fusion protein composed of extracellular domain of human CTLA-4 and constant region of human IgG, was a potential immune modulator in humoral, transplantation rejection
and
antitumor immuneresponses. This study focused on prokaryotic expression of human CTLA-4 gene with potential clinical application in neat future. Plasmid Pgex-kg, Pgex-2T and Phk412 were three vectors employed in this experiment. The former two
were
glutathione S- transferase(GST) and the latter one wasβ-galactosidase(lacZ) fusion systems respectively. By using these three vectors four gene fusions inserted with total or various fragments of human CTLA-4 cDNA were constructed. Recombinant
plasmid
pGEX-KG-CTA4. Pgex-2T-CTLA4pvull, Pgex-2T-CTLA4rsal and pHK412-CTLA4Hindlll-BamHI were the four fusion constructs made by and used in this experiment. Three CLONES OF Escherichia coli transformed with pGEX-KG, pGE-2T and pHK412 were showed normal
growth
pattern and rate regardless of adding IPTG to induce gene carried on the vectors. And expression of 26 KD GST and 121 KD lacZ protein were inducible upon IPTG addition into the culture. It was impossible to induce GST-CTLA4 fusion protein in dkdk
-KG-CTLA4, pGEX-2T-CTLA4pvull, and pGEX-2T-CTLA4Rsal transformants. And the IPTG induction of fusion proteins in these three transformants was extremely toxic to host bacteria carrying total or fragments of human CTLA-4 gene. Contrary to the GST
fusion
system, the lacZ fused Phk412-CTLA4 Hindlll-BamHI transformant was successfully induced to produce Cro-CTLA4Hindlll-BamHI-lacZ fusion protein upon IPTG addition to the culture. But the growth rate of this transformant decreased down-to 50-70% of
Phk412
transformant by adding IPTG. This findings imply that expression of total or partial fragment of human CTLA-4 protein is extremely toxic to host bacteria. And the toxicity of this protein is unlikely due to its hydrophobicity, The result of this
study
suggests that one should adopt more than two expression vector systems for the prokaryotic expression of mammalian gene to get a successful outcome.
|