Construction of CTLA4 extracellular domain expression vectors and screening chromosomal integrants in Pichia pastoris

Objective A 400bp cDNA fragment of the CTLA4 extracellular domain coding sequence was amplified by PCR from mRNA of actived human T cells, and then inserted into the P. pastoris expression vector pPIC9 and pPIC9K.Methods Thus the gene's expression was under the alcohol oxidase(AOX1) promoter control and the coding sequence was fused to the alpha mating factor signal.Results Following electroporation of the DNA into the P. pastoris strain GS115(his 4),His+ transformants were recovered and plated on medium containing increasing concentrations of the G418.High copy member of heterogenes was identified by means of PCR and southern blotting to be integrated in chromosome of P pastoris. Conclusion This work offers efficient P pastoris stains for production of CTLA4 extracellular protein for structure/function analyses.