High-level expression of mutant CTLA4 extracellular domain in Pichia Pastoris

Objective To get high-level expression of human cytotoxic T lymphocyte associated antigen-4 (CT-LA4) extracellular protein in Pichia Pastoris by designing and modification of CTLA4 extracellular domain coding sequence and construction of a secretion expression vector based on the codon usage bias of Pichia Pastoris. Methods The low frequency usage codon was changed into high frequency usage synonymous codon without changing the ami-no acid sequence by PCR-based site-directed mutagenesis. After DNA sequence analysis, the high frequency usage synonymous codon was inserted into the Pichia Pastoris expression vector pPIC9K. Following electroporation of the Sac I -linearized DNA into the Pichia Pastori strain GS115 (his-4) , His + transformants were recovered and plated in medium containing increasing concentrations of the G418. The target gene integration was identified by PCR and the expression was induced by methanol. The induced protein expression was identified by SDS-PAGE, Western blotting and peptide mass fingerprint analysis. Results The human CTLA4 extracellular domain coding sequence was successfully modified as expected. The mutant expression vector successfully expressed CTLA4 extracellular protein at high level in the fermentation supernatant. Conclusion This study provides a foundation for the studies of high-level expression of human CTLA4 extracellular protein, its functions and clinical application.