RESUMO
The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.
Assuntos
Pichia/química , Pichia/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/biossíntese , Inibidor Secretado de Peptidases Leucocitárias/química , Técnicas de Cultura de Células , Fermentação , Glicosilação , Humanos , Pichia/genética , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Inibidor Secretado de Peptidases Leucocitárias/genética , Inibidor Secretado de Peptidases Leucocitárias/isolamento & purificação , Transfecção , Tripsina/metabolismoRESUMO
The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan(r) gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANalpha B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.
