Intracellular Expression of Human Pulmonary Surfactant Associated Protein in Methyltrophic Yeast, Pichia Pastoris

Abstract:

OBJECTIVE: To study the expression of pulmonary sufactant associated protein A1 (SP A1) in Methyltrophic Yeast, Pichia pastoris. METHODS: The cDNA encoding SP A1 derived from the healthy adult's lungs was cloned into the pPIC3.5K, an intracellular expression vector of Pichia pastoris, and then transformed into Pichia pastoris by electroporation. The PCR fast detection method and G418 resistance method were used to screen the positive transformants and the multiple copy transformants, respectively. SP A1 intracellular expression was induced by methanol. SDS PAGE and Westernblotting were applied to detect the characteristics of the product. RESULTS: SP A1 gene was highly expressed in Pichia pastoris. The molecular weight of the protein was 32 kD by SDS PAGE analysis. Anti SP A polyclonal antibodies could specifically recognize SP A1 protein. CONCLUSIONS:The human tissue specific SP A1 protein can be highly expressed in Methyltrophic Yeast, Pichia pastoris and the products can show antigenicity.