Expression And Purification Of Human Surfactant Protein A1 In Pichia Pastoris

Respiratory distress syndrome ( RDS ) is the chief factor contributing to premature neonatal death and related to genetic background of individual. Replacement therapy with pulmonary surfacrant ( PS ) is the only effective path for the treatment of RDS. Like RDS,alteration in pulmonary surfactant function or composition has been associated with a number of other respiratory disease such as adult respiratory distress syndrome ( ARDS ),idiopathic pulmonary fibrosis ( IFF ),pulmonary bacterial and viral infection. Till now,PS was extracted from animal lungs and amniotic fluids,the products of PS can not meet the neets of emergency medicine due to the difficult purification and expensiveness. Synthetic lipids and genetic engineering products of surfacrant-associated protein ( SP ) are the most suitable alternative.PS is a lipoprotein complex synthesized and secreted by alveolar type II cells. SP-A is the most aboundant protein of PS and attributes to multiple physiologic functions. Human SP-A is encoded by two very similar but nonidentical genes,SP-A1 and SP-A2. The two genes are 94% identical at the nucleotide level and encode proteins with 96% identity. The Human SP-A locus has been mapped to 10q22-23 and also contains a nonfunctional pseudogene. Human SP-A comprises of 248aa and the expressed product of monomeric form is about 26-28kDa.As a eukaryote,Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing,protein folding,and posttranslational modification,while being as easy to manipulate as E.coli or Saccharomyces cerevisiae. It is faster,easier and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture,and generally gives higher expression levels. It can express heterologous soluble protein and secrete out of cell. Thesefeatures make Pichia very useful as a protein expression systems.A methylotrophic yeast Pichia pastoris was used to produce SP-A1 protein. The recombined secreted vector pPIC9K/SP-Al and intracellular vector pPIC3.5K/SP-Al were cloned and transformed into P. pastorises respectively. The gene was subsequently integrated into genomic DNA of P.pastoris strains GS115 and KM71. Resisrance to G418 conferred by the kanamycin gene present on pPIC3.5K and pPIC9K is used as a screen for multicopy integrants. Through optimization of the growth conditions,under the induction by methanol,the expressed SP-A1 protein was secreted into fermentation broth and reached a level of approximately 200ml/L of cultured medium. The strain was screened with high expressed of SP-A1 and then was cultured in fermentation tank.Secreted soluble SP-A1 was produced in fermentation tank after induced by methanol,which was 32kDa in molecular weight. The rough products were recovered by centrifuged and ultro filtration and purified by Size exclusion chromatography and High Performance Liqid Chromatography ( HPLC ). The purity of the production was above 95 percent by HPLC,the expressed SP-Alwas secreted into medium at the level of 930mg/L.The SP-A1 protein expressed in pichia pastoris was efficient in enhancing the phagocytosis of E.coli J5 by alveolar macrophages. The research showed that SP-A1 proteins were highly expressed in Pichia pastoris,the purified products showed satisfied antigenicity and significant enhancement of the phagocytosis of E.coli J5 by alveolar macrophages.