Studies On Expression Of Human Laminin α4 LG4-5 CDNA In Pichia Yeast

The Laminins constitute a family of heterotrimeric glycoproteins with crucial functions in basement membrane assembly and function. Laminins consist of three different subunits, α,β,andγchains. So far, five α, three β, and three γchains have been identified, and at least 15 isoforms(Laminin-1to -15) are formed by various combinations of each subunit. Laminins have diverse activities, including promotion of cell adhesion, migration, neurite ortgrowth, tumor metastasis, and angiogenesis. The changes of laminins in serum have been the criterion or reference in clinic for some diseases including the part of lung, liver, tumor, and diabetes etc. Laminin-1 which is the most extesively characterized laminin isoform, has been analyzed for biological activity using proteolytic fragments, remcombinant proteins, and synthetic peptides. The C-terminal globular domains (G domains) of Laminin αchains consist of five laminin globular domain-like modules(LG1 to LG5) that play a critical role in the biological functions of laminins. The main methods of studying laminin G domains is proteolytic fragments, recombinant proteins, and synthetic peptides etc. So far, the reserch of Laminin α4 chain focus on mouse laminin α4 chain. The recombinant mouse laminin. α4 chain LG4-5 protein is able to bind to heparin, integrins, sulfatides, syndecan-2 and syndecan-4,and increase cell attachment activity. But human laminin α4 chain LG4-5 gene has little been reseached. The research makes use of the yeast expression system to express human laminin α4 chain LG4-5 (hLG4-5) gene, and detect the biological activity of hLG4-5 fusion protein. We designed forward primer ( 5′-GTCACTCGAGAAAAGACACC TTTCCAACAGCCCTAGAG-3′) and reverse primer (5′-GTTAGCGG CCGCGGCTGCTGGACAGGAGTTGAT-3′) by human laminin α4 chain LG4-5 cDNA gene sequence in Genbank, and gained LG4-5 cDNA gene sequence using gene cloning technique, and construct the pichia yeast expression vector(pGAPZαA-LG4-5). The forward primer has the XhoⅠsite and the Kex2 cleavage site. The reverse primer has the NotⅠsite and not stop code. The C-terminal of The gained fusion protein with two tags( the myc protein with 10 peptides and the His protein with 6 His) is convenient for the identification and purification of the fusion protein. The secretion signal peptide of Its N-terminal can be cleaved on Kex2 cleavage site, and becomes a native N-terminal. After the linearized recombinant plasmid pGAPZαA-LG4-5 had been electrotransformed into the pichia pastoris SMD1168. The positive strains is identified by Zeocin and PCR. The recombinant protein is identified by SDS-PSGE and Western-blot. The best expression strain and time are confirmed by Dot-blot and ELISA. The result showed the recombinant protein LG4-5 can be secreted and expressed in pichia pastoris SMD1168. The best expression time is 72 hour. Finally, the function of the purified recombinant protein LG4-5 by Hitrap Chelating HP affinity chromatography was detected by cell attachment assay. The result showed the recombinant protein LG4-5 has biological activity. This study is basic work to find the biologically active sequences of human...