title: Cloning and functional expression of a human lysozyme gene (hly) from             human leukocytes in Pichia pastoris.
abstract: hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His+, Mut+) with multiple hly copies. One transformant resistant to 4.0 mg/ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol.
authors.affiliation: School of Medicine and Pharmaceutics, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
authors.forename: Jing-Tu
authors.initials: JT
authors.lastname: Wei
authors.forename: Cun-Duo
authors.initials: CD
authors.lastname: Tang
authors.forename: Min-Chen
authors.initials: MC
authors.lastname: Wu
authors.forename: Gao-Lei
authors.initials: GL
authors.lastname: Liu
authors.forename: Hong-Ling
authors.initials: HL
authors.lastname: Shi
authors.forename: Jian-Fang
authors.initials: JF
authors.lastname: Li
mesh_terms: D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D009113:Muramidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D009113:Muramidase