Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

Molecular Medicine Reports
Jing-Tu WeiJianfang Li

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 w...Continue Reading

Citations

Jan 17, 2014·Journal of Biotechnology·Huiqing YuGuoxiang Cheng
Mar 27, 2020·Frontiers in Bioengineering and Biotechnology·Huahua HeLi Yi
Nov 21, 2020·International Journal of Molecular Sciences·Seung-Gyun WooDae-Hee Lee

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