High-level heterologous expression of an alkaline lipase gene from Penicillium cyclopium PG37 in Pichia pastoris

Abstract

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT-PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut ? ) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His ? , Mut ? ). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5 0 -and 3 0 -AOX1 primers. SDS-PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0-10.5 and at a temperature of 30°C or below.

A Unique Mono- and Diacylglycerol Lipase from Penicillium cyclopium: Heterologous Expression, Biochemical Characterization and Molecular Basis for Its Substrate Selectivity

“…The lip I gene was efficiently expressed in P. pastoris GS115 by induction under optimized conditions [7]. In this work, the cDNA encoding PcMdl was cloned and functionally expressed in P. pastoris .…”

Determinants for the improved thermostability of a mesophilic family 11 xylanase predicted by computational methods

“…The protein expression level, however, was not directly proportional to the concentration of G418 [19]. Therefore, all P. pastoris transformants, resistant to 1.0, 2.0, and 4.0 mg mL -1 of G418, were picked out for flask expression tests.…”

Enhanced Anti-Tumor (Anti-Proliferation) Activity of Recombinant Human Interleukin-29 (IL-29) Mutants Using Site-Directed Mutagenesis Method

“…Screening and Expression of P. pastoris Transformants P. pastoris transformants that could resist higher concentrations of geneticin G418 might have integrated multiple copies of heterologous genes into its genome, which could potentially lead to higher expression levels of heterologous proteins [14]. Based on the manual of Multi-Copy Pichia Expression Kit, 20 transformants of each mutant resistant to 2.0 and 4.0 mg/mL of G418 were picked out respectively for flask expression tests.…”