Recombinant expression of glycerol-3-phosphate dehydrogenase using the Pichia pastoris system

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 65; no. 1; pp. 128 - 132
• Main Authors: de Freitas Sanches Peres, Maristela, Silva, Viviane Cristina, Valentini, Sandro Roberto, de Lucca Gattás, Edwil Aparecida
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 01-08-2010; Elsevier
• Subjects: Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Glycerol-3-phosphate dehydrogenase (GPDH); Heterologous protein expression; Methods. Procedures. Technologies; Pichia pastoris; Glycerol-3-phosphate dehydrogenase (GPDH); Heterologous protein expression; Pichia pastoris; Ascomycota; Fungi; Glycerol-3-phosphate dehydrogenase; Enzyme; Oxidoreductases; Recombinant protein; Gene expression; GPDH
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2010.01.008

In the present study, the GPD2 gene from
Saccharomyces cerevisiae, which codifies for the enzyme glycerol-3-phosphate dehydrogenase (GPDH), was cloned from the pPICZ-α expression vector and used with the purpose of inducing the extracellular expression of the glycerol-3-phosphate dehydrogenase under the control of the methanol-regulated AOX promoter. The presence of the GPD2 insert was confirmed by PCR analysis.
Pichia pastoris X-33 (Mut
+) was transformed with linearized plasmids by electroporation and transformants were selected on YPDS plates containing 100
μg/mL of zeocin. Several clones were selected and the functionality of this enzyme obtained in a culture medium was assayed. Among the mutants tested, one exhibited 3.1
×
10
−2
U/mg of maximal activity. Maximal enzyme activity was achieved at 6 days of growth. Medium composition and pre-induction osmotic stress influenced protein production. Pre-induction osmotic stress (culturing cells in medium with either 0.35
M sodium chloride or 1.0
M sorbitol for 4
h prior to induction) led to an increase in cell growth with sorbitol and resulted in a significant increase in GPDH productivity with sodium chloride in 24
h of induction approximately fivefold greater than under standard conditions (without pre-induction).