Secretory expression and purification of a soluble NADH cytochrome b5 reductase enzyme from Mucor racemosus in Pichia pastoris based on codon usage adaptation.
The genome of Mucor racemosus was analyzed to determine the relative levels of codon usage. The codon bias differed from that of Escherichia coli. The active, soluble isoform of NADH cytochrome b5 reductase containing 228 amino acids was successfully overexpressed and secreted using alpha factor in Pichia pastoris under the control of the alcohol oxidase promoter and finally purified. The culture medium and incubation time were optimized, and the maximum expression level observed was about 23 U/ml using X-33 recombinant yeast grown for 120 h with 0.5% (v/v) methanol in complex media.
Mirzaei
S A
SA
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran.
Yazdi
M Tabatabaei
MT
Sepehrizadeh
Z
Z
D000595:Amino Acid Sequence; D003062:Codon; D003470:Culture Media / Q000737:chemistry; D042966:Cytochrome-B(5) Reductase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013997:Time Factors
D003062:Codon; D003470:Culture Media; D004271:DNA, Fungal; D011994:Recombinant Proteins; D042966:Cytochrome-B(5) Reductase