Secretory expression and purification of a soluble NADH cytochrome b5 reductase enzyme from Mucor racemosus in Pichia pastoris based on codon usage adaptation

• Published in: Biotechnology letters 2010-11, Vol.32 (11), p.1705-1711
• Main Authors: Mirzaei, S. A, Yazdi, M. Tabatabaei, Sepehrizadeh, Z
• Format: Article
• Language: English
• Subjects: Active control; Amino Acid Sequence; Amino acids; Biological and medical sciences; Biotechnology; Codon; Culture Media - chemistry; Cytochrome; Cytochrome b5 reductase; Cytochrome-B Reductase - genetics; Cytochrome-B Reductase - isolation & purification; Cytochrome-B Reductase - secretion; Cytochromes; DNA, Fungal - chemistry; DNA, Fungal - genetics; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors; Genomes; Genomics; Methanol; Methyl alcohol; Molecular Sequence Data; Mucor - enzymology; Mucor - genetics; Mucor racemosus; NADH; Pichia - genetics; Pichia pastoris; Promoter Regions, Genetic; Protein purification; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - secretion; Reductases; Relative adaptiveness; Secretory expression; Sequence Analysis, DNA; Time Factors; Yeasts
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-010-0348-z

The genome of Mucor racemosus was analyzed to determine the relative levels of codon usage. The codon bias differred 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.