The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter.
We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris. This gene was isolated from a P. pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases. The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids. The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms. There is a 64% identity between amino acid sequences of P. pastoris Icl and Saccharomyces cerevisiae Icl. Northern blot analyses showed that, as in S. cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth. Expression in P. pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P(ICL1) is a good alternative for the expression of heterologous proteins in this methylotrophic yeast. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ272040.
Menendez
Javier
J
División de Vacunas, Centro de Ingeniería Genética y Biotecnología, La Habana, Cuba. javier.menendez@utoronto.ca
Valdes
Iris
I
Cabrera
Nelson
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003910:Dextranase / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D007522:Isocitrate Lyase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D016415:Sequence Alignment; D014158:Transcription, Genetic / Q000502:physiology*
D005656:Fungal Proteins; D003910:Dextranase; D007522:Isocitrate Lyase