Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter.
We report the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris. The gene is predicted to encode a 35.4-kDa protein with significant sequence similarity to glyceraldehyde-3-phosphate dehydrogenases from other organisms. Promoter studies in P. pastoris using bacterial beta-lactamase as a reporter showed that the GAP promoter (P(GAP)) is constitutively expressed, although its strength varies depending on the carbon source used for cell growth. Expression of beta-lactamase under control of P(GAP) in glucose-grown cells was significantly higher than under control of the commonly employed alcohol oxidase 1 promoter (P(AOX1)) in methanol-grown cells. As an example of the use of P(GAP), we showed that beta-lactamase synthesized under transcriptional control of P(GAP) is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal. P(GAP) has been successfully utilized for synthesis of heterologous proteins from bacterial, yeast, insect and mammalian origins, and therefore is an attractive alternative to P(AOX1) in P. pastoris.
Waterham
H R
HR
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Digan
M E
ME
Koutz
P J
PJ
Lair
S V
SV
Cregg
J M
JM
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D008830:Microbodies / Q000378:metabolism; D008969:Molecular Sequence Data; D019301:Oleic Acid / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D019301:Oleic Acid; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D001618:beta-Lactamases; D005947:Glucose; D005990:Glycerol; D000432:Methanol