Novel Pichia methanolica secretory signal polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods of using are disclosed. Methods of producing large amounts of recombinant proteins by employing DNA constructs having a polypeptide of interest preceded by a novel Pichia methanolica secretory signal sequence.

Methods for preparing Pichia methanolica cells containing foreign DNA constructs and methods for producing foreign polypeptides in Pichia methanolica cells are disclosed. The cells are transformed with a DNA construct comprising a first DNA segment comprising a transcription promoter of a methanol-inducible P. methanolica gene, a second DNA segment encoding a higher eukaryotic polypeptide, a third DNA segment comprising a P. methanolica gene transcription terminator, and a selectable marker. For...

Novel Pichia methanolica secretory signal polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods of using are disclosed. Methods of producing large amounts of recombinant proteins by employing DNA constructs having a polypeptide of interest preceded by a novel Pichia methanolica secretory signal sequence.

Methods for transforming Pichia methanolica, and DNA molecules useful in transformation of P. methanolica, are disclosed. P. methanolica cells are exposed, in the presence of DNA molecules, to a pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a pulse duration of from 1 to 40 milliseconds, whereby the DNA molecules are introduced into the cells. The DNA molecules may comprise an expression unit that includes a transcription promoter of a P. methanolica gene operably lin...

Methods for altering a selected chromosomal locus in P. methanolica cells and cells comprising such altered loci are disclosed. A linear DNA construct comprising (i) a segment comprising a portion of the target locus in which at least one nucleotide pair is altered and (ii) a selectable marker that complements adenine auxotrophy is introduced into cells auxotrophic for adenine. The cells are cultured under selective conditions, and cells in which the linear DNA construct has been chromosomally i...

Novel Pichia methanolica secretory signal polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods of using are disclosed. Methods of producing large amounts of recombinant proteins by employing DNA constructs having a polypeptide of interest preceded by a novel Pichia methanolica secretory signal sequence.

Methods for preparing Pichia methanolica cells having auxotrophic mutations are disclosed. The methods comprise the steps of (a) exposing P. methanolica cells to mutagenizing conditions, (b) culturing the cells from step (a) in a rich medium to allow mutations to become established and replicated in at least a portion of the cells, (c) culturing the cells from step (b) in a culture medium deficient in assimilable nitrogen to deplete cellular nitrogen stores, (d) culturing the cells from step (c)...

Protease-deficient strains of the methylotrophic yeast Pichia methanolica and materials and methods for generating such strains are disclosed. The strains have a functional deficiency in a vacuolar protease, such as proteinase A or proteinase B. The strains are useful as hosts for the expression of heterologous genes encoding proteins of commercial or other interest.

DNA constructs, Pichia methanolica cells, and methods for producing polypeptides using the DNA constructs and cells are disclosed. The DNA constructs comprise a transcription promoter of a Pichia methanolica gene, a DNA segment encoding a polypeptide heterologous to P. methanolica, a transcription terminator of a P. methanolica gene, and a selectable marker. P. methanolica cells containing the DNA constructs are used within methods for producing polypeptides heterologous to P. methanolica. DNA m...

DNA constructs, Pichia methanolica cells, and methods for producing polypeptides using the DNA constructs and cells are disclosed. The DNA constructs comprise a transcription promoter of a Pichia methanolica gene, a DNA segment encoding a polypeptide heterologous to P. methanolica, a transcription terminator of a P. methanolica gene, and a selectable marker. P. methanolica cells containing the DNA constructs are used within methods for producing polypeptides heterologous to P. methanolica. DNA m...