Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P11021 (BiP)
 2,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source.
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PMID:Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. 1475 54

Efficient production of heterologous proteins with yeasts and other eukaryotic hosts is often hampered by inefficient secretion of the product. Limitation of protein secretion has been attributed to a low folding rate, and a rational solution is the overexpression of proteins supporting folding, like protein disulfide isomerase (Pdi), or the unfolded protein response transcription factor Hac1. Assuming that other protein factors which are not directly involved in protein folding may also support secretion of heterologous proteins, we set out to analyze the differential transcriptome of a Pichia pastoris strain overexpressing human trypsinogen versus that of a nonexpressing strain. Five hundred twenty-four genes were identified to be significantly regulated. Excluding those genes with totally divergent functions (like, e.g., core metabolism), we reduced this number to 13 genes which were upregulated in the expression strain having potential function in the secretion machinery and in stress regulation. The respective Saccharomyces cerevisiae homologs of these genes, including the previously characterized secretion helpers PDI1, ERO1, SSO2, KAR2/BiP, and HAC1 as positive controls, were cloned and overexpressed in a P. pastoris strain expressing a human antibody Fab fragment. All genes except one showed a positive effect on Fab fragment secretion, as did the controls. Six out of these novel secretion helper factors, more precisely Bfr2 and Bmh2 (involved in protein transport), the chaperones Ssa4 and Sse1, the vacuolar ATPase subunit Cup5, and Kin2 (a protein kinase connected to exocytosis), proved their benefits for practical application in laboratory-scale production processes by increasing both specific production rates and the volumetric productivity of an antibody fragment up to 2.5-fold in fed-batch fermentations of P. pastoris.
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PMID:Transcriptomics-based identification of novel factors enhancing heterologous protein secretion in yeasts. 1776 60