Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.8 (phytase)
 1,997 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for super-transformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-gamma and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L(-1) range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFalpha1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.
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PMID:Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha. 1761 19

Combining ease of genetic manipulation and fermentation with the ability to secrete and to glycosylate proteins in the basic eukaryotic manner, Arxula adeninivorans provides an attractive expression platform. Based on a redesign of the basic vector, a new Arxula vector system, Xplor 2, for heterologous gene expression was established, which allows (1) the construction of expression plasmids for supertransformation of A. adeninivorans strains secreting target proteins of biotechnological interest and (2) the integration of small vector cassettes consisting of yeast DNA sequences only. For this purpose, a set of modules including the ATRP1m selection-marker module, expression modules for constitutive expression of the genes phyK (Klebsiella-derived phytase) and IFNalpha2a (human interferon alpha), the HARS (Hansenula polymorpha autonomous replication sequence) for autonomous replication and the chaperone module AHSB4 promoter -HpCNE1 gene (calnexin) -PHO5 terminator to improve secretion efficiency were constructed and integrated in various combinations in the basic vector Xplor 2. After removal of the complete Escherichia coli-based plasmid parts (resistance marker, ColE1 ori and f1(-) origin), the remaining yeast-based linear vector fragment with or without rDNA targeting sequences were transformed as yeast rDNA integrative expression cassettes and yeast integrative expression cassettes (YICs), respectively, and the resulting strains were tested for their capacity to secrete PhyK or IFNalpha2a. Maximal expression levels were consistently obtained using YICs for transformation irrespective of whether or not they carry HARS and/or calnexin modules. It is recommended that at least 50 such transformants be analyzed to ensure selection of the best transformants.
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PMID:Xplor 2--an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans. 1967 89