Topic
Pichia pastoris
About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.
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TL;DR: To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed.
Abstract: Sea raven type II antifreeze protein (SRAFP) is one of three diAerent fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimen- sional structure of SRAFP, study its ice-binding mech- anism, and as a basis for engineering these molecules, an eAcient system for its biosynthetic production was de- veloped. Several diAerent expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake- flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four diAerent strate- gies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut s clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut + clones. The mixed- feeding/Mut s strategy proved to be the most eAcient with SRAFP yields reaching 30 mg/l.
69 citations
TL;DR: The deuteration procedures examined here should facilitate economical expression of 2H/13C/15N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.
Abstract: Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D2O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D2O medium with protiated methanol as carbon source, or in 95% D2O medium containing deuterated methanol. A high level of uniform Calpha deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [1H/15N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues. including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of 2H/13C/15N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.
69 citations
TL;DR: Recombinant tLH stimulated the release of 11-ketotestosterone from mature testes, whereas its release from immature testes was less pronounced, and was found to possess only N-linked carbohydrates.
69 citations
TL;DR: The first heterologous expression of human CBG is described, showing that the human enzyme has significant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, flavonoids, simple phenolics and cyanogens with higher apparent affinities and specificities for dietary Xenobiotics than for other aryl-glycosides.
Abstract: Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic β-glucosidase (CBG) that hydrolyses various β-d-glycosides, but whose physiological function is not known. Here, we describe the first heterologous expression of human CBG, a system that facilitated␣a detailed assessment of the enzyme specificity towards dietary glycosides. A full-length CBG cDNA (cbg-1) was cloned from a human liver cDNA library and expressed in the methylotrophic yeast Pichia pastoris at a secretion yield of ≈ 10 mg·L−1. The recombinant CBG (reCBG) was purified from the supernatant using a single chromatography step and was shown to be similar to the native enzyme isolated from human liver in terms of physical properties and specific activity towards 4-nitrophenyl-β-d-glucoside. Furthermore, the reCBG displayed a broad specificity with respect to the glycone moiety of various aryl-glycosides (β-d-fucosides, α-l-arabinosides, β-d-glucosides, β-d-galactosides, β-l-xylosides, β-d-arabinosides), similar to the native enzyme. For the first time, we show that the human enzyme has significant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, flavonoids, simple phenolics and cyanogens with higher apparent affinities (Km) and specificities (kcat/Km) for dietary xenobiotics than for other aryl-glycosides. These data indicate that human CBG hydrolyses a broad range of dietary glucosides and may play a critical role in xenobiotic metabolism.
69 citations
TL;DR: The xylanase gene, xynAM6, was isolated from the genomic DNA library of Streptomyces megasporus DSM 41476 using colony PCR screening method and showed broad temperature adaptability, had good thermostability at 60°C and 70°C, remained stable at pH 4.0-11.0, and was resistant to most proteases.
69 citations