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.

Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris

Abstract: Laccases are strong oxidizing enzymes that oxidize chlorinated phenols, synthetic dyes, pesticides, polycyclic aromatic hydrocarbons as well as a very wide range of other compounds with high redox potential. Based on the bias of genetic codons between fungus and yeast, we synthesized a laccase gene GlLCCI, originated from Ganoderma lucidum using optimized codons and a PCR-based two-step DNA synthesis method. The recombinant laccase, GlLCCI was successfully over-expressed in yeast, Pichia pastoris, with an alcohol oxidase1 promoter. The recombinant GlLCCI has a molecular mass of approximately 58 kDa. The K m values of GlLCCI for 2-2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol were 0.9665, and 1.1122 mM, respectively. The V max of GlLCCI for both substrates was 3,024 and 82.13 μM mg−1 min−1. When ABTS was used as a substrate, the enzyme had an optimal temperature of approximately 55°C. The enzyme was detected over pH values from 2 to 8. The enzyme was strongly activated by K+, Na+, Cu2+ and mannitol. Six amino acids (alanine, histidine, glycine, arginine, aspartate and phenylalanine) increased the catalytic ability of the enzyme. The activity of laccase was obviously inhibited by Fe2+, Fe3+, sodium hydrosulphite, and sodium azide. Additionally, under optimal conditions, GlLCCI decolorized 37.62 mg l−1 of azo dye methyl orange (MO) in cultural medium. With a high MO degradation ability, GlLCCI may have potential in the treatment of industrial effluent containing azo dye MO.

Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50

Abstract: In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast.

Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification, and characterization.

Abstract: Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.

Mammalian glutaminyl cyclases and their isoenzymes have identical enzymatic characteristics

Abstract: Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate residues at the N-terminus of several peptides and proteins from plants and animals. Recently, isoenzymes of mammalian QCs have been identified. In order to gain further insight into the biochemical characteristics of isoQCs, the human and murine enzymes were expressed in the secretory pathway of Pichia pastoris. Replacement of the N-terminal signal anchor by an alpha-factor prepropeptide from Saccharomyces cerevisiae resulted in poor secretion of the protein. Insertion of an N-terminal glycosylation site and shortening of the N-terminus improved isoQC secretion 100-fold. A comparison of different recombinant isoQC proteins did not reveal an influence of mutagenic changes on catalytic activity. An initial characterization showed identical modes of substrate conversion of human isoQC and murine isoQC. Both proteins displayed a broad substrate specificity and preference for hydrophobic substrates, similar to the related QC. Likewise, a determination of the zinc content and reactivation of the apo-isoQC revealed equimolar zinc present in QC and isoQC. Far-UV CD spectroscopic analysis of murine QC and isoQC indicated virtually identical structural components. The present investigation provides the first enzymatic characterization of mammalian isoQCs. QC and isoQC represent very similar proteins, which are both present in the secretory pathway of cells. The functions of QCs and isoQC probably complement each other, suggesting a pivotal role of pyroglutamate modification for protein and peptide maturation.