Protein Expression In Pichia Pastoris Research Articles

Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris

Secretion efficiency of the 85-amino acid Sacchromyces cerevisiae alpha signal peptide and the 25-amino acid Candida antarctica lipase B signal (nsB) peptide were compared. Three reporter proteins used for the study are C. antarctica lipase A (CalA), lipase B (CalB) and hGMCSF. The copy number of recombinant α-CalB and nsB-CalB clones was determined by qPCR and clones with equivalent gene copies were used for comparative analysis. About threefold increased CalB production corresponding to an activity of 480Uml−1 was obtained with its native signal peptide, whereas with the alpha signal peptide the maximum activity was 160Uml−1. Also, CalB was secreted as a mature protein with native N-terminus when fused to its own signal peptide, while unprocessed CalB with N-terminal extension was detected with the alpha signal peptide. Real time PCR analysis of CalB strains indicated that the difference in protein expression was not at the transcriptional level. The nsB signal sequence was also effective in secreting CalA enzyme and its secretion efficiency was on par with the alpha signal sequence. Further, hGMCSF fused inframe with the nsB signal peptide was also efficiently secreted into the medium. These results indicate that the nsB signal peptide can be a better alternative to alpha signal peptide for heterologous protein expression in Pichia pastoris.

Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5′ untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5′UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5′UTR clearly decreased expression of a β-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5′UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5′UTR optimized for a higher level of expression may be challenging.

Incomplete protein disulphide bond conformation and decreased protein expression result from high cell growth during heterologous protein expression in Pichia pastoris

Previous report has shown that the expression of recombinant human consensus interferon-α mutant (cIFN) in Pichia pastoris in bioreactor is limited with respect to the incorrectly folded cIFN with incomplete disulfide bond, which lead to the degradation and aggregation of cIFN. In this study, the origin of incorrectly folded cIFN is firstly studied. Fed-batch fermentation in bioreactor shows that the incorrectly folded cIFN is formed intramolecularly and secreted to the extracellular environment. Further chemostat cultures indicate that the specific growth rate is the critical factor for the production of incorrect cIFN. In addition, cell shows reduced expression level of cIFN at high specific growth rate. We also demonstrate that the incorrectly folded cIFN could form aggregates intracellularly and these aggregates are non-covalent forms. Taken together, these results suggest that the efficient heterologous expression of cIFN is limited by high cell growth that is unique from expression limitations seen for soluble proteins. A balance has to be found between the increase for high efficient expression of heterologous proteins and requirement of the high cell growth during the expression of recombinant proteins in P. pastoris.

Efficient vectors for expression cloning of large numbers of PCR fragments in P. pastoris

The yeast vectors described, pYEV and pYEVB, were designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) and immediate protein expression in Pichia pastoris. The pYEV vector was used to clone PCR fragments obtained by using Taq or similar polymerase mixes, which leave an A-base overhang. The other vector pYEVB, with the same features for blunt-end ligation of PCR products, was developed to be complementary to pYEV. These two plasmids were linearized using the restriction enzymes BfuI and SchI, respectively. The purified PCR products, without any other treatments, were cloned into the linearized vectors, followed by selection on plates supplemented with X-gal. Only desired recombinants carrying the target gene in the correct orientation can give typical blue colonies. This screening technique is based on a lacO reconstruction strategy that can produce a full-length lacO to exhaust endogenous LacI and switch on the transcription of lacZ in the host. The recombinant plasmids extracted from the blue colonies can be linearized by SalI and transformed into P. pastoris for immediate expression. By using these two vectors, researchers could be saved from the tedious and time-consuming conventional cloning procedures.

Molecular Mechanisms of Yeast Cell Wall Glucan Remodeling

Yeast cell wall remodeling is controlled by the equilibrium between glycoside hydrolases, glycosyltransferases, and transglycosylases. Family 72 glycoside hydrolases (GH72) are ubiquitous in fungal organisms and are known to possess significant transglycosylase activity, producing elongated beta(1-3) glucan chains. However, the molecular mechanisms that control the balance between hydrolysis and transglycosylation in these enzymes are not understood. Here we present the first crystal structure of a glucan transglycosylase, Saccharomyces cerevisiae Gas2 (ScGas2), revealing a multidomain fold, with a (betaalpha)(8) catalytic core and a separate glucan binding domain with an elongated, conserved glucan binding groove. Structures of ScGas2 complexes with different beta-glucan substrate/product oligosaccharides provide "snapshots" of substrate binding and hydrolysis/transglycosylation giving the first insights into the mechanisms these enzymes employ to drive beta(1-3) glucan elongation. Together with mutagenesis and analysis of reaction products, the structures suggest a "base occlusion" mechanism through which these enzymes protect the covalent protein-enzyme intermediate from a water nucleophile, thus controlling the balance between hydrolysis and transglycosylation and driving the elongation of beta(1-3) glucan chains in the yeast cell wall.

The effect of ethanol and acetate on protein expression in Pichia pastoris

Pichia pastoris is an excellent host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. P. pastoris produces a small amount of ethanol as a by-product during the glycerol fed-batch phase and the mixed-feed induction phase (glycerol-methanol) of high cell density fermentations, regardless of the phenotype (Mut +, Mut s, or Mut −). We have investigated ethanol repression of the AOX1 promoter using strains, GS115 (Mut +) and MC100-3 (Mut −), expressing an AOX1-lacZ fusion. The addition of 10 mg l −1 ethanol at the start of methanol induction delayed β-galactosidase production and methanol utilization for four hours in shake flask experiments. When ethanol and acetate were added together, all of the ethanol was converted to acetate, which also represses the AOX1 promoter. The effects of ethanol and acetate on protein expression in P. pastoris at shake flask and fermentor conditions are discussed.

The Effect of Ethanol and Acetate on Protein Expression in Pichia pastoris.

Pichia pastoris is an excellent host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. P. pastoris produces a small amount of ethanol as a by-product during the glycerol fed-batch phase and the mixed-feed induction phase (glycerol-methanol) of high cell density fermentations, regardless of the phenotype (Mut+, Mut(s), or Mut-). We have nvestigated ethanol repression of the AOX1 promoter using strains, GS115 (Mut+) and MC100-3 (Mut-), expressing an AOX1-lacZ fusion. The addition of 10 mg l(-1) ethanol at the start of methanol induction delayed beta-galactosidase production and methanol utilization for four hours in shake flask experiments. When ethanol and acetate were added together, all of the ethanol was converted to acetate, which also represses the AOX1 promoter. The effects of ethanol and acetate on protein expression in P. pastoris at shake flask and fermentor conditions are discussed.

A system for dual protein expression in Pichia pastoris and Escherichia coli.

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.

Small vectors for expression based on dominant drug resistance with direct multicopy selection.

The most commonly used vectors for heterologous protein expression in Pichia pastoris carry its wild-type HIS4 gene and the bacterial ampicillin resistance gene as selectable markers (1–6). The HIS4 gene is relatively large (3 kb), with ill-defined functional boundaries, and its use limits the vectors to selection in his4 auxotrophic strains. HIS4-based vectors are large, typically between 7 and 10 kb in size, creating difficulties for routine cloning manipulations, especially when generating constructions with large or multiple insertions. The Escherichia coli kanamycin gene (from Tn 903) present on some P. pastoris expression vectors functions as a second marker on some HIS4-based vectors to identify multicopy (i.e., G418 hyperresistant) recombinant strains. However, it cannot be used efficiently as a direct selectable marker for transformation of P. pastoris to G418 resistance, and its presence leads to a further increase in vector size. The combination of the HIS4, ampicillin, and kanamycin genes adds up to 5.5 kb of “space” in a typical expression vector.

Overview of Protein Expression in Pichia pastoris

Pichia pastoris is a methylotrophic yeast and can be used as a heterologous expression system. This microorganism is as easy to manipulate as Escherichia coli, but has many of the advantages of eukaryotic expression (e.g., protein processing, folding, and post-translational modifications), and it is faster, easier, and cheaper to use than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture. It also generally yields higher expression levels. This overview discusses important considerations for the use of Pichia pastoris, including strains for expression, expression plasmids, transformation by integration, and post-translational modifications. Examples of expression are given and finally, legal issues regarding patent rights for heterologous protein expression in Pichia pastoris are described.