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A dual-expression vector allowing expression in E. coli and P. pastoris, including new modifications
... Genes that are cloned inframe to this sequence will code for enzymes that are secreted extracellularly into the media. Cahill and colleagues also developed a series of P. pastoris episomal expression vectors (Lueking et al., 2000(Lueking et al., , 2003. However, their expression vectors did not contain a signal sequence. ...
... This feature is in contrast to the inducible promoters employed by Cahill and colleagues to design their episomal expression vectors. They used the AOX (methanol inducible) (Lueking et al., 2000) and the CUP (copper inducible) (Lueking et al., 2003) promoters. ...
... Although this zeocin selection marker can also be used in E. coli, we chose to include the ampicillin-resistance gene (ampR) because the ampicillin antibiotic is more stable and economical than zeocin. The inclusion of the ampicillin-resistance gene is another feature of pBGP1 that is distinct from the vectors designed by Cahill and colleagues (Lueking et al., 2000(Lueking et al., , 2003. ...
We describe here the construction of Gateway-compatible vectors, pBGP1-DEST and pPICZα-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. Both vectors direct the synthesis of fusion proteins consisting of the N-terminal signal and pro-sequences of Saccharomyces cerevisiae α-factor, the recognition sites for Kex2 and Ste13 processing proteases, the mature region of a foreign protein flanked by attB1- and attB2-derived sequences at N- and C-termini, respectively, and myc plus hexahistidine tags added at the extreme C-terminus. To test the usefulness of these vectors, production of endo-glucanases and xylanases from termite symbionts, as well as a fungal glucuronoyl esterase, was performed. Enzyme activities were detected in the culture supernatants, indicating that the chimeric proteins were synthesized and secreted as designed.
... Genes that are cloned inframe to this sequence will code for enzymes that are secreted extracellularly into the media. Cahill and colleagues also developed a series of P. pastoris episomal expression vectors (Lueking et al., 2000Lueking et al., , 2003). However, their expression vectors did not contain a signal sequence. ...
... This feature is in contrast to the inducible promoters employed by Cahill and colleagues to design their episomal expression vectors. They used the AOX (methanol inducible) (Lueking et al., 2000 ) and the CUP (copper inducible) (Lueking et al., 2003) promoters. The GAP, AOX, and CUP promoters have comparable activities (Koller et al., 2000; Waterham et al., 1997). ...
... Although this zeocin selection marker can also be used in E. coli, we chose to include the ampicillin-resistance gene (ampR) because the ampicillin antibiotic is more stable and economical than zeocin. The inclusion of the ampicillin-resistance gene is another feature of pBGP1 that is distinct from the vectors designed by Cahill and colleagues (Lueking et al., 2000Lueking et al., , 2003). ...
... Genes that are cloned inframe to this sequence will code for enzymes that are secreted extracellularly into the media. Cahill and colleagues also developed a series of P. pastoris episomal expression vectors (Lueking et al., 2000(Lueking et al., , 2003. However, their expression vectors did not contain a signal sequence. ...
... This feature is in contrast to the inducible promoters employed by Cahill and colleagues to design their episomal expression vectors. They used the AOX (methanol inducible) (Lueking et al., 2000) and the CUP (copper inducible) (Lueking et al., 2003) promoters. ...
... Although this zeocin selection marker can also be used in E. coli, we chose to include the ampicillin-resistance gene (ampR) because the ampicillin antibiotic is more stable and economical than zeocin. The inclusion of the ampicillin-resistance gene is another feature of pBGP1 that is distinct from the vectors designed by Cahill and colleagues (Lueking et al., 2000(Lueking et al., , 2003. ...
Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screening projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the alpha-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants.
... The low cost of culture media, the ease of scaling up from small cultures to fermentation cultures, and high biomass production in fermenters (up to 400 g/L) combine to make Pichia a very useful protein expression system. Several thousand heterologous proteins have now been expressed in P. pastoris (Cregg 2007), including many eukaryotic and integral membrane proteins that were nonfunctional when expressed in E. coli (Lueking et al. 2003;Parcej and Eckhardt-Strelau 2003;Long et al. 2005;Andre et al. 2006;Aller et al. 2009); see also Introduction: Considerations for Membrane Protein Purification (Kielkopf et al. 2021g). ...
Obtaining high quantities of a specific protein directly from native sources is often challenging, particularly when dealing with human proteins. To overcome this obstacle, many researchers take advantage of heterologous expression systems by cloning genes into artificial vectors designed to operate within easily cultured cells, such as Escherichia coli, Pichia pastoris (yeast), and several varieties of insect and mammalian cells. Heterologous expression systems also allow for easy modification of the protein to optimize expression, mutational analysis of specific sites within the protein and facilitate their purification with engineered affinity tags. Some degree of purification of the target protein is usually required for functional analysis. Purification to near homogeneity is essential for characterization of protein structure by X-ray crystallography or nuclear magnetic resonance (NMR) and characterization of the biochemical and biophysical properties of a protein, because contaminating proteins almost always adversely affect the results. Methods for producing and purifying proteins in several different expression platforms and using a variety of vectors are introduced here.
... Various vectors have been developed for expression in P. pastoris; however, most of them cannot be maintained as extra-chromosomal elements, due to lack of autonomous origin of replication sequence [27][28][29]. On the other hand, available vectors which contain autonomous replication sequences lack a signal sequence for the secretion of recombinant protein products into the fermentation medium, and can therefore only perform cytosolic expression [30,31]. So far, only two episomal Pichia pastoris vectors which contain a signal sequence for secretory expression have been reported [13,15]. ...
Aim and objective:
This study describes the design and evaluation of an expression vector for Pichia pastoris (pPICZαBHF), which is based on the commercial vector construct pPICZαB.
Material and method:
The performance of pPICZαBHF was evaluated with red fluorescence protein as a reporter. Additional His- and Flag-tags on the N-terminal ensure a simplified protein purification procedure. Transformation efficiency, expression level and plasmid maintenance were studied in order to test the functionality and usefulness of the constructed vector.
Results:
We found that high transformation efficiencies were achieved using pPICZαBHF plasmid for yeast cell transformation in comparison with the commercial vector pPICZαB, which has to be integrated into the Pichia genome. However, expression levels of the recombinant protein were generally lower compared to the commercial construct. Recombinant plasmids were shown to be maintained in cells for approximately five days.
Conclusion:
Although pPICZαBHF may not suitable for the production of high levels of recombinant protein, the simplicity of this P. pastoris expression system may be still of interest for the expression of proteins involved in cofactor regeneration or the expression of reporter genes. In addition, high transformation efficiency of pPICZαBHF may be beneficial for the applications such as the high-throughput screening of mutant gene libraries.
... Komagataella phaffii X-33 was transformed by electroporation following the protocol described in the Pichia Expression Kit (Invitrogen). Transformation with pYRCre2 was carried out as previously described for the auto-replicative pPICHOLI vector [51]. ...
Background
A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele.
Results
A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth.
Conclusions
In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-017-0715-8) contains supplementary material, which is available to authorized users.
... Este trabalho contrastou com os resultados de Sears et al. (1998) que estudaram os níveis de expressão da enzima β-glucuronidase sob controle dos promotores AOX1, GAP e YPT1 sendo que o primeiro foi bem superior aos outros (AOX1 587 U/mL; GAP 70,4 U/mL e YPT1 1,67 U/mL). Nestes exemplos, é evidente que o tipo de promotor não é o único fator que determina os altos níveis de expressão heteróloga, mas sim, a combinação de vários fatores, como tipo vetor de expressão empregado, linhagem hospedeira, local de integração do cassete de expressão, composição do meio de cultura e propriedades da proteína a ser expressa (Lueking et al., 2003). O conjunto destes estudos mostra, pois, que as condições ótimas de expressão devem ser analisadas caso a caso, e que os dados obtidos com um determinado clone não são, geralmente, transferíveis para a expressão de outra proteína e a otimização do processo deve ser buscada para cada novo produto (Hong et al., 2006) ...
Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2008. A levedura metilotrófica Pichia pastoris tem sido utilizada com sucesso para a expressão de várias proteínas heterólogas sendo que diversos vetores já foram desenvolvidos para este sistema. Os principais vetores de expressão de P. pastoris são baseados no promotor do gene AOX1 que codifica a enzima álcool oxidase 1. Alguns estudos têm sido realizados para o uso de promotores alternativos uma vez que o sistema AOX1 apresenta algumas desvantagens. Em nosso laboratório foi isolado e caracterizado o promotor do gene PGK1 de P. pastoris (PPGK1), um promotor forte e constitutivo, que representa uma alternativa promissora para a construção de novos vetores. O objetivo deste estudo foi determinar a região promotora mínima do PPGK1 por meio de deleções controladas utilizando o gene da α-amilase de Bacillus subtilis como gene repórter. Quatro deleções foram obtidas sendo que a menor (PPGKΔ3), com ~400 pb, ainda manteve atividade promotora. Foi demonstrado que esta seqüência é capaz de dirigir a integração de um vetor no genoma de P. pastoris e 51% dos clones transformantes tiveram mais de uma cópia integrada. O uso deste promotor propiciou a expressão heteróloga de α-amilase em altos níveis (250 U.mL-1). Também foram construídos vetores para expressão intracelular baseados no promotor PGK que, todavia, ainda necessitam ser aprimorados para uso em P. pastoris. _______________________________________________________________________________ ABSTRACT The methylotrophic yeast Pichia pastoris has been successfully used for the expression of heterologous proteins and several different expression vectors have been developed for this system. The main expression vectors for P. pastoris are based on the alcohol oxidase 1 gene promoter, AOX1. Some studies have been carried out aiming at the use of alternative promoters once the AOX1 system shows some disadvantages. Our laboratory has isolated and characterized the P. pastoris promoter from the PGK1 gene (PPGK1), a strong and constitutive promoter, which represents a promising alternative for the construction of new vectors. The aim of this study was to determine the minimal promoter sequences from PPGK1 through controlled deletions using the α-amylase gene from Bacillus subtilis as a reporter gene. Four deletions were obtained and the smallest (PPGK Δ3) with ~ 400 bp, still maintained promoter activity. We have demonstrated that this sequence was able to direct vector integration into the P. pastoris genome and 51% of the transformants showed more than one copy integrated. The use of this promoter allowed high level expression of α-amylase (250 UmL-1). Also, vectors for intracellular expression were based on the PGK promoter were also constructed which however still need to be improved for use in P. pastoris.
... To identify the protein-protein interactions responsible for this regulation, we synthesized a biotin-tagged peptide corresponding to this sequence and used it as a probe to identify high affinity protein interactions dependent on this motif. From a high content, high density protein array derived from a redundant human brain cDNA expression library (37,200 clones) (43,44,46), we identified a number of novel, potential integrinregulating proteins (Fig. 1A). Nineteen clones, corresponding to thirteen different proteins, bound significantly and specifically to the biotin-tagged KVGFFKR peptide on the array, but not to a control peptide, biotin KAAAAAR. ...
A critical role for the conserved alpha-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin alpha(IIb)beta(3). To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with alpha(IIb)beta(3) by surface plasmon resonance. The affinity of this interaction was 82.2 +/- 24.4 nm in a cell free assay. ICln co-immunoprecipitates with alpha(IIb)beta(3) in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 microm to 5 mm), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10-100 microm) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.
A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P. pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P. pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http:// www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P. pastoris. HR547, HR919, and HR1697 hurnan proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P. pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P. pastoris.
The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.
Die methylotrophe Hefe Pichia pastoris wurde als eukaryotisches Expressions- und Sekretionssystem zur Evaluierung einer Funktionellen Protein Technologie (FunProTec) getestet. Die humanen Phosphodiesterasen 10A und 1B1 (kPDE10A, PDE1B1), das humane Lungensurfactant Protein D (SP-D(N/CRD)), die alkalische Phosphatase (PhoA) aus E. coli K-12 und die Lipase A (LipA) aus B. subtilis 168M wurden als Beispielproteine für die heterologe Genexpression gewählt. Außer für kPDE10A wurde die Expression und Sekretion aller Proteine in das Medium gezeigt. Bei der Expression von rekombinantem SP-D(N/CRD) wurde gezeigt, dass die Expressionsprodukte proteolytisch degradiert waren. Es wurden mehrere physio-logische und molekularbiologische Strategien zur Minimierung der Protein-degradation von rekombinantem SP-D(N/CRD) verfolgt. Durch den Einsatz von Pepstatin (15 µg/mL) wurde bei der Schüttelkolbenexpression ein Rückgang der Degradation von SP-D(N/CRD) erzielt. Dieses Experiment zeigte auch, dass mög-licherweise eine Aspartat-Peptidase an der Degradation von SP-D(N/CRD) betei-ligt war. Ausgehend von der P. pastoris Genomsequenz wurden acht Aspartat-Peptidasen identifiziert: Proteinase A und sieben weitere, vorher nicht beschriebene, Aspartat-Peptidasen (Aspartat-Peptidase 1 bis Aspartat-Peptidase 7). Diese neu identifizierten Aspartat-Peptidasen wurden kloniert und mittels bioinformatischer Studien untersucht. Es konnten mögliche Aussagen über wichtige Strukturelemente getroffen werden. Die Aspartat-Peptidasen 3 und 5 wurden in dem P. pastoris-Stamm X-33 durch homologe Rekombination deletiert. Die Expression von rekombinantem SP-D(N/CRD) erfolgte in beiden Protease-defizienten Stämmen und zeigte vor allem bei dem Stamm P. pastoris X-33deltaAP-5-SP-D(N/CRD) ein verändertes Degradationsprofil im Vergleich zum Wildtyp-Stamm. The methylotrophic yeast Pichia pastoris was used as a possible eukaryotic expression and secretion system for the evaluation of a functional protein tech-nology (FunProTec). Human phosphodiesterase 1B1 and 10A, a truncated form of human lung surfactant protein D (SP-D(N/CRD)), lipase A from Bacillus subtilis 168M and alkaline phosphatase from E. coli K-12 were chosen as examples for heterologous gene expression. Gene expression and protein secretion was ob-served for all proteins except for phosphodiesterase 10A. The analysis of the expression products of SP-D(N/CRD) showed that the recom-binant protein was proteolytically degraded. Several experiments, including physiological and molecular approaches, were performed to avoid proteolytic deg-radation of recombinant SP-D(N/CRD). Only when the inhibitor of aspartic pro-teinases pepstatin (15 µg/mL) was added to the medium a significant decrease of protein degradation could be observed. This experiment also showed that proba-bly al least one aspartic proteinase was involved in the proteolytic degradation of SP-D(N/CRD). Based on the preliminary P. pastoris genome sequence data proteinase A and seven yet undescribed aspartic proteinases destinated aspartic proteinase 1 to aspartic proteinase 7 were identified. The sequence data of these proteinases were verified by cloning experiments and subjected to bioinformatic studies so that conclusions concerning the primary structure of the proteinases were drawn. Protease-deficient strains of aspartic proteinase 3 and aspartic proteinase 5 were constructed in P. pastoris X-33. Expression of recombinant SP-D(N/CRD) was carried out in the designed protease-deficient strains and showed to be different in comparison to the wildtype strain.
There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not previously available. Here we show a glass chip containing 2413 nonredundant purified human fusion proteins on a polymer surface, where densities up to 1600 proteins/cm(2) on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions under nondenaturing conditions. Such screenings require only 200-microl sample volumes, illustrating their potential for high-throughput applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity; and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first application, we have incubated these protein chips with anti-RGSHis(6), anti-GAPDH, and anti-HSP90beta antibodies. In an initial proof of principle study for the second application, we have screened serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins to generate novel diagnostic markers may be possible.
The human genome has been sequenced and the challenges of understanding the function of the newly discovered genes have been addressed. High-throughput technologies such as DNA microarrays have been developed for the profiling of gene expression patterns in whole organisms or tissues. Protein arrays are emerging to follow DNA chips as possible screening tools. Here, we review the generation and application of microarray technology to obtain more information on the regulation of proteins, their biochemical functions and their potential interaction partners. Already, a large variety of assays based on antibody-antigen interactions exists. In addition, the medical relevance of protein arrays will be discussed.
The Bacillus subtilis genes scoA and scoB encode subunits of the heteromeric enzyme ScoAB, a putative succinyl-CoA:acetoacetate coenzyme A transferase. High-throughput, ligation-independent cloning (LIC) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. Transfer of the LIC region of vector pMCSG7 (L. Stols, M. Gu, L. Dieckman, R. Raffen, F.R. Collart, M.I. Donnelly. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. (2002) 25, 8-15) into commercial vectors with alternative, compatible origins of replication allowed introduction of standard LIC PCR products into the vectors by uniform protocols. Replacement of the His-tag encoding region of pMCSG7 with a sequence encoding the S-tag enabled selective purification of interacting proteins based on the His-tag associated with one member of the complex. When expressed separately and mixed, the ScoAB subunits failed to interact productively; no transferase activity was detected, and S-tagged ScoB failed to co-purify with His-tagged ScoA. Co-expression, in contrast, generated active transferase that catalyzed the predicted reaction. The ScoAB complex was purified by standard high-throughput metal-ion affinity chromatography procedures, crystallized robotically, and its structure was determined by molecular replacement.
We report the construction of a Pichia pastoris integrating vector which contains the inducible CUP1 promoter from Saccharomyces cerevisiae. We show that the promoter is indeed inducible by copper when used in P. pastoris and that the level of induction is dependent on the amount of copper in the medium.