共查询到20条相似文献,搜索用时 15 毫秒
1.
《Molecular membrane biology》2013,30(1):15-31
AbstractAquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism – including humans. These proteins are vital molecules and their malfunction can lead to several severe disorders and diseases. Hence, an increased understanding of their structure, function and regulation is of the utmost importance for developing current and future drugs. Heading towards this goal, the first problem to overcome is to acquire the proteins in sufficient amounts to enable functional and structural characterization. Using a suitable host organism, large amounts of target molecules can possibly be produced, but for membrane proteins limitations are frequently encountered. In the work described here, we have produced the 13 human aquaporins (hAQPs) in one of the most successful hosts for recombinant overproduction of eukaryotic proteins; the yeast Pichia pastoris, in order to explore the underlying bottleneck to a successful membrane protein production experiment. Here we present exceptional yield of hAQP1, whereas some other hAQPs were below the threshold needed for scaled up production. In the overproduction process, we have established methods for efficient production screening as well as for accurate determination of the initial production yield. Furthermore, we have optimized the yield of low producing targets, enabling studies of proteins previously out of reach, exemplified with hAQP4 as well as the homologue PfAQP. Taken together, our results. present insight into factors directing high production of eukaryotic membrane proteins together with suggestions on ways to optimize the recombinant production in the yeast P. pastoris. 相似文献
2.
Nyblom M Oberg F Lindkvist-Petersson K Hallgren K Findlay H Wikström J Karlsson A Hansson O Booth PJ Bill RM Neutze R Hedfalk K 《Protein expression and purification》2007,56(1):110-120
Eukaryotic--especially human--membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. 相似文献
3.
Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme that transfers sialic acids among glycoproteins and glycolipids both from the host cell surface and its own surface. This enzyme, called trans-sialidase, is different from higher eukaryotic sialyltransferases in that it does not accept cytidine 5′-monophospho-N-acetylneuraminic acid as a donor substrate. Also, the common glycosyltransferase structure is not present. To study this enzyme, an active member was cloned and expressed in higher eukaryotic cells. Expression of recombinant enzyme was achieved in the methylotrophic yeast Pichia pastoris. The N-terminal fusion of a secretion signal and the C-terminal addition of an epitope tag resulted not only in high expression levels, but also enabled easy detection and purification. Using P. pastoris, we obtained about 5 mg of enzymatically active trans-sialidase per liter of induced culture medium. 相似文献
4.
M. V. Padkina L. V. Parfenova A. E. Gradoboeva E. V. Sambuk 《Applied Biochemistry and Microbiology》2010,46(4):409-414
The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed.
There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed
that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested
that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features
of its metabolism. 相似文献
5.
Labarre C van Tilbeurgh H Blondeau K 《Extremophiles : life under extreme conditions》2007,11(2):403-413
We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative
on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence α-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3′-end of the targeted genes to allow immunodetection of the recombinant
proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest
and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation
for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch
cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted,
solubilized and partially purified. Large-scale purification will be necessary for further structural work. 相似文献
6.
Erin Quartley Andrei Alexandrov Maryann Mikucki Frederick S. Buckner Wim G. Hol George T. DeTitta Eric M. Phizicky Elizabeth J. Grayhack 《Journal of structural and functional genomics》2009,10(3):233-247
High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many
proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability,
and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that
are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins
that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated
with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield
highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation
that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification
that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor. 相似文献
7.
Angelika Lueking Caterina Holz Christine Gotthold Hans Lehrach Dolores Cahill 《Protein expression and purification》2000,20(3):372
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. 相似文献
8.
Marlene Lopes Carla Oliveira Lucília Domingues Manuel Mota Isabel Belo 《Biotechnology progress》2014,30(5):1040-1047
Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β‐galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1‐inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β‐galactosidase‐specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1040–1047, 2014 相似文献
9.
The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins.
The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which
is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous
expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts
at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields
of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera
Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives
in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in
P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin. 相似文献
10.
Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation 总被引:5,自引:0,他引:5
Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P.pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P.pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P.pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10–100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N- labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment. 相似文献
11.
Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Pornkamol Unrean 《Biotechnology progress》2014,30(1):28-37
This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol–methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:28–37, 2014 相似文献
12.
Myrsini Michou Angelos Stergios Georgios Skretas 《Biotechnology and bioengineering》2020,117(8):2434-2445
The bacterium Escherichia coli is among the most popular hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently generated the specialized MP-producing E. coli strain SuptoxD, which upon co-expression of the effector gene djlA, is capable of alleviating two major bottlenecks in bacterial recombinant MP production: it suppresses the toxicity that frequently accompanies the MP-overexpression process and it markedly increases the cellular accumulation of membrane incorporated and properly folded recombinant MP. Combined, these two positive effects result in dramatically enhanced volumetric yields for various recombinant MPs of both prokaryotic and eukaryotic origin. Based on the observation that djlA is found in the genomes of various pathogenic bacteria, the aim of the present work was to investigate (a) whether other naturally occurring DjlA variants can exert the MP toxicity-suppressing and production-promoting effects similarly to the E. coli DjlA and (b) if we can identify a DjlA variant whose efficiency surpasses that of the E. coli DjlA of SuptoxD. We report that a quite surprisingly broad variety of homologous DjlA proteins exert beneficial effects on recombinant MP when overexpressed in E. coli. Furthermore, we demonstrate that the Salmonella enterica DjlA is an even more potent enhancer of MP productivity compared with the E. coli DjlA of SuptoxD. Based on this, we constructed a second-generation SuptoxD strain, termed SuptoxD2.0, whose MP-production capabilities surpass significantly those of the original SuptoxD, and we anticipate that SuptoxD2.0 will become a broadly utilized expression host for recombinant MP production in bacteria. 相似文献
13.
M. C. Loewen X. Liu P. L. Davies A. J. Daugulis 《Applied microbiology and biotechnology》1997,48(4):480-486
Sea raven type II antifreeze protein (SRAFP) is one of three different 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-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. Several different 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 different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and
dissolved oxygen, (2) mixed feeding of methanol and glycerol with Muts clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut+ clones. The mixed-feeding/Muts strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l.
Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997 相似文献
14.
《朊病毒》2013,7(5):347-354
ABSTRACTThe yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve. 相似文献
15.
The human bradykinin B2 receptor (B2R) fused with green fluorescent protein (GFP) at the C-terminal has been expressed in the methylotrophic yeast of Pichia pastoris. In the expression vector, B2R gene was drove under the highly inducible promoter of alcohol oxidase 1 gene of P. pastoris. By fluorescence activated cell sorting (FACS) analysis and western blot analysis, it was proved that B2R recombinant receptor proteins were expressed at high level in the yeast. Further more, the transformants of P. pastoris were monitored with confocal microscopy, a strong green fluorescence was checked out. The recombinant B2R receptor proteins were mainly located on the plasma membrane proved by immunofluorescence microscopy. 相似文献
16.
Ezzine A M'hirsi El Adab S Bouhaouala-Zahar B Hmila I Baciou L Marzouki MN 《Biotechnology and applied biochemistry》2012,59(1):15-21
Most large-scale microbial production of recombinant proteins are based on Escherichia coli, yeasts, or filamentous fungi systems. Using eukaryotic hosts, antibody fragments are generally expressed by targeting to the secretory pathway. This enables not only efficient disulfide bond formation but also secretion of soluble and correctly folded product. For this goal, a recombinant vector was constructed to produce a single-domain antibody (NbAahI'22) directed against AahI' scorpion toxin using the methylotrophic yeast Pichia pastoris. The corresponding complementary DNA was cloned under control of the alcohol oxidase promoter in frame with the Saccharomyces α-factor secretion signal and then transferred to P. pastoris cell strain X-33. Using Western blot, we detected the expression of the recombinant NbAahI'22 exclusively in the culture medium. Targeting to the histidine label, the secreted nanobody was easily purified on nickel-nitrilotriacetic acid resin and then tested in enzyme-linked immunosorbent assay. Interestingly, the production level of the NbAahI'22 in its new glycosylated form reached more than sixfold that obtained in E. coli. These findings give more evidence for the utilization of P. pastoris as a heterologous expression system. 相似文献
17.
Stable isotope labeling for proteins of interest is an important technique in structural analyses of proteins by NMR spectroscopy. Escherichia coli is one of the most useful protein expression systems for stable isotope labeling because of its high-level protein expression and low costs for isotope-labeling. However, for the expression of proteins with numerous disulfide-bonds and/or post-translational modifications, E. coli systems are not necessarily appropriate. Instead, eukaryotic cells, such as yeast Pichia pastoris, have great potential for successful production of these proteins. The hemiascomycete yeast Kluyveromyces lactis is superior to the methylotrophic yeast P. pastoris in some respects: simple and rapid transformation, good reproducibility of protein expression induction and easy scale-up of culture. In the present study, we established a protein expression system using K. lactis, which enabled the preparation of labeled proteins using glucose and ammonium chloride as a stable isotope source. 相似文献
18.
Gil DF García-Fernández R Alonso-del-Rivero M Lamazares E Pérez M Varas L Díaz J Chávez MA González-González Y Mansur M 《FEMS yeast research》2011,11(7):575-586
Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris. 相似文献
19.
Bretthauer RK 《Trends in biotechnology》2003,21(11):459-462
The yeast Pichia pastoris is used extensively as the host cell for large-scale production of secreted recombinant proteins. Many proteins of pharmaceutical importance are N-glycosylated, and therefore require an expression host that yields N-linked oligosaccharides that are structurally and functionally identical to the human counterpart. The recent report by Choi et al. describes the use of combinatorial genetic libraries to alter the N-glycosylation pathway in P. pastoris to yield N-linked oligosaccharides with hybrid structures that are the same as the intermediates of mammalian-protein N-glycosylation. In view of recent progress in this area, the production of complex human glycans in yeasts is anticipated. 相似文献
20.
Protein secretion in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and advances in protein production 总被引:1,自引:0,他引:1
Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the
growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room
for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons
for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene
dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production. 相似文献