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1.
A Pichia pastoris system was used to express a single-chain variable fragment (scFv) antibody targeted against Metolcarb. The specific scFv gene was amplified from the phage-display scFv library and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C-scFv, was linearized and transformed into P. pastoris strain X-33. A transformant named X-33-Pp-SMW-12-6, which showed strong expression of antibodies, was isolated, and the culture conditions, including methanol induction concentrations, inoculum densities, and pH, were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of the scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against Metolcarb are comparable to those of scFv antibodies produced in E. coli. Recoveries of Metolcarb demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of Metolcarb residue in environmental and agricultural samples using a competitive inhibition enzyme-linked immunosorbent assay. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against Metolcarb for downstream applications. 相似文献
2.
The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene
was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino
acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained
the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass
of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml
was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after
the hydrolysis of inulin with the crude recombinant inulinase. 相似文献
3.
To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated
into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut+ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant
up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant
proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV
system, which was 3.3×105 IU/mL.
Foundation items: The National ‘973’ Basic Research Program (2002CB111302); The National Natural Science Foundation of China
(30370807) 相似文献
4.
High level expression of human endostatin in Pichia pastoris using a synthetic gene construct 总被引:1,自引:0,他引:1
Su Z Wu X Feng Y Ding C Xiao Y Cai L Feng W Li X 《Applied microbiology and biotechnology》2007,73(6):1355-1362
Endostatin, a 20-kDa C-terminal fragment derived from type XVIII collagen, is a potent angiogenesis inhibitor and an antitumor
factor. To improve the production of recombinant human endostatin on increasing demand in clinical practice, we constructed
an artificial gene encoding its mature peptide sequence in human collagen XVIII. The synthetic gene consisted of 20 codons
in preference in methylotropic yeast—Pichia pastoris and was cloned into expression vector pPICZαA; and the recombinant protein was expressed in P. pastoris strain SMD1168 and purified to near homogeneity using heparin affinity chromatography. The amount of expressed recombinant
protein in cultural media using described strategy was 80 mg/l in shake flask cultivation and 435 mg/l in high-density bioreactor
fermentation. Methylthiazolium assay demonstrated that human endostatin expressed in P. pastoris using artificial synthetic gene of preference in P. pastoris was able to inhibit the acidic fibroblast growth factor-induced proliferation of endothelial cells in vitro. 相似文献
5.
A new lipase gene designated as SlLipA was isolated from Serratia liquefaciens S33 DB-1 by the genomic-walking method. The cloned gene contained an open reading frame (ORF) of 1,845 bp encoding 615 amino
acids with a conserved GXSXG motif. Genome sequence analysis showed that an aldo/keto reductase gene closed to the SlLipA gene. The lipase gene was cloned into the expression vector pPICZαA and successfully integrated into the heterologous host,
methylotrophic yeast Pichia pastoris GS115. Five transformants could be expressed as secreted recombinant proteins with the high activity on Triglyceride–Agarose
plate and as candidates to produce the recombinant enzyme. A C-terminal His tag was used for its purification. The lipase
activity of different transformants against substrate para-nitrophenyl laurate (p-NPL) varied from 14 to 16 U ml−1. For the substrates para-nitrophenyl caprate (p-NPC), p-NPL, para-nitrophenyl myristate (p-NPM), para-nitrophenyl palmitate (p-NPP), and para-nitrophenyl stearate (p-NPS), the specific activity was shown to be preferred to long acyl chain length of p-NPS. 相似文献
6.
Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis> 总被引:5,自引:0,他引:5
The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast,
Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature
LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed
into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE
analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS
analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression
of other human defensins without resorting to fusion protein constructions. 相似文献
7.
Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in <Emphasis Type="Italic">Pichia pastoris</Emphasis> 总被引:3,自引:0,他引:3
Damasceno LM Anderson KA Ritter G Cregg JM Old LJ Batt CA 《Applied microbiology and biotechnology》2007,74(2):381-389
In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l−1 in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP)
and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression
of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI
had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase
and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression
of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased
single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show
that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former
is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER. 相似文献
8.
B-Cell activating factor (BAFF) is critical for B cell survival and maturation; excessive expression of it corrupts B-cell
tolerance and may lead to autoimmunity. The gene, scFv-Fc, coding for the antibody of BAFF was inserted into the eukaryotic
expression vector, pPICZαA, and transformed into Pichia pastoris. A high-level expression strain was obtained using a ‘yeastern blotting’ method. The scFv-Fc antibody was purified and 56 mg
was obtained from 1 l of culture supernatant. It retained high binding activity to both soluble BAFF and membrane-bound BAFF. 相似文献
9.
Cai H Chen L Wan L Zeng L Yang H Li S Li Y Cheng J Lu X 《Applied microbiology and biotechnology》2009,82(1):41-48
The administration of antibodies against the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a promising approach
in the upregulation of immune responses in many cancers and infectious diseases. The single-chain variable fragment of antibody
against CTLA4 is also useful in developing immunotoxins that might be used in the treatment of cancer, transplant rejection,
and autoimmune diseases. Here, we report the production of a soluble and functional scFv antibody against CTLA4 by using Pichia pastoris as the expression system. The gene encoding scFv hS83 with an additional 6His-tag at the 5’-end was inserted into the expression
vector pPIC9K. Then, the transformants were double-screened on plates containing 0.25 mg/mL and 1.5 mg/mL of neomycin G418
and many clones with different levels of G418-resistance were selected for further studies on expression. After induction
by the addition of methanol, various levels of hS83 were detected in the supernatant of P. pastoris containing pPIC9K-hS83. Clones with low G418-resistance produced more hS83 than those with higher G418-resistance. Under
the optimized conditions (initial inoculum, 40 A600nm AU/mL; pH 6.0; methanol concentration, 3.0%; induction time, 72 h), approximately 16–20 mg protein could be recovered from
1 L of the culture. The purified hS83 had a stronger binding ability towards CTLA4-positive Raji cells than CTLA4-negative
ECV304 cells. This finding indicates that the antibody produced by P. pastoris is functional and may be used in immunotherapy for cancer, infection, transplant rejection, and autoimmune diseases.
Huawei Cai and Lihong Chen contributed equally to this work. 相似文献
10.
Huifang Tan Guoqing Zhang Guangyu Zheng Fumian Cui Shijun Qian 《Frontiers of Biology in China》2008,3(3):287-292
Endo-β-glucanase II (EG II) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR. It was cloned into the expression vector pGAPZαA. The resultant recombinant plasmid was introduced into
Pichia pastoris GS115 by electroporation after being linearized by BspHI digestion. The recombinant Pichia pastoris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation
condition and the characteristics of the recombinant EG II were also explored.
__________
Translated from Microbiology, 2006, 33(6): 68273 [译自: 微生物学 通报] 相似文献
11.
β-Mannanase (EC 3.2.1.78) is a key enzyme to hydrolyze the β-mannosidic linkages in mannan and heteromannan. The expression of a wild type β-mannanase (manWT) of Aspergillus sulphureus in Pichia pastoris is not high enough for its application in feed supplement. To earn a high expression level, the manWT gene was firstly optimized
to manM according to the code bias of P. pastoris, which was then inserted into pPICzαA and transformed into P. pastoris strain X-33. In the induction by methanol, β-mannanase was expressed in high level with 32% increase in comparison with the manWT gene expressed in P. pastoris in shaken flask. In a 10-L fermenter, the manM was expressed in 9-fold higher level than that in shaken flask, which yielded
the enzyme activity of 1100 U/mL. This is the first study on codon bias effect on the β-mannanase gene expression level, which helps to achieve high β-mannanase yield and enzymatic activity in P. pastoris. 相似文献
12.
The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the -factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg–1 protein which was more than a recombinant P. pastoris GS115 (552 U mg–1 protein) or KM71H (539 U mg–1 protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg–1 protein by P. pastoris GS115, 1176 U mg–1 protein by P. pastoris KM71H and 1522 U mg–1 protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 °C) than the wild-type PLC from B. cereus. Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co2+ and Mn2+ etc., also influenced the activity of the recombinant PLCs. 相似文献
13.
Blanchard V Gadkari RA George AV Roy S Gerwig GJ Leeflang BR Dighe RR Boelens R Kamerling JP 《Glycoconjugate journal》2008,25(3):245-257
The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating
hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using
NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for
an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the
site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed
in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type
N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor
assays, and in vitro bioassays.
Véronique Blanchard and Rupali A. Gadkari contributed equally. 相似文献
14.
Min-A Kwon Hyun Suk Kim Taek Ho Yang Bong Keun Song Jae Kwang Song 《Protein expression and purification》2009,68(1):104-109
High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZαA with the Saccharomyces cerevisiae α-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additional sequences of the c-myc epitope and (His)6-tag of the vector were fused to the C-terminus of cutinase, while the other expression vector was constructed without any additional sequence. P. pastoris expressing the non-tagged cutinase exhibited about two- and threefold higher values of protein amount and cutinase activity in the culture supernatant, respectively. After simple purification by diafiltration process, both cutinases were much the same in the specific activity and the biochemical properties such as the substrate specificity and the effects of temperature and pH. In conclusion, the high-level secretion of F. solani cutinase in P. pastoris was demonstrated for the first time and would be a promising alternative to many expression systems previously used for the large-scale production of F. solani cutinase in Saccharomyces cerevisiae as well as Escherichia coli. 相似文献
15.
Expression of a rabbit liver carboxylesterase has been achieved in several different model systems including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda, and COS7 cells. Although, recombinant protein was observed in E. coli sonicates, little or no enzymatic activity was detected. Similarly, no activity was observed following expression in S. cerevisiae. In contrast, active protein was produced in P. pastoris from S. frugiperda, following baculoviral infection and in COS7 cells following transient transfection of plasmid DNA. For the preparation of
small amounts of protein for kinetic and biochemical studies, enzyme expressed in P. pastoris has proved sufficient. However, to produce large amounts of carboxylesterase for structural studies, baculoviral-mediated
expression of a secreted form of the protein in S. frugiperda was the most efficient. Using this system, we have generated and purified milligram quantities of essentially pure protein.
These results demonstrate that the choice of in vitro system for the generation of large amounts of active carboxylesterase,
and probably most endoplasmic reticulum processed proteins, is crucial for high level expression and subsequent purification. 相似文献
16.
The endo-β-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase
chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an α-mating
factor in a constitutive expression vector pGAPzαA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast–peptone–dextrose
liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed
with a yield of 120 units/mL under the flask culture at 28°C for 3 days. The enzyme showed optimal activity at 50°C and pH 2.4–3.4.
Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80°C
for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60°C for
30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na2HPO4-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na+, Mg2+, Ca2+, K+, Ba2+, Zn2+, Fe2+, and Mn2+) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed
industry as an additive. 相似文献
17.
To obtain the protein expression of a hybrid xylanase in yeast, the gene encoding it was modified according to the codon bias
of Pichia pastoris and expressed extracellularly in this yeast as an active xylanase, MBtx, exhibited a molecular mass of approximately 35 kDa
on SDS–PAGE. The pH behavior of MBtx in terms of both activity and stability was similar to that of Btx, original gene product
in Escherichia coli, while a certain difference was observed in optimal temperature for activity and in thermal stability. HPLC analysis revealed
the xylan in wheat could be hydrolyzed by MBtx and the major hydrolysis product was xylotriose. These results showed codon
usage played a key role in regulating the expression of the hybrid xylanase in P. pastoris and the recombinant hybrid xylanase, MBtx, produced by P. pastoris could be potentially useful in feed industry. 相似文献
18.
Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with
its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an
enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial
peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy
and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after
proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR. 相似文献
19.
Expression of <Emphasis Type="Italic">Escherichia coli</Emphasis> AppA2 phytase in four yeast systems 总被引:4,自引:0,他引:4
To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ A), and one constitutive system: P. pastoris (pGAPZA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml–1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml–1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower Kmfor sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.Revisions requested 18 November 2004; Revisions received 7 January 2005 相似文献
20.
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