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1.
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
2.
Panjideh H Coelho V Dernedde J Fuchs H Keilholz U Thiel E Deckert PM 《Bioprocess and biosystems engineering》2008,31(6):559-568
Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins. 相似文献
3.
Pichia pastoris is an important eukaryotic organism for the expression, processing, and secretion of recombinant proteins. Here, the secretion
of enhanced green fluorescent protein (EGFP) in P. pastoris by using three novel secretion signals originating from the HFBI and HFBII class 2 hydrophobins of Trichoderma reesei was investigated. EGFP was fused to the carboxyl terminus of hydrophobin secretion signals and expressed under the control
of the constitutive GAP promoter. In every case, recombinant EGFP entered the secretory pathway of P. pastoris. SDS-polyacrylamide gel electrophoresis, Western blot analysis of the cells' supernatant, and fluorescence measurements on
single-cell level via flow cytometry confirmed the efficient secretion of EGFP mediated by the novel secretion sequences.
In conclusion, the data clearly show that the secretion sequences derived from HFBI and HFBII of T. reesei have the potential to achieve an efficient secretion of heterologous proteins in P. pastoris. Due to the small size of the hydrophobin-derived secretion signals, their coding sequence can be easily introduced to the
gene of interest by PCR. 相似文献
4.
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
5.
Huiming Duan Sirajo Umar Yiping Hu Jinchun Chen 《World journal of microbiology & biotechnology》2009,25(10):1779-1783
We constructed a Pichia pastoris expression vector with two strongly inducible promoters (an alcohol oxidase 1 promoter and a formaldehyde dehydrogenase 1
promoter) based on pPIC9 k. To test the function of these promoters, the vector was used to co-express two genes that encode
for green fluorescent protein (GFP) and a portion of a gelatin gene (an intra- and extracellular protein). The gelatin gene
was placed under the control of PAOX1, while the GFP was under the control of PFLD1. The two proteins were simultaneously expressed upon induction with 0.5% (v/v) methanol. The two promoters functioned effectively
and their coexistence on one vector did not affect their efficiency in protein expression. Thus, it was possible to simultaneously
induce the expression of at least two proteins from one vector, using two different promoters. 相似文献
6.
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
7.
Andrelisse Arruda Viviane Castelo Branco Reis Vinícius Daniel Ferreira Batista Bruno Sahim Daher Luiza Cesca Piva Janice Lisboa De Marco Lidia Maria Pepe de Moraes Fernando Araripe Gonçalves Torres 《Biotechnology letters》2016,38(3):509-517
Objectives
To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.Results
P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.Conclusions
A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.8.
Zhongbiao Tan Jianfang Li Minchen Wu Cunduo Tang Huimin Zhang Junqing Wang 《World journal of microbiology & biotechnology》2011,27(12):2767-2774
A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI,
was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His+, Mut+). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration
of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular
weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that
(10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at
a broad pH range of 7.0–10.5 and at a temperature of 30°C or below. 相似文献
9.
Carnicer M Canelas AB Ten Pierick A Zeng Z van Dam J Albiol J Ferrer P Heijnen JJ van Gulik W 《Metabolomics : Official journal of the Metabolomic Society》2012,8(2):284-298
Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies
of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and
washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without
causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation
by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these
protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are
whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was
used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat
cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60%
v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under
glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for
the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism. 相似文献
10.
Objectives
To characterize the genes responsible for ethanol utilization in Pichia pastoris.Results
ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.Conclusion
The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.11.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
12.
Bevan KS Chung Suresh Selvarasu Andrea Camattari Jimyoung Ryu Hyeokweon Lee Jungoh Ahn Hongweon Lee Dong-Yup Lee 《Microbial cell factories》2010,9(1):50
Background
Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism. 相似文献13.
A versatile vector was developed for heterologous proteins display on the cell surface of Pichia pastoris using the C-terminal half of alpha-agglutinin from Saccharomyces cerevisiae as a membrane anchor under the control of the alcohol oxidase 1 promoter (pAOX1). Multiple cloning sites and the sequence encoding the Xpress epitope (-Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys-) were introduced into the vector for insertion of heterologous genes and selective cleavage of target proteins. Enhanced green fluorescence protein (EGFP) was used as a model protein to check the function of this vector. The expression of EGFP on the P. pastoris surface was confirmed by confocal laser scanning microscopy. Fluorescence microscopy and western blot analysis confirmed that EGFP can be successfully cleaved from the cell surface by treating with enterokinase. 相似文献
14.
Jungoh Ahn Min-Jung Jang Kok Siong Ang Hongweon Lee Eui-Sung Choi Dong-Yup Lee 《Biotechnology letters》2016,38(12):2137-2143
Objectives
To evaluate different codon optimization parameters on the Saccharomyces cerevisiae-derived mating factor α prepro-leader sequence (MFLS) to improve Candida antarctica lipase B (CAL-B) secretory production in Pichia pastoris.Results
Codon optimization based on the individual codon usage (ICU) and codon context (CC) design parameters enhanced secretory production of CAL-B to 7 U/ml and 12 U/ml, respectively. Only 3 U/ml was obtained with the wild type sequence while the sequence optimized using both ICU and CC objectives showed intermediate performance of 10 U/ml. These results clearly show that CC is the most relevant parameter for the codon optimization of MFLS in P. pastoris, and there is no synergistic effect achieved by considering both ICU and CC together.Conclusion
The CC optimized MFLS increased secretory protein production of CAL-B in P. pastoris by fourfold.15.
J. F. Li Y. Z. Hong Y. Z. Xiao Y. H. Xu W. Fang 《World journal of microbiology & biotechnology》2007,23(5):741-745
The coding sequence of a laccase isozyme from Trametes sp. AH28-2 was cloned in pPIC9K vector and heterologously overexpressed in the yeast Pichia
pastoris strain GS115. In the minimal medium containing 0.3 mM CuSO4 and 0.6% alanine, the maximum yield of the recombinant laccase rLacB reached 32,000 U/l (1,012 U/mg), slightly higher than
that of the native enzyme nLacB (∼30,000 U/l, 1,356 U/mg). The enzymatic properties of rLacB were different from those of
nLacB as well. Regardless of the inferior thermal stability, rLacB had much better stability at both neutral and basic pH
range compared to nLacB. In addition, the dye decolorization potential of rLacB was similar to that of nLacB. 相似文献
16.
Sygmund C Gutmann A Krondorfer I Kujawa M Glieder A Pscheidt B Haltrich D Peterbauer C Kittl R 《Applied microbiology and biotechnology》2012,94(3):695-704
Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications
in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression
hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening
for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L
scale, which gave a volumetric yield of 223 mg L−1 PDH or 1,330 U L−1 d−1 in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated
recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially
identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression
system together with a simplified purification scheme for easy high-yield purification is shown. 相似文献
17.
Sauer M Branduardi P Gasser B Valli M Maurer M Porro D Mattanovich D 《Microbial cell factories》2004,3(1):17
Background
Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. 相似文献18.
Jinjia Wang Xiaolong Wang Lei Shi Yuanxing Zhang Xiangshan Zhou Menghao Cai 《Journal of industrial microbiology & biotechnology》2018,45(1):25-30
High oxygen consumption and heat release caused by methanol catabolism usually bring difficulties to industrial scale-up and cost for protein expression driven by methanol-induced AOX1 promoter in Pichia pastoris. Here, reduced methanol feeding levels were investigated for expression of insulin precursor in a trans-acting elements engineered P. pastoris strain MF1-IP. Insulin precursor expression level reached 6.69 g/(L supernatant) at the methanol feeding rate of 6.67 mL/(h·L broth), which was 59% higher than that in the wild-type strain WT-IP at the methanol feeding rate of 12 mL/(h·L broth). Correspondingly, the insulin precursor expression level in fermentation broth and maximum specific insulin precursor production rate was 137 and 77% higher than the WT-IP, respectively. However, oxygen consumption and heat evolution were reduced, and the highest oxygen consumption rate and heat evolution rate of the MF1-IP were 18.0 and 37.7% lower than the WT-IP, respectively. 相似文献
19.
20.
Pedersen MH Borodina I Moresco JL Svendsen WE Frisvad JC Søndergaard I 《Applied microbiology and biotechnology》2011,90(6):1923-1932
Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic
interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis
of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins
in Pichia pastoris and present fed-batch fermentation yields of 200–300 mg/l fermentation broth. Protein bands of expected sizes were detected
by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified
using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA
as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB
was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this
ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant
RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge
the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy
and fed-batch production using P. pastoris may be transferred to hydrophobins from other species. 相似文献