共查询到20条相似文献,搜索用时 15 毫秒
1.
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
2.
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
3.
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
4.
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.5.
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.6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
A cell surface display system was developed in Pichia pastoris using the gene TIP1, encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae (ScTIP). Human lactoferrin cDNA (hLf) was fused to a full-length TIP1 DNA (ScTIP
630
) or a short-TIP1 fragment (ScTIP
120
) encoding the 40 C-terminal amino acids of ScTIP. Both hLf-ScTIP fusion genes were expressed in P. pastoris SMD 1168. The fused protein was detected by western blotting after extraction of the lysed recombinant cells with Triton
X-100, urea, and Triton X-100 plus urea, suggesting that the hLf is associated with the membrane. The localization of surface-displayed
hLf was confirmed by immunofluorescence confocal microscopy and flow cytometric analysis using FITC-labeled anti-hLf antibody,
suggesting that hLf was successfully located at the surface of P. pastoris. The intact recombinant cells and cell lysates showed antibacterial activity against target microorganisms, meaning that
the expressed hLf was biologically active. The results indicated that the ScTIP anchoring motif is useful for cell surface
display of foreign proteins in P. pastoris. 相似文献
13.
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14.
The sequence of an endo-chitosanase gene (CSN) from Aspergillus fumigatus was optimized based on the preferred codons of Pichia pastoris and synthesized in vitro through overlapping PCR (CSN-P). The gene was cloned into a yeast expression vector, pHBM905A, and secretorily expressed in Pichia pastoris GS115. The yield of CSN-P reached ~3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was
~25,000 U/ml. The enzyme had half-lives of 2.5 h at 80°C, 1 h at 90°C and 32 min at 100°C. It retained 70% activity after
incubation with 10 M urea at room temperature for 30 min. This enzyme was used for a large-scale preparation of oligosaccharides:
3 g enzyme converted 200 kg chitosan into oligosaccharides in 24 h at 60°C. 相似文献
15.
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
16.
A novel lipase gene, lipJ08, was cloned from Candida rugosa ATCC14830, along with the already reported five lipase genes (lip1–lip5). Nucleotide sequencing indicated that the lipJ08 gene contains a 1650 bp open reading frame (ORF) without introns. The deduced amino acid sequence corresponds to 534 amino
acid residues, including a putative signal sequence of 15 amino acid residues. Seventeen of the non-universal serine codons
(CTG) of lipJ08 were converted into universal serine codons (TCT) by PCR-based mutagenesis. The native and codon-optimized lipJ08 genes were expressed in Pichia
pastoris. The hydrolytic activity of the recombinant LIPJ08 was 4.7 U/ml, whereas the activity of the recombinant wild-type lipase
could not be detected. 相似文献
17.
Gao Z Li Z Zhang Y Huang H Li M Zhou L Tang Y Yao B Zhang W 《Biotechnology letters》2012,34(3):507-514
The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75%
maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve
high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml−1 (2.5 g protein l−1) in a 3 l fermentor—410% higher than GOD-w (148 U ml−1), and thus is a low-cost alternative for the bread baking industry. 相似文献
18.
Dan Wu Xiao Wei Yu Tong Chun Wang Rui Wang Yan Xu 《Biotechnology and Bioprocess Engineering》2011,16(2):305-311
Rhizopus lipases have been successfully expressed in Pichia pastors and different fermentation strategies have been investigated. However, there is no sufficient study on the effects of methanol
concentration on the production of Rhizopus lipases in P. pastors. In this study, the lipase from Rhizopus chinensis CCTCC M20102 was expressed under different fed-batch fermentation conditions at methanol concentrations ranging from 0.5
to 3.5 g/L. The lipase activity, stability, and productivities were analyzed. The optimum methanol concentration was 1 g/L,
with the highest lipase activity of 2,130 U/mL, without degradation. Additional information was obtained from the analysis
of methanol consumption and production rates. The results also suggested that the cell concentration at the end of the glycerol
fed-batch phase was very important for cell viability and protease activity. 相似文献
19.
To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter
was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in
flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity
for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry
cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic
acid, 15 mmol/L cysteine, and 15 mmol/L glycine). 相似文献
20.
The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and
pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation
strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation.
The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells,
controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate
high-level product. A stable lipase activity of approximately 14,000 IU ml−1 and a cell wet weight of ca. 500 g l−1 at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further
scale-up for industrial lipase production. 相似文献