共查询到20条相似文献,搜索用时 0 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.
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
3.
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.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.
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
6.
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
7.
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
8.
Bent Larsen Petersen Jack Egelund Iben Damager Kirsten Faber Jacob Krüger Jensen Zhang Yang Eric Paul Bennett Henrik Vibe Scheller Peter Ulvskov 《Glycoconjugate journal》2009,26(9):1235-1246
Two Arabidopsis xylosyltransferases, designated RGXT1 and RGXT2, were recently expressed in Baculovirus transfected insect
cells and by use of the free sugar assay shown to catalyse transfer of D-xylose from UDP-α-D-xylose to L-fucose and derivatives
hereof. We have now examined expression of RGXT1 and RGXT2 in Pichia pastoris and compared the two expression systems. Pichia transformants, expressing soluble, secreted forms of RGXT1 and RGXT2 with an N- or C-terminal Flag-tag, accumulated recombinant,
hyper-glycosylated proteins at levels between 6 and 16 mg protein • L-1 in the media fractions. When incubated with 0.5 M L-fucose and UDP-D-xylose all four RGXT1 and RGXT2 variants catalyzed transfer
of D-xylose onto L-fucose with estimated turnover numbers between 0.15 and 0.3 sec-1, thus demonstrating that a free C-terminus is not required for activity. N- and O-glycanase treatment resulted in deglycosylation of all four proteins, and this caused a loss of xylosyltransferase activity
for the C-terminally but not the N-terminally Flag-tagged proteins. The RGXT1 and RGXT2 proteins displayed an absolute requirement
for Mn2+ and were active over a broad pH range. Simple dialysis of media fractions or purification on phenyl Sepharose columns increased
enzyme activities 2-8 fold enabling direct verification of the product formed in crude assay mixtures using electrospray ionization
mass spectrometry. Pichia expressed and dialysed RGXT variants yielded activities within the range 0.011 to 0.013 U (1 U = 1 nmol conversion of substrate
• min-1 • μl medium-1) similar to those of RGXT1 and RGXT2 expressed in Baculovirus transfected insect Sf9 cells. In summary, the data presented
suggest that Pichia is an attractive host candidate for expression of plant glycosyltransferases. 相似文献
9.
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
10.
Anjali Apte-Deshpnade Goutam Mandal Sudheerbabu Soorapaneni Bhaskarjyoti Prasad Jitendra Kumar Sriram Padmanabhan 《Biotechnology letters》2009,31(6):811-817
Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced
after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (~1 g/l) of extracellular glycosylated rSAK (~18 kDa) with
negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated
and highly active rSAK (~15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous
rSAK of >95% purity which suitable for future structural and functional studies. 相似文献
11.
Pandee P Summpunn P Wiyakrutta S Isarangkul D Meevootisom V 《Journal of microbiology (Seoul, Korea)》2011,49(2):257-264
A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced
amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible
disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C.
Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum
pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity
after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The
enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K
m and V
max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested,
including Fe2+, Fe3+, and Al3+. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained.
However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin
at a trypsin/phytase ratio of 0.01 (U/U). 相似文献
12.
Constitutive expression of human angiostatin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture 总被引:1,自引:0,他引:1
Zhang AL Zhang TY Luo JX Chen SC Guan WJ Fu CY Peng SQ Li HL 《Journal of industrial microbiology & biotechnology》2007,34(2):117-122
A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression
of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method
by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained
at 25–30% and pH was controlled at 5 by the addition of 7 M NH4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption
of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16
melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression
of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale
expression of angiostatin and other heterologous proteins.
Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal
Government of China (99-Z-004-001). 相似文献
13.
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
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.
Helena Marešová Zdena Marková Renáta Valešová Jan Sklenář Pavel Kyslík 《BMC biotechnology》2010,10(1):7
Background
Penicillin G acylase of Escherichia coli (PGAEc) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. 相似文献16.
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
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.
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
19.
D. G. Kozlov S. E. Cheperegin A. V. Chestkov V. N. Krylov Yu. D. Tsygankov 《Russian Journal of Genetics》2010,46(3):300-307
Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted
proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form. 相似文献
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