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Biochemical and Biomolecular Engineering Commons™
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- Keyword
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- Pichia pastoris (8)
- Chlamydomonas reinhardtii (3)
- AOXl promoter (2)
- Carbon catabolite repression (2)
- 5-bisphosphate carboxylase/oxygenase (Rubisco) (1)
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- Alcohol oxidase regulation (1)
- Anaplerosis. (1)
- Ancylostoma caninum anticoagulant peptide (rAcAP-5) (1)
- C/N-assimilation (1)
- CO2-concentrating mechanism (1)
- CO2-concentrating mechanism (CCM) (1)
- Cell-bank characterization (1)
- Fed-batch fermentation (1)
- Fed-batch growth (1)
- Fermentation optimization (1)
- Filtration (1)
- Glycolate excretion (1)
- Green microalgae (1)
- Growth modeling (1)
- Homogenized (1)
- Hydrophobic charge induction chromatography (1)
- Interferon-H (1)
- Ion-exchange chromatography (1)
- Low-CO2 responsive gene (1)
- Methanol (1)
- Methylotrophic yeast (1)
- Na-ASP1 (1)
- Necator americanus secretory protein (1)
- PEP carboxylase (PEPC [Ppc]) (1)
- Pgp1-1 mutation (1)
Articles 1 - 15 of 15
Full-Text Articles in Biochemical and Biomolecular Engineering
Cell Bank Characterization And Fermentation Optimization For Production Of Recombinant Heavy Chain C-Terminal Fragment Of Botulinum Neurotoxin Serotype E (Rbonte(HC): Antigen E) By Pichia Pastoris, Jayanta Sinha, Mehmet Inan, Sarah Fanders, Shinichi Taoka, Mark Gouthro, Todd Swanson, Rick Barent, Ardis Barthuli, Bonnie M. Loveless, Leonard A. Smith, Theresa Smith, Ian Henderson, John Ross, Michael M. Meagher
Cell Bank Characterization And Fermentation Optimization For Production Of Recombinant Heavy Chain C-Terminal Fragment Of Botulinum Neurotoxin Serotype E (Rbonte(HC): Antigen E) By Pichia Pastoris, Jayanta Sinha, Mehmet Inan, Sarah Fanders, Shinichi Taoka, Mark Gouthro, Todd Swanson, Rick Barent, Ardis Barthuli, Bonnie M. Loveless, Leonard A. Smith, Theresa Smith, Ian Henderson, John Ross, Michael M. Meagher
Papers in Biochemical Engineering
A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(Hc)in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(Hc) gene inserted into pHILD4 Escherichia coli—P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(Hc) sequence. Expression of rBoNTE(Hc) from the Mut+ HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in …
Enhancement Of Protein Secretion In Pichia Pastoris By Overexpression Of Protein Disulfide Isomerase, Mehmet İnan, Dinesh Aryasomayajula, Jayanta Sinha, Michael M. Meagher
Enhancement Of Protein Secretion In Pichia Pastoris By Overexpression Of Protein Disulfide Isomerase, Mehmet İnan, Dinesh Aryasomayajula, Jayanta Sinha, Michael M. Meagher
Papers in Biochemical Engineering
A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na- ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the α-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the …
Purification And Scale-Up Of A Recombinant Heavy Chain Fragment C Of Botulinum Neurotoxin Serotype E In Pichia Pastoris Gs115, Michael P. Dux, Rick Barent, Jayanta Sinha, Mark Gouthro, Todd Swanson, Ardis Barthuli, Mehmet İnan, John T. Ross, Leonard A. Smith, Theresa J. Smith, Robert Webb, Bonnie Loveless, Ian Henderson, Michael M. Meagher
Purification And Scale-Up Of A Recombinant Heavy Chain Fragment C Of Botulinum Neurotoxin Serotype E In Pichia Pastoris Gs115, Michael P. Dux, Rick Barent, Jayanta Sinha, Mark Gouthro, Todd Swanson, Ardis Barthuli, Mehmet İnan, John T. Ross, Leonard A. Smith, Theresa J. Smith, Robert Webb, Bonnie Loveless, Ian Henderson, Michael M. Meagher
Papers in Biochemical Engineering
A recombinant C-terminus heavy chain fragment from Botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(Hc), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(Hc) protein was purified from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development …
The Phosphoglycolate Phosphatase Gene And The Mutation In The Phosphoglycolate Phosphatase-Deficient Mutant (Pgp1-1) Of Chlamydomonas Reinhardtii, Kensaku Suzuki, Hidenobu Uchida, Tarlan G. Mamedov
The Phosphoglycolate Phosphatase Gene And The Mutation In The Phosphoglycolate Phosphatase-Deficient Mutant (Pgp1-1) Of Chlamydomonas Reinhardtii, Kensaku Suzuki, Hidenobu Uchida, Tarlan G. Mamedov
Papers in Biochemical Engineering
The sequences of the phosphoglycolate phosphatase (PGPase) gene Pgp1 and the 5′-upstream region from Chlamydomonas reinhardtii wildtype 2137 and the pgp1-1 mutant N142 that lacks the activity of PGPase (PGP1) were determined. The comparison revealed the alteration of a G to A at position 98 relative to the start codon. This destroyed the “GT” splice donor site at the beginning of the first intron of this gene, resulting in an extension of the first exon to 49 translatable codons followed by a stop codon, containing the codons corresponding to whole transit peptide for the chloroplast stroma and the first four …
Identification And Expression Analysis Of Two Inorganic C-And N-Responsive Genes Encoding Novel And Distinct Molecular Forms Of Eukaryotic Phosphoenolpyruvate Carboxylase In The Green Microalga Chlamydomonas Reinhardtii, Tarlan G. Mamedov, Eric R. Moellering, Raymond Chollet
Identification And Expression Analysis Of Two Inorganic C-And N-Responsive Genes Encoding Novel And Distinct Molecular Forms Of Eukaryotic Phosphoenolpyruvate Carboxylase In The Green Microalga Chlamydomonas Reinhardtii, Tarlan G. Mamedov, Eric R. Moellering, Raymond Chollet
Papers in Biochemical Engineering
Phosphoenolpyruvate carboxylase (PEPC [Ppc]) has been previously purified and characterized in biochemical and immunological terms from two green microalgae, Chlamydomonas reinhardtii and Selenastrum minutum. The findings indicate that these algae possess at least two distinct PEPC enzyme-forms, homotetrameric Class1 and heteromeric Class-2 that differ significantly from each other and their plant and prokaryotic counterparts. Surprisingly, however, green-algal PEPC has been unexplored to date in molecular terms. This study reports the molecular cloning of the two Ppc genes in C. reinhardtii (CrPpc1, CrPpc2), each of which is transcribed in vivo and encodes a fully active, recombinant PEPC that lacks the regulatory, …
Crystallization And Preliminary X-Ray Analysis Of Na-Asp-1, A Multi-Domain Pathogenesis-Related-1 Protein From The Human Hookworm Parasite Necator Americanus, Mehmet İnan, Oluwatoyin A. Asojo, Alex Lucas, Rick Barent, Jicai Huang, Brad Plantz, Amber Swanson, Mark Gouthro, Michael M. Meagher, Peter J. Hotez
Crystallization And Preliminary X-Ray Analysis Of Na-Asp-1, A Multi-Domain Pathogenesis-Related-1 Protein From The Human Hookworm Parasite Necator Americanus, Mehmet İnan, Oluwatoyin A. Asojo, Alex Lucas, Rick Barent, Jicai Huang, Brad Plantz, Amber Swanson, Mark Gouthro, Michael M. Meagher, Peter J. Hotez
Papers in Biochemical Engineering
Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis related- 1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na …
Ranque–Hilsch Vortex Tube Thermocycler For Fast Dna Amplification And Real-Time Optical Detection, Ryan J. Ebmeier, Scott E. Whitney, Amitabha Sarkar, Michael Nelson, Nisha Padhye, George Gogos, Hendrik J. Viljoen
Ranque–Hilsch Vortex Tube Thermocycler For Fast Dna Amplification And Real-Time Optical Detection, Ryan J. Ebmeier, Scott E. Whitney, Amitabha Sarkar, Michael Nelson, Nisha Padhye, George Gogos, Hendrik J. Viljoen
Papers in Biochemical Engineering
An innovative polymerase chain reaction (PCR) thermocycler capable of performing real-time optical detection is described below. This device utilizes the Ranque–Hilsch vortex tube in a system to efficiently and rapidly cycle three 20 mL samples between the denaturation, annealing, and elon-gation temperatures. The reaction progress is displayed real-time by measuring the size of a fluo-rescent signal emitted by SYBR green/double-stranded DNA complexes. This device can produce significant reaction yields with very small amounts of initial DNA, for example, it can amplify 0.25 fg (,5 copies) of a 96 bp bacteriophage l-DNA fragment 2.731011-fold by performing 45 cycles in less than …
Causes Of Proteolytic Degradation Of Secreted Recombinant Proteins Produced In Methylotrophic Yeast Pichia Pastoris: Case Study With Recombinant Ovine Interferon-Τ, Mehmet İnan, Michael M. Meagher, Bradley A. Plantz, Jayanta Sinha
Causes Of Proteolytic Degradation Of Secreted Recombinant Proteins Produced In Methylotrophic Yeast Pichia Pastoris: Case Study With Recombinant Ovine Interferon-Τ, Mehmet İnan, Michael M. Meagher, Bradley A. Plantz, Jayanta Sinha
Papers in Biochemical Engineering
It was observed that during fermentative production of recombinant ovine interferon-τ (r-oIFN-τ) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were …
Pichia Pastoris Fermentation With Mixed-Feeds Of Glycerol And Methanol: Growth Kinetics And Production Improvement, Wenhui Zhang, Karen J. Hywood Potter, Bradley A. Plantz, Vicki L. Schlegel, Leonard A. Smith, Michael M. Meagher
Pichia Pastoris Fermentation With Mixed-Feeds Of Glycerol And Methanol: Growth Kinetics And Production Improvement, Wenhui Zhang, Karen J. Hywood Potter, Bradley A. Plantz, Vicki L. Schlegel, Leonard A. Smith, Michael M. Meagher
Papers in Biochemical Engineering
Fed-batch fermentation of a methanol utilization plus (Mut+) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies …
Non-Repressing Carbon Sources For Alcohol Oxidase (Aoxi) Promoter Of Pichia Pastoris, Mehmet İnan, Michael M. Meagher
Non-Repressing Carbon Sources For Alcohol Oxidase (Aoxi) Promoter Of Pichia Pastoris, Mehmet İnan, Michael M. Meagher
Papers in Biochemical Engineering
The growth of Pichia pastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C1, utilizing enzymes being repressed. Therefore, these carbon sources can not be used as a mixture with methanol to simultaneously grow P. pastoris and induce C1, utilizing enzymes, especially in shake flask cultures of AOX-deficient P. pastoris. Among the alternative carbon sources tested, alanine, sorbitol, mannitol and trehalose, did not repress β-gal production when methanol was used as an inducer in mut- strain of P. pastoris. Our results show that either one of alanine, sorbitol, …
Characteristics And Sequence Of Phosphoglycolate Phosphatase From A Eukaryotic Green Alga Chlamydomonas Reinhardtii, Tarlan G. Mamedov, Kensaku Suzuki, Kenji Miura, Ken-Ichi Kucho, Hideya Fukuzawa
Characteristics And Sequence Of Phosphoglycolate Phosphatase From A Eukaryotic Green Alga Chlamydomonas Reinhardtii, Tarlan G. Mamedov, Kensaku Suzuki, Kenji Miura, Ken-Ichi Kucho, Hideya Fukuzawa
Papers in Biochemical Engineering
Phosphoglycolate phosphatase (PGPase), a key en¬zyme of photorespiration in photosynthetic organisms, was purified from Chlamydomonas reinhardtii. The en¬zyme was an ~65-kDa homodimer with a pI value of 5.1 composed of ~32-kDa subunits not connected by any S–S bridges. It was also highly specific for phosphoglycolate with a Km value of 140 μM and an optimal pH between 8 and 9. The activity was strongly inhibited by CaCl2, and it recovered competitively following the addition of MgCl2 or EGTA. A mobility shift was observed in SDS-polyacrylamide gel electrophoresis by the addition of CaCl2, indicating that the enzyme binds to Ca2+ . …
The Effect Of Ethanol And Acetate On Protein Expression In Pichia Pastoris, Mehmet İnan, Michael M. Meagher
The Effect Of Ethanol And Acetate On Protein Expression In Pichia Pastoris, Mehmet İnan, Michael M. Meagher
Papers in Biochemical Engineering
Pichia pastoris is an excellent host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. P. pastoris produces a small amount of ethanol as a by-product during the glycerol fed-batch phase and the mixed-feed induction phase (glycerol-methanol) of high cell density fermentations, regardless of the phenotype (Mutf, Mut" or Mut-). We have investigated ethanol repression of the AOXl promoter using strains, GS115 (Mutf) and MC100-3 (Mut-), expressing an AOXI-lac2 fusion. The addition of 10 mg t1 ethanol at the start of methanol induction delayed P-galactosidase production and methanol utilization …
Fermentation Strategies For Recombinant Protein Expression In The Methylotrophic Yeast Pichia Pastoris, Wenhui Zhang, Mehmet Inan, Michael M. Meagher
Fermentation Strategies For Recombinant Protein Expression In The Methylotrophic Yeast Pichia Pastoris, Wenhui Zhang, Mehmet Inan, Michael M. Meagher
Papers in Biochemical Engineering
Fermentation strategies for recombinant protein production in Pichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, application of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.
A Mutant Of Chlamydomonas Reinhardtii With Reduced Rate Of Photorespiration, Kensaku Suzuki, Tarlan G. Mamedov, Toraoyoshi Ikawa
A Mutant Of Chlamydomonas Reinhardtii With Reduced Rate Of Photorespiration, Kensaku Suzuki, Tarlan G. Mamedov, Toraoyoshi Ikawa
Papers in Biochemical Engineering
Photorespiration rates under air-equilibrated conditions (0.04% CO2 and 21% O2) were measured in Chlamydomonas reinhardtii wild-type 2137, a phosphoglycolate-phosphatase-deficient (pgpl) mutant and a suppressor double mutant (7FR2N) derived from the pgpl mutant. In both cells grown under 5% CO2 and adapted air for 24 h in the suppressor double mutant, the maximal rate of photorespiration (phosphoglycolate synthesis) was only about half of that in either the wild type or the pgpl mutant (18-7F) cells. In the progeny, the reduced rate of photorespiration was accompanied by increased photosynthetic affinity for inorganic carbon and the capacity for growth …
Optimization Of Temperature-Glycerol-Ph Conditions For Fed-Batch Fermentation Process For Recombinant Hookworm (Ancylostoma Caninum) Anticoagulant Peptide (Acap-5) Production By Pichia Pastoris, Mehmet İnan, Vijay Chiruvolu, Kent M. Eskridge, George P. Vlasuk, Kenneth Dickerson, Scott Brown, Michael M. Meagher
Optimization Of Temperature-Glycerol-Ph Conditions For Fed-Batch Fermentation Process For Recombinant Hookworm (Ancylostoma Caninum) Anticoagulant Peptide (Acap-5) Production By Pichia Pastoris, Mehmet İnan, Vijay Chiruvolu, Kent M. Eskridge, George P. Vlasuk, Kenneth Dickerson, Scott Brown, Michael M. Meagher
Papers in Biochemical Engineering
This study was undertaken to determine the optimum pH, temperature and glycerol feed rate for the production of recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (rAcAP-5) by Pichia pastoris using response surface methodology (RSM). A central composite design was used as an experimental design for allocation of treatment combinations in three blocks. The variables selected for study were pH, temperature and glycerol feed rate. pH was the most important variable affecting yield, specific yield and specific activity of rAcAP-5. Glycerol feed rate had a significant effect on the specific activity of rAcAP-5 (% of total secreted protein) while temperature …