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Full-Text Articles in Biochemical and Biomolecular Engineering
Optimal Conditions Of Fermentation Of The Pichia Pastoris Yeast Expressing The Recombinant Pres2-S Protein (M-Hbsag) Of Hepatitis B Virus (Hbv), Sasmakоv Sobirdjan, Ashirоv Оybek, Abdurakhmanоv Jaloliddin, Xasanоv Shuhrat
Optimal Conditions Of Fermentation Of The Pichia Pastoris Yeast Expressing The Recombinant Pres2-S Protein (M-Hbsag) Of Hepatitis B Virus (Hbv), Sasmakоv Sobirdjan, Ashirоv Оybek, Abdurakhmanоv Jaloliddin, Xasanоv Shuhrat
CHEMISTRY AND CHEMICAL ENGINEERING
The aim of this study is determination of optimal fermentation conditions of the Pichia pastoris yeast expressing the recombinant Pres2-S protein of hepatitis B virus (HBV). For this purpose we investigated the main factors influencing for the cultivation of recombinant strain of the yeast Pichia pastoris pPIC3,5-S-HBsAg - the composition of the nutrient medium, pH and dissolved oxygen (DO). As a result, by enzyme-linked immunosorbent assay (ELISA) the optimal concentration of methanol ≈1.0 %, which is the initiator and the only source of carbon for protein expression in the Mut+ phenotype Pichia pastoris yeast cells was determined. In the selected …
Study Of Recombinant Protein Fractions From The Bombyx Mori And Pichia Pastoris Using "Native Page" Method, Abdurhakhmanov Jaloliddin, Sasmakov Sobirdjan, Khasanov Shuhrat, Ashirov Оybek
Study Of Recombinant Protein Fractions From The Bombyx Mori And Pichia Pastoris Using "Native Page" Method, Abdurhakhmanov Jaloliddin, Sasmakov Sobirdjan, Khasanov Shuhrat, Ashirov Оybek
CHEMISTRY AND CHEMICAL ENGINEERING
We have studied the recombinant protein fractions of ̴ 24-34 kDa obtained from Bombyx mori - silkworm larvae and yeast Pichia pastoris. Fractions, containing the recombinant proteins by gel-electrophoresis using the “Native PAGE” method, which allows for differentiation of proteins according to their natural (surface) charge and conformational properties were studied. The results showed that the recombinant protein fraction from Bombyx mori (̴ 34 kDa) consisted of four components and fraction from Pichia pastoris (̴ 24 kDa) consisted one component. The most optimal conditions for the “Native PAGE”- the main gel: Tris HCl (pH-8.8) 244 mM, Bis-Acrylamide 10%, TEMED 0.08%, …
Study Of Recombinant Protein Fractions From The Bombyx Mori And Pichia Pastoris Using "Native Page" Method, Abdurhakhmanov Aloliddin, Sasmakov Sobirdjan, Khasanov Shuhrat, Ashirov Оybek
Study Of Recombinant Protein Fractions From The Bombyx Mori And Pichia Pastoris Using "Native Page" Method, Abdurhakhmanov Aloliddin, Sasmakov Sobirdjan, Khasanov Shuhrat, Ashirov Оybek
CHEMISTRY AND CHEMICAL ENGINEERING
We have studied the recombinant protein fractions of ̴ 24-34 kDa obtained from Bombyx mori - silkworm larvae and yeast Pichia pastoris. Fractions, containing the recombinant proteins by gel-electrophoresis using the “Native PAGE” method, which allows for differentiation of proteins according to their natural (surface) charge and conformational properties were studied. The results showed that the recombinant protein fraction from Bombyx mori (̴ 34 kDa) consisted of four components and fraction from Pichia pastoris (̴ 24 kDa) consisted one component. The most optimal conditions for the “Native PAGE”- the main gel: Tris HCl (pH-8.8) 244 mM, Bis-Acrylamide 10%, TEMED 0.08%, …
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 …
Causes Of Proteolytic Degradation Of Secreted Recombinant Proteins Produced In Methylotrophic Yeast Pichia Pastoris: Case Study With Recombinant Ovine Interferon-T, Jayanta Sinha, Bradley A. Plantz, Mehmet Inan, Michael Meagher
Causes Of Proteolytic Degradation Of Secreted Recombinant Proteins Produced In Methylotrophic Yeast Pichia Pastoris: Case Study With Recombinant Ovine Interferon-T, Jayanta Sinha, Bradley A. Plantz, Mehmet Inan, Michael Meagher
Papers in Biotechnology
It was observed that during fermentative production of recombinant ovine interferon-H (r-oIFN-H ) 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 in-creasing 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 …
Optimization Of Cell Density And Dilution Rate In Pichia Pastoris Continuous Fermentations For Production Of Recombinant Proteins, Wenhui Zhang, Chih-Ping Liu, Mehmet Inan, Michael M. Meagher
Optimization Of Cell Density And Dilution Rate In Pichia Pastoris Continuous Fermentations For Production Of Recombinant Proteins, Wenhui Zhang, Chih-Ping Liu, Mehmet Inan, Michael M. Meagher
Papers in Biomolecular Engineering
This paper provides an approach for optimizing the cell density (Xc) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon τ (INF-τ). The objective was to maximize the volumetric productivity (Q, mg INF-τ I-1 h-1), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of Xc and D within the ranges 150 ≤ Xc ≤ 450 g cells (wet weight) l-1 and 0.1 μm ≤ D …
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 …
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, …
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.
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 …