POLY (1-TRIMETHYSILYL-1-PROPYNE) MEMBRANE REGENERATION PROCESS

A novel process for the regeneration of PTMSP membranes is disclosed. The regeneration process includes washing a fouled PTMSP membrane with a solution containing at least about 50% butanol in water for a time sufficient to increase the flux and/or selectivity of the membrane. This regeneration process with a butanol containing solution significantly improves both the flux and selectivity of the PTMSP membrane and can regenerate the flux of the PTMSP membrane to the level of a previously unused membrane

Production and Purification of the Heavy Chain Fragment C of Botulinum Neurotoxin, Serotype A, Expressed in the Methylotrophic Yeast \u3ci\u3ePichia pastoris\u3c/i\u3e

A recombinant Hc fragment of botulinum neurotoxin, serotype A (rBoNTA(Hc)), has been successfully expressed in a Mutt strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to achieve high cell density. Induction times were short to maximize rBoNTA(Hc) production while minimizing proteolytic degradation. Concentration of rBoNTA(Hc) in yeast cell lysates was generally 1-2% of the total protein based on ELISA analysis. The Hc fragment was purified from cell lysates using a multistep ion-exchange (IEC) chromatographic process, including SP, Q, and HS resins. The zwitterionic detergent Chaps was included in the buffer system to combat possible interactions, such as protein-protein or protein-DNA interactions. Following IEC was a hydrophobic interaction chromatography (HIC) polishing step, using phenyl resin. The Hc fragment was purified to \u3e95% purity with yields up to 450 mg/kg cells based on ELISA and Bradford protein assay. The purified Hc fragment of serotype A was stable, elicited an immune response in mice, and was protected upon challenge with native botulinum type A neurotoxin

Modeling Pichia pastoris Growth on Methanol and Optimizing the Production of a Recombinant Protein, the Heavy-Chain Fragment C of Botulinum Neurotoxin, Serotype A

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut+ expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(Hc)], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consump-tion rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (μ) determined from the model was 0.08 h−1 at a methanol concentration of 3.65 g/L, while the actual maximum μ was 0.0709 h−1. The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or metha-nol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (YX/MeOH) was lower and the maintenance coefficient (mMeOH) was higher than compared to limited methanol conditions. The YX/MeOH decreased and mMeOH increased with increasing methanol concen-tration under methanolinhibited conditions. BoNT/A(Hc) content in cells (a) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum a of 1.72 mg/g WCW was achieved at a μ of 0.0267 h−1 and induction time of 12 h

Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate

<p>Abstract</p> <p>Background</p> <p>Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.</p> <p>Results</p> <p>Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 <it>Pichia pastoris </it>strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man₅GlcNAc₂N-glycosylation of the secreted proteins is achieved at all specific growth rates tested.</p> <p>Conclusions</p> <p>Together, these data illustrate that the GlycoSwitch-Man5 <it>P. pastoris </it>is a robust production strain for homogenously N-glycosylated proteins.</p

Crystallization and preliminary X-ray analysis of \u3ci\u3eNa\u3c/i\u3e-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite \u3ci\u3eNecator americanus\u3c/i\u3e

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-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 A ° have been collected from a crystal that belongs to the monoclinic space group P21 with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit

Crystallization and preliminary X-ray analysis of \u3ci\u3eNa\u3c/i\u3e-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite \u3ci\u3eNecator americanus\u3c/i\u3e

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-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 A ° have been collected from a crystal that belongs to the monoclinic space group P21 with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit

Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus. Corrigendum

A correction to the paper by Asojo et al. [(2005), Acta Cryst. F61, 391–394]

Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H\u3csub\u3ec\u3c/sub\u3e): Antigen E) by \u3ci\u3ePichia pastoris\u3c/i\u3e

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 batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(Hc). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(Hc) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(Hc) per gram wet cell mass as determined by HPLC and Western blot analysis