High-Level Expression and Purification of Coffee Bean α-Galactosidase Produced in the YeastPichia pastoris

Zhu, Alex; Monahan, Catherine; Zhang, Zhanfan; Hurst, Rosa; Leng, Lin; Goldstein, Jack

doi:10.1006/abbi.1995.9928

Cited by 57 publications

(18 citation statements)

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“…Either way, the trend of higher protease activity at higher pH i s indicates that the enhancement of MSP3 expression at pH i 6.8 was not due to protease inhibition in the culture supernatants at this pH. Secretion of proteases into culture media was reported to be responsible for low expression by product degradation in P. pastoris culture supernatants (Adrio et al, 2001;Hong et al, 2002;Inan et al, 1999;Jahic et al, 2003;Sinha et al, 2003;Zhu et al, 1995). Changing the medium pH away from 5, the optimum pH for growth, is a tactic often used to avoid protease activity in the fermentation medium (Clare et al, Supplements were added at 3 h prior to induction and 24 h post-induction to all cultures induced at pH i 6.8.…”

Section: Discussionmentioning

confidence: 87%

“…Shi et al (2003) found low levels of protease activity at pH 3.0, 5.0, 6.0, and 8.0; however, low pH values of 3.0 and 5.0 were the least conducive to protein expression in BMMY media. Zhu et al (1995) and Inan et al (1999) both reported that the highest yields of their recombinant proteins were obtained at a similar pH of 6.5 and 7. However, they attributed this enhancement to inhibition of proteases at these pHs, though no detection of protease was described.…”

Section: Discussionmentioning

confidence: 94%

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Improved yield of recombinant merozoite Surface protein 3 (MSP3) from Pichia pastoris using chemically defined media

Wang

Nguyen

Glen

et al. 2005

Biotech & Bioengineering

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Plasmodium falciparum merozoite surface protein 3 (MSP3) is a leading blood-stage malaria vaccine candidate. Vaccination with Pichia pastoris derived recombinant MSP3 protected Aotus nacymai monkeys from the parasite's lethal challenge and the post-challenge antibody titer against MSP3 correlated with protection. In our preliminary attempts to produce this vaccine in fermentors, little or no expression of MSP3 was observed in chemically defined media, although the same P. pastoris strain produced MSP3 in complex media. Our goal is to develop a Phase I/II clinical manufacturing process in completely chemically defined media because of the concern of potential prion contamination in complex media containing animal derived products. Here, we report our investigations into various factors to improve the yield of MSP3 in defined media. We found that an induction pH (pH(i)) 6.8 yielded MSP3 at 434 mg/L whereas there was no product at pH(i)< or = 5, though cell growth was the same in all pH(i) levels examined. High levels of NH(4) (+) consumed at pH(i) 6.8 were directly correlated to the enhanced MSP3 production. Furthermore, an additional 3.5-fold increase in the yield of MSP3 was obtained by addition of casamino acids at pH(i) 6.8. No direct correlation was observed between protease activity in the culture supernatants and lack of MSP3 expression. Neither high P. pastoris biomass generated at a high specific growth rate (0.04/h) nor low induction temperatures during induction improved yield. Nitrogen source was the most important factor affecting expression of MSP3 in defined media.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 94%

Improved yield of recombinant merozoite Surface protein 3 (MSP3) from Pichia pastoris using chemically defined media

Wang

Nguyen

Glen

et al. 2005

Biotech & Bioengineering

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“…Table 1. The following enzymes were used: none (0), ϳ0.2 g of BfGal110A (1), BfGal110B (2), BtGal110A (3), BtGal110B (4), SaGal110A (5), and SgGal110A (6), or 1.2 g of recombinant coffee bean ␣-galactosidase (C1) (44) or Elizabethkingia meningoseptica ␣-N-acetylgalactosaminidase (C2) (1). Arrows indicate the mobilities of substrates (S) and products (P).…”

Section: Resultsmentioning

confidence: 99%

Identification of a GH110 Subfamily of α1,3-Galactosidases

Liu

Yuan

Bennett

et al. 2008

Journal of Biological Chemistry

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In search of ␣-galactosidases with improved kinetic properties for removal of the immunodominant ␣1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of ␣-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454 -464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Gal␣1-3(Fuc␣1-2)Gal, whereas linear oligosaccharides terminated by ␣1,3-linked galactose such as the immunodominant xenotransplantation epitope Gal␣1-3Gal␤1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific ␣1,3-galactosidases that act equally well on both branched blood group B and linear ␣1,3Gal structures. We determined by one-dimensional 1 H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known ␣-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant ␣3Gal xenotransplantation epitope.

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“…Coffee bean α-galactosidase also has been successfully cloned in this system (Zhu et al 1996). Human tumor necrosis factor (TNF) was able to produce 10 g of TNF per liter of culture (Sreekrishna et al 1989).…”

Section: Introductionmentioning

confidence: 98%

Cloning, Expression, and Characterization of Rice α-Galactosidase

2008

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We have successfully cloned an α-galactosidase gene from a rice cDNA library and transformed it into Escherichia coli BL21. It was subsequently cloned to the pPIC9K vector and expressed in Pichia pastoris. A selected clone was found to result in high production yield of the galactosidase enzyme. The secreted enzyme was purified, and it revealed as a major protein band on an SDS-PAGE gel. The optimal pH value, enzyme stabilities, and substrate specificity were studied. The enzyme specificity toward the terminal α1→6, 1→4, and 1→3 linked galactosyl residue from various substrates was investigated. By determining the Michelis constant (Km) of the enzyme for melibiose, raffinose, and stachyose, our results showed that melibiose was hydrolyzed faster than raffinose, whereas the published data reported a reversed sequence, raffinose > melibiose. The enzyme also showed the ability of converting B red blood cells into O red cells. The objective of this work is to develop the Pichia system to produce a large quantity of enzyme for blood cell conversion for transfusion.

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High-Level Expression and Purification of Coffee Bean α-Galactosidase Produced in the YeastPichia pastoris

Cited by 57 publications

References 0 publications

Improved yield of recombinant merozoite Surface protein 3 (MSP3) from Pichia pastoris using chemically defined media

Improved yield of recombinant merozoite Surface protein 3 (MSP3) from Pichia pastoris using chemically defined media

Identification of a GH110 Subfamily of α1,3-Galactosidases

Cloning, Expression, and Characterization of Rice α-Galactosidase

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