Increasing the thermal stability of an oligomeric protein, beta-glucuronidase

Flores, Humberto; Ellington, Andrew D.

doi:10.1006/jmbi.2001.5223

Cited by 66 publications

(47 citation statements)

References 46 publications

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“…1BHG). The domain structure of the protein model is very similar to that predicted for E. coli (Flores and Ellington, 2002) and the mutations mapped to a loop near the putative catalytic residues (Fig. 5).…”

Section: Sequence Analysis Of the Modified Gusa Genesmentioning

confidence: 54%

“…In addition, background activity is rarely encountered in plants, animal or microbial systems. Molecular characterization of Escherichia coli and human GusA proteins has revealed critical residues that determine enzyme activity, specificity and stability (Islam et al, 1999;Matsumura et al, 1999;Matsumura and Ellington, 2001;Flores and Ellington, 2002). To our knowledge, there have been no functional studies of other β-glucuronidase enzymes.…”

Section: Introductionmentioning

confidence: 99%

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Modification of Lactobacillus β-glucuronidase activity by random mutagenesis

Callanan

Klaenhammer

2007

Gene

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Section: Sequence Analysis Of the Modified Gusa Genesmentioning

confidence: 54%

Section: Introductionmentioning

confidence: 99%

Modification of Lactobacillus β-glucuronidase activity by random mutagenesis

Callanan

Klaenhammer

2007

Gene

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“…The crystal structure of human GusA has been determined and suggests that the enzyme is likely to function as a 273-kDa homotetramer (3) with the catalytic site formed from a large cleft at the interface of two monomers (20). Presumably, the E. coli enzyme, which shares 50% amino acid identity with the human version, is also a homotetramer (10). To attempt the functional display of E. coli GusA at the surface of B. subtilis spores, the uidA gene was fused to the 3Ј-end of the oxdD gene with, as for the oxdD-phy fusion (see above), an interspacing 10 amino acid-linker (Fig.…”

Section: Vol 76 2010 Display Of Recombinant Proteins On B Subtilismentioning

confidence: 99%

Display of Recombinant Proteins on Bacillus subtilis Spores, Using a Coat-Associated Enzyme as the Carrier

Potot¹,

Serra

Henriques

et al. 2010

Appl Environ Microbiol

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The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric ␤-glucuronidase enzyme from Escherichia coli.Under extreme nutrient deprivation, Bacillus subtilis has the ability to enter a complex differentiation process that culminates with the formation of an extremely resistant spore.

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“…Flores et al indicated that the quaternary structure disruption is a characteristic of the loss of enzyme activity of GUS. 29 The freeze-drying procedure might interfere with chemical stability of monomer 75 KDa and further interfere with the interactions between the monomers. 30 Therefore, degradation and particularly quaternary structural changes were 2 of the possible reasons for the enzyme activity loss.…”

Section: Influences Of Lyophilization and Jet-milling On Liposome Encmentioning

confidence: 99%

Liposomal dry powders as aerosols for pulmonary delivery of proteins

Hickey

2005

AAPS PharmSciTech

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The purpose of this research was to develop liposomal dry powder aerosols for protein delivery. The delivery of stable protein formulations is essential for protein subunit vaccine delivery, which requires local delivery to macrophages in the lungs. β-Glucuronidase (GUS) was used as a model protein to evaluate dry powder liposomes as inhaled delivery vehicles. Dimyristoyl phosphatylcholine:cholesterol (7:3) was selected as the liposome composition. The lyophilization of liposomes, micronization of the powders, aerosolization using a dry powder inhaler (DPI), and in vitro aerodynamic fine particle fraction upon collection in a twinstage liquid impinger were evaluated. After lyophilization and jet-milling, the total amount of GUS and its activity, representing encapsulation efficiency and stability, were evaluated. The GUS amount and activity were measured and compared with freshly-prepared liposomes in the presence of mannitol, 43% of initial GUS amount, 29% of GUS activity after lyophilization and 36% of GUS amount, 22% of activity after micronization were obtained. Emitted doses from dry powder inhaler were 53%, 58%, 66%, and 73% for liposome powder:mannitol carrier ratios of 1:0, 1:4, 1:9, and 1:19. Fifteen percent of the liposome particles were less than 6.4 μm in aerodynamic diameter. The results demonstrate that milled liposome powders containing protein molecules can be aerosolized effectively at a fixed flow rate. Influences of different cryoprotectants on lyophilization of protein liposome formulations are reported. The feasibility of using liposomal dry powder aerosols for protein delivery has been demonstrated but further optimization is required in the context of specific therapeutic proteins.

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Increasing the thermal stability of an oligomeric protein, beta-glucuronidase

Cited by 66 publications

References 46 publications

Modification of Lactobacillus β-glucuronidase activity by random mutagenesis

Modification of Lactobacillus β-glucuronidase activity by random mutagenesis

Display of Recombinant Proteins on Bacillus subtilis Spores, Using a Coat-Associated Enzyme as the Carrier

Liposomal dry powders as aerosols for pulmonary delivery of proteins

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