Stability and a-complementation of M15 B-galactosidase from escherichia coli

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dc.contributor.advisorHuber, R. Eugene
dc.contributor.authorGallagher, Clare
dc.date.accessioned2005-07-29T23:08:49Z
dc.date.available2005-07-29T23:08:49Z
dc.date.issued1994
dc.descriptionBibliography: p. 198-205.en
dc.description.abstractM15 B-Galactosidase from Escherichia coli is an inactive variant of B­galactosidase with a deletion of residues 11-41. The protein can become active by the addition of a peptide derived from the N-terminal end of the wild type protein, by a process termed a-complementation. A fusion protein system was developed which allowed for the production and purification of a-peptide. This significantly decreased the time required to obtain pure peptide. The peptide produced in this way was also found to complement to a higher activity than the chemically cleaved (by cyanogen bromide) peptide from wild type B-galactosidase. Factors which affected the a-complementation of M15 B-galactosidase were investigated for their effects on activation (a-complementation). It was found that NaCl and B-mercaptoethanol increased the activation. Mg2+ increased the activity at low concentration but inhibited activation at high concentrations. The equilibrium between monomers and dimers was investigated. From experiments done at a series of protein concentrations, the Kd for monomer-dimer equilibrium was found to be 2.78 ± 0.76 x 10-7 M. The concentration of the protein was important in the equilibrium. Mg2+, NaCl and B-mercaptoethanol were shown to lower the Kd. The pH also affected the monomer-dimer equilibrium. The stability of M15 B-galactosidase was examined by thermal denaturation. The uncomplemented protein was found to have a lower temperature of activity loss than wild type protein. The complemented protein was more stable to heat than the uncomplemented M15 B-galactosidase if Mg2+ was present. The complemented enzyme lost activity non-cooperatively. The conformational stability of the M15 B-galactosidase protein was examined by urea denaturation. Unfolding intermediates of wild type B­galactosidase were stabilized by Mg2+ and NaCl. For the M15 B­galactosidase, NaCl stabilizes the intermediate structures but there was minimal stabilization by Mg2+. Complementation of the M15 B-galactosidase did not appear to increase the AG(H2O). The binding of a-peptide to the protein was found to be a 1:1 ratio of peptide per monomer ofM15 B-galactosidase with both dimers and tetramers. It was also shown that the peptide derived from the fusion protein formed distinct states of tetramers. The a-complementation process did not alter the binding of substrate to enzyme. The catalytic activity was, however, altered.
dc.format.extentxxi, 205 leaves : ill. ; 30 cm.en
dc.identifier.citationGallagher, C. (1994). Stability and a-complementation of M15 B-galactosidase from escherichia coli (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/17223en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/17223
dc.identifier.isbn0612031020en
dc.identifier.lccQP 603 G3 G35 1994en
dc.identifier.urihttp://hdl.handle.net/1880/30470
dc.language.isoeng
dc.publisher.institutionUniversity of Calgaryen
dc.publisher.placeCalgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subject.lccQP 603 G3 G35 1994en
dc.subject.lcshBeta-galactosidase
dc.subject.lcshEscherichia coli - Genetics
dc.subject.lcshComplementation
dc.titleStability and a-complementation of M15 B-galactosidase from escherichia coli
dc.typedoctoral thesis
thesis.degree.disciplineUniversity Biochemistry Group
thesis.degree.grantorUniversity of Calgary
thesis.degree.nameDoctor of Philosophy (PhD)
ucalgary.item.requestcopytrue
ucalgary.thesis.accessionTheses Collection 58.002:Box 973 520538283
ucalgary.thesis.notesoffsiteen
ucalgary.thesis.uarcreleaseyen
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