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STRUCTURAL CHANGING OF ACETOHYDROXY ACID ISOMEROREDUCTASE FROM ESCHERICHIA COLI AS THE EFFECT OF SLOW TIGHT BINDING INHIBITOR IDENTIFIED BY SITE DIRECTED MUTAGENESIS

Jamonnak, Nuttara
http://rave.ohiolink.edu/etdc/view?acc_num=akron1144861157

Abstract Details

2006, Master of Science, University of Akron, Chemistry.
Acetohydroxy acid isomeroreductase (AHIR) is the second enzyme common to the biosynthetic pathway for the branched chain amino acids. Acetohydroxy acid isomeroreductase involved in isomerization and reduction of á-acetolactate which is converted to á, â-dihydroxyisovalerate, or it converts á-aceto-á-hydroxybutyrate to á, â-dihydroxy â-methylvalerate. The ilvC gene encoding AHIR was subcloned from plasmid DNA pRWB2 . The gene and its associated promoter/operator were contained in a Bgl II-Hind III fragment and this fragment was conveniently ligated into pSP72 vector. In order to subclone the coding region of the ilvC gene into an expression vector like pET to get high level expression. The internal NcoI site of this gene was eliminated using a PCR based site directed mutagenesis procedure. The possibility to construct a plasmid that contains a variant of the ilvC gene suitable for subcloning into expression vectors that contain an Nco I site at an appropriate distance from the ribosome binding site has been examined. The ability to express acetohydroxy acid isomeroreductase in this format will allow a straightforward method for the PCR site directed mutagenesis followed by the production of large quantities of the enzyme to facilitate the search for sites in the enzyme that affect the rate of tight binding inhibition.
Kim Calvo (Advisor)
115 p.
Chemistry, Biochemistry
ACETOHYDROXY ACID ISOMEROREDUCTASE

Recommended Citations

Citations

  • Jamonnak, N. (2006). STRUCTURAL CHANGING OF ACETOHYDROXY ACID ISOMEROREDUCTASE FROM ESCHERICHIA COLI AS THE EFFECT OF SLOW TIGHT BINDING INHIBITOR IDENTIFIED BY SITE DIRECTED MUTAGENESIS [Master's thesis, University of Akron]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=akron1144861157

    APA Style (7th edition)

  • Jamonnak, Nuttara. STRUCTURAL CHANGING OF ACETOHYDROXY ACID ISOMEROREDUCTASE FROM ESCHERICHIA COLI AS THE EFFECT OF SLOW TIGHT BINDING INHIBITOR IDENTIFIED BY SITE DIRECTED MUTAGENESIS. 2006. University of Akron, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=akron1144861157.

    MLA Style (8th edition)

  • Jamonnak, Nuttara. "STRUCTURAL CHANGING OF ACETOHYDROXY ACID ISOMEROREDUCTASE FROM ESCHERICHIA COLI AS THE EFFECT OF SLOW TIGHT BINDING INHIBITOR IDENTIFIED BY SITE DIRECTED MUTAGENESIS." Master's thesis, University of Akron, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=akron1144861157

    Chicago Manual of Style (17th edition)

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© 2006, all rights reserved.
This open access ETD is published by University of Akron and OhioLINK.