کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2021460 1069247 2007 11 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression and characterization of α-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression and characterization of α-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv
چکیده انگلیسی

Glycogen branching enzyme (GlgB, EC 2.4.1.18) catalyzes the third step of glycogen biosynthesis by the cleavage of an α-(1,4)-glucosidic linkage and subsequent transfer of cleaved oligosaccharide to form a new α-(1,6)-branch. A single glgB gene Rv1326c is present in Mycobacterium tuberculosis. The predicted amino acid sequence of GlgB of M. tuberculosis has all the conserved regions of α-amylase family proteins. The overall amino acid identity to other GlgBs ranges from 48.5 to 99%. The glgB gene of M. tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity using metal affinity and ion exchange chromatography. The recombinant protein is a monomer as evidenced by gel filtration chromatography, is active as an enzyme, and uses amylose as the substrate. Enzyme activity was optimal at pH 7.0, 30 °C and divalent cations such as Zn2+ and Cu2+ inhibited activity. CD spectroscopy, proteolytic cleavage and mass spectroscopy analyses revealed that cysteine residues of GlgB form structural disulfide bond(s), which allow the protein to exist in two different redox-dependent conformational states. These conformations have different surface hydrophobicities as evidenced by ANS-fluorescence of oxidized and reduced GlgB. Although the conformational change did not affect the branching enzyme activity, the change in surface hydrophobicity could influence the interaction or dissociation of different cellular proteins with GlgB in response to different physiological states.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 51, Issue 2, February 2007, Pages 198–208
نویسندگان
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