Expression, single-step purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific beta-glucosidase.
Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein. Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)(6)Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies. Native (His)(6)Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies. In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed. In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated. The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies. Gain of authentic biological activity and quaternary structure after the refolding process was confirmed by K(m) determination and electrophoretic mobility under native conditions. The yield of properly refolded protein was assessed based on the specific activity of the refolded product.
Zouhar
J
J
Laboratory of Plant Molecular Physiology, Faculty of Science, Masaryk University, Kotlárská 2, Brno, CZ-61137, Czech Republic.
Nanak
E
E
Brzobohatý
B
B
D002846:Chromatography, Affinity; D003583:Cytokinins; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005960:Glucosides; D002479:Inclusion Bodies / Q000201:enzymology; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics*; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D003583:Cytokinins; D005960:Glucosides; D011993:Recombinant Fusion Proteins; C071257:cytokinin-beta-glucosidase; D001617:beta-Glucosidase