Heterologous expression, purification and characterization of heterodimeric monellin.
Monellin is an intensely sweet-tasting protein present in the berry of Dioscoreophyllum cumminsii. Naturally occurring monellin (double chain monellin) is a heterodimer of two subunits commonly referred to as chain A and chain B. Monellin is a good model system for structural and dynamic studies of proteins. Single chain monellin has been generated by covalently linking the two subunits of naturally occurring double chain monellin, and has been used extensively for folding and unfolding studies, as well as for protein aggregation studies. There are, however, relatively few reports on such studies with double chain monellin. The primary difficulty associated with studies using double chain monellin appears to be the lack of a standard purification method. Here, a simple method for the purification of double chain monellin is presented. The genes encoding the two chains of monellin were cloned into a modified pETDUET vector under separate T7 promoters. The expression vector containing the genes of the two chains was expressed in E. coli BL21 Star (DE3). The expressed protein was purified using two steps of chromatography, ion exchange chromatography and gel filtration chromatography. This expression system consistently produced 40 mg of pure double chain monellin per litre of E. coli culture, in the correctly folded native state. The purity of the protein was confirmed by mass spectrometry and SDS-PAGE analysis. The purified protein was characterized using different spectroscopic methods, and the spectra obtained were in good agreement with the published spectra of naturally occurring double chain monellin.
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.
Nilesh
N
Aghera
Jayant B
JB
Udgaonkar
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C002541:monellin protein, Dioscoreophyllum cumminsii