Expression and purification of glycine N-methyltransferases in Escherichia coli.
Expression and purification of recombinant mouse, rat, and human glycine N-methyltransferases (GNMTs) in pTYB and pET expression vectors was done in order to prepare the proteins for structure studies of the enzymes from different sources. When human and mouse GNMTs were expressed in pTYB vector as a fusion protein with intein and the chitin binding domain, an unusual cleavage of intein was found. This cleavage takes place at two sites near the N-terminus of intein. This resulted in the appearance of an abnormal GNMT protein after on-column cleavage of the fusion protein, which could not be separated from normal GNMT. For this reason expression of mouse, rat, and human GNMTs was done in the pET-17b expression vector, resulting in the expression of soluble protein at about 20-40mg/L of culture. A new procedure for GNMT isolation after expression in the pET vector was developed. This included only two steps, ammonium sulfate precipitation and ion-exchange chromatography, and resulted in preparations containing 95-97% pure protein. All expressed proteins were tetrameric with molecular weights of 130kDa as determined by size-exclusion chromatography. Activity in Tris buffer at pH 9 of mouse, rat, and human GNMTs was found to be 255, 260, and 540U/mg, respectively. This implies that expressed and purified GNMT proteins are biologically active and suitable for biochemical and structural studies.
Luka
Zigmund
Z
Department of Biochemistry, Vanderbilt University School of Medicine, 620 Light Hall, Vanderbilt University, Nashville, TN 37232, USA.
Wagner
Conrad
C
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D050938:Glycine N-Methyltransferase; D006801:Humans; D008780:Methyltransferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; C494510:GNMT protein, human; D050938:Glycine N-Methyltransferase; C494511:Gnmt protein, mouse; C494512:Gnmt protein, rat