Cloning, expression, and purification of rat brain protein L-isoaspartyl methyltransferase.
Protein L-isoaspartyl methyltransferase (PIMT) methylates isoaspartyl residues in peptides and proteins using S-adenosyl-L-methionine as the methyl donor. A cloned source of this enzyme should be useful in the identification of cellular substrates and for quantitation and localization of isoaspartyl sites in purified proteins, including recombinant proteins used as pharmaceuticals. Rat brain PIMT cDNA was amplified using the polymerase chain reaction. The reaction product was directionally cloned into the expression vector p delta blue (M. E. Brandt and L. E. Vickery, Arch. Biochem. 294, 735-740, 1992). The vector contains the strong promoter lambda pL and allows for the direct expression of cloned genes. After transformation, Escherichia coli cells containing the plasmid constitutively produced recombinant rat brain PIMT (rrPIMT) at levels between 2 and 3% of total soluble protein. Recombinant enzyme was purified to homogeneity by ammonium sulfate precipitation of the crude extract followed by anion-exchange chromatography. The specific activity was 14,000 pmol methyl groups transferred/min/mg protein at 30 degrees C using bovine gamma-globulin as the methyl acceptor. A typical yield was 12 mg of purified rrPIMT per liter of bacterial culture. Subsequent dye ligand chromatography increased the specific activity of the preparation to 16,800 pmol methyl groups transferred/min/mg protein with an overall yield of 5 mg per liter of bacterial culture. Using isoaspartyl delta sleep-inducing peptide as the methyl acceptor, rrPIMT exhibited normal Michaelis-Menten kinetics that yielded the following constants: Km (S-adenosyl-L-methionine) = 1.1 microM, Km (peptide) = 16 microM, Vmax = 60,000 pmol/min/mg.
David
C L
CL
Department of Molecular Biology and Biochemistry, University of California at Irvine 92717-3900, USA.
Aswad
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003701:Delta Sleep-Inducing Peptide / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012436:S-Adenosylmethionine / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003701:Delta Sleep-Inducing Peptide; D012436:S-Adenosylmethionine; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase