Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.
A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked by 12 bp at the 5' end and 287 bp at the 3' end. The full-length cDNA was then cloned into a pET22b vector as a NdeI-EcoRI insert. HMS174(DE3) cells were transformed with this plasmid and, after induction with isopropyl beta-D-thiogalactopyranoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed rabbit enzyme lacking the first methionine residue. Spectral characteristics of the bound pyridoxal phosphate and kinetic constants for the natural substrates L-serine and tetrahydrofolate were essentially identical to the values obtained previously for the rabbit cytosolic enzyme. The pattern of bands shown by the pure recombinant enzyme on an isoelectric focusing gel containing 6 M urea showed a major band and a minor band representing about 15-20% of the protein. Upon incubation of the recombinant enzyme at pH 7.3 and 37 degreesC, three new bands were observed on isoelectric focusing with the concomitant formation of isoaspartyl residues, as determined by reactivity with protein isoaspartyl methyltransferase. These results are consistent with deamidation of Asn residues to isoaspartyl during the in vitro incubation. The enzyme purified from rabbit liver has previously been shown to contain isoaspartyl residues.
di Salvo
M L
ML
Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università La Sapienza, Rome, Italy.
Delle Fratte
S
S
De Biase
D
D
Bossa
F
F
Schirch
V
V
D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing; D007536:Isomerism; D007700:Kinetics; D011732:Pyridoxal Phosphate / Q000032:analysis; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013053:Spectrophotometry; D013763:Tetrahydrofolates / Q000378:metabolism
D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D001224:Aspartic Acid; C030371:5,6,7,8-tetrahydrofolic acid; D012694:Serine; D011732:Pyridoxal Phosphate; D001216:Asparagine; D012696:Glycine Hydroxymethyltransferase