Expression, purification, and characterization of human cytosolic serine hydroxymethyltransferase.
A cDNA which codes for human cytosolic serine hydroxymethyltransferase (Garrow et al., 1993, J. Biol. Chem. 268, 11910-11916) has been cloned into a pT7-7 vector as a NdeI-EcoRI insert. HMS174 (de3) cells were transformed with this plasmid and, after induction with isopropyl thiogalactoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed human enzyme by N-terminal amino acid sequencing. About 225 mg of pure enzyme can be obtained from a 20-liter culture. Spectral characteristics of the bound pyridoxal phosphate were essentially identical to the spectral properties of rabbit cytosolic serine hydroxymethyltransferase. Kinetic constants for the natural substrates L-serine and tetrahydrofolate were also similar to the values obtained previously for the rabbit cytosolic enzyme.
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614, USA.
H
H
Kruschwitz
S
S
Ren
M
M
Di Salvo
V
V
Schirch
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D017886:Durapatite; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D017886:Durapatite; D012696:Glycine Hydroxymethyltransferase