Expression of human cystathionine beta-synthase in Escherichia coli: purification and characterization.
Cystathionine beta-synthase (CBS) purification from mammalian tissues is complicated by proteolysis and enzyme aggregation. To surmount these difficulties, we cloned human CBS cDNA in tandem with the beta-galactosidase sequence of the fusion vector, pAX5-, then expressed the fusion protein, beta-galactosidase/CBS, in transformed Escherichia coli cells. Proteolytic treatment of the ammonium sulfate fraction of bacterial lysates with endoproteinase Xa liberated CBS which could then be separated from its fusion partner by DEAE-cellulose chromatography. This nearly homogeneous enzyme preparation was purified 140-fold over the crude bacterial lysate with nearly 50% recovery, and its specific activity, 210 U/mg protein, was comparable to that purified from human liver. The purified enzyme contained pyridoxal 5'-phosphate and exhibited positive cooperativity toward S-adenosyl-L-methionine (Hill coefficient = 5.2; Kact = 34 microM). Km values of the cloned enzyme in the absence of AdoMet are 3.1 and 1.1 mM for serine and homocysteine, respectively. They are virtually identical to those from human hepatic CBS. A Soret absorbance band (lambda max = 428 nm) which shifted to 448 nm after reduction with sodium dithionite revealed the presence of heme in the enzyme. Expression of the fusion protein in E. coli with subsequent purification represents the first time this enzyme has been isolated in sufficient quantities for biophysical and biochemical investigation.
Bukovska
G
G
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262.
Kery
V
V
Kraus
J P
JP
D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D003541:Cystathionine beta-Synthase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D006710:Homocysteine / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012436:S-Adenosylmethionine / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D013053:Spectrophotometry; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D006710:Homocysteine; D012694:Serine; D012436:S-Adenosylmethionine; D001616:beta-Galactosidase; D015951:Factor Xa; D003541:Cystathionine beta-Synthase