High-level expression and purification of human xylosyltransferase I in High Five insect cells as biochemically active form.
Human xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to consensus serine residues of proteoglycan core proteins. Expression of a soluble form of recombinant histidine-tagged XT-I (rXT-I-HIS) was accomplished at a high level with High Five/pCG255-1 insect cells in suspension culture. The recombinant protein was purified to homogeneity by a combination of heparin affinity chromatography and metal (Ni(2+)) chelate affinity chromatography. Using the modern technique of perfusion chromatography, a rapid procedure for purification of the rXT-I-HIS from insect cell culture supernatant was developed. The purified, biologically active enzyme was homogeneous on SDS-PAGE, was detected with anti-XT-I-antibodies, and had the expected tryptic fragment mass spectrum. N-terminal amino acid sequencing demonstrated that the N-terminal signal sequence of the expressed protein was quantitatively cleaved. The total yield of the enzyme after purification was 18% and resulted in a specific XT-I activity of 7.9mU/mg. The K(m) of the enzyme for recombinant [Val(36),Val(38)](delta1),[Gly(92),Ile(94)](delta2)bikunin was 0.8microM. About 5mg purified enzyme could be obtained from 1L cell culture supernatant. The availability of substantial quantities of active, homogeneous enzyme will be of help in future biochemical and biophysical characterization of XT-I and for the development of a immunological XT-I assay.
Kuhn
Joachim
J
Institut für Laboratoriums und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany. jkuhn@hdz-nrw.de
Müller
Sandra
S
Schnölzer
Martina
M
Kempf
Tore
T
Schön
Sylvia
S
Brinkmann
Thomas
T
Schöttler
Manuela
M
Götting
Christian
C
Kleesiek
Knut
K
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D008970:Molecular Weight; D009036:Moths / Q000378:metabolism*; D010430:Pentosyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015202:Protein Engineering / Q000379:methods*; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D000097608:UDP Xylose-Protein Xylosyltransferase
D010430:Pentosyltransferases; D011994:Recombinant Proteins; D000097608:UDP Xylose-Protein Xylosyltransferase