TY  - THES
AB  - UDP-D-xylose: proteoglycan-core-protein [beta]-D-xylosyltransferase (EC 2.4.2.26, XylT) initiates the biosynthesis of glycosaminoglycan chains in proteoglycans by transferring xylose from UDP-xylose to specific serine residues of a core protein. Xylosyltransferase II (XylT-II) is a protein homologue to XylT-I, whose enzymatic activity was recently identified.
In this study, the putative catalytic domain of XylT-II was heterologously expressed in the methylotrophic yeast Pichia pastoris. It was produced as a soluble active protein and biochemically characterized. The substrate specificity for various acceptors was investigated and a modified bikunin peptide was shown to be the optimal XylT-II acceptor (K_M = 1.9 µM). The core protein/sugar-linkage type, the influence of nucleotide derivatives, the temperature optimum, the stability, the monofunctionality and the ion-dependency were investigated and among others the necessity for magnesium and manganese ions for the enzymatic activity was confirmed. An inhibition of XylT-II by its biosynthesis pathway end products in particular heparin was detected and an influence of basic proteins like histones and protamines was figured out.
The in-gel detection through purification of xylosyltransferase II was achieved from 40 liters P. pastoris-supernatant by a combination of ammonium sulphate precipitation, heparin affinity chromatography and ion exchange chromatography. With a final yield of 3 per cent, the protein was purified over 7000-fold compared to the original protein sample. The purified product was identified as XylT-II by mass spectra.
Further the heparin-binding sequence of xylosyltransferase II should be identified. Prerequisite for that was the production of a sufficient amount of purified protein. Because XylT-I binds independently of its structure to heparin, E. coli was chosen to produce soluble fusion proteins consisting of the solubility enhancement protein maltose-binding-protein (MBP) and XylT-II fragments. The fusion proteins were purified and the binding to heparin was analysed. Although the binding of various XylT-II fragments was immunologically detected, sufficient purified protein could not be produced for further analysis due to the fact that the codon usage of the human XylT-II mRNA was not optimized for proper E. coli processing.
DA  - 2008
KW  - , Xylosyltransferase , Proteoglykan , Glykosyltransferase , Glykosaminoglykane ,
LA  - ger
PY  - 2008
TI  - Rekombinante Expression von humanen Xylosyltransferasen in der methylotrophen Hefe Pichia pastoris und in dem Bakterium Escherichia coli
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-14351
Y2  - 2024-11-25T01:03:08
ER  -