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Title: High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris
Authors: Fierens, Katleen ×
Geudens, Nele
Brijs, Kristof
Courtin, Christophe
Gebruers, Kurt
Robben, Johan
Van Campenhout, Steven
Volckaert, Guido
Delcour, Jan #
Issue Date: Aug-2004
Series Title: Protein expression and purification vol:37 issue:1 pages:39-46
Abstract: Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.
ISSN: 1046-5928
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Centre for Food and Microbial Technology
Thoracic Surgery
Division of Gene Technology (-)
Biochemistry, Molecular and Structural Biology Section
× corresponding author
# (joint) last author

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