PEDS Advance Access originally published online on April 13, 2004
Protein Engineering Design and Selection 2004 17(3):213-221; doi:10.1093/protein/gzh026
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© 2004 Oxford University Press
A carbohydrate binding module as a diversity-carrying scaffold
1Department of Immunotechnology, Lund University, PO Box 7031, S-220 07 Lund, 2Department of Biotechnology, Lund University, PO Box 124, S-221 00 Lund and 3Alligator Bioscience, Scheelevägen 19 A, S-223 70 Lund, Sweden
4 To whom correspondence should be addressed. e-mail: mats.ohlin{at}immun.lth.se
The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6x106 clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 ± 5.3°C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
Received November 30, 2003; revised March 11, 2004; accepted March 30, 2004 Edited by Arne Skerra
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