PEDS Advance Access published online on April 13, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh026
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1 Department of Immunotechnology, Lund University, P.O. Box 7031, S-220 07 Lund, Sweden
* To whom correspondence should be addressed. E-mail: mats.ohlin{at}immun.lth.se.
The growing field of biotechnology is in a 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, (1.6 x 106 clones), variants specific towards different carbohydrate polymers (birchwood xylan, AvicelTM 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 659 mg purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.76±56.3°C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
Keywords:
binding specificity / carbohydrate-binding module / phage selection / molecular library / scaffold
Revised March 11, 2004
Accepted March 30, 2004
Oxford University Press 1741-0134
Article
A carbohydrate binding module as a diversity-carrying scaffold
2 Department of Biotechnology, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
3 Alligator Bioscience, Scheelevägen 19 A, S-223 70 Lund, Sweden
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