A carbohydrate binding module as a diversity-carrying scaffold

Abstract

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 (1.6 x 10(6) 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 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

“…In another study, a combinatorial library was created by introducing restricted variations in defined positions in CBM4-2, originating from Rhodothermus marinus. This library was then used to select CBM variants that would interact with different carbohydrates and with human immunoglobulin G, thus demonstrating that a CBM is a suitable scaffold for creating binding modules for different substrates (37). A similar approach was taken by Lehtio et al (114) when screening for ␣-amylase inhibition in a combinatorial library of a CBM scaffold that was displayed on phage.…”

Molecular Evolution of Specific Human Antibody against MUC1 Mucin Results in Improved Recognition of the Antigen on Tumor Cells

“…After amplification with Pfu polymerase (Fermentas, Burlington, Ont., Canada), the remaining product was digested with Not I and Sfi I restriction enzymes (NEB), using conditions recommended by the supplier. The resulting fragments carrying the mutated scFv-encoding genes were subsequently ligated into a modified version of the pFAB5c.His vector [31] , using T4 DNA ligase (NEB). The product was used to transform electrocompetent Escherichia coli TOP10F' (Invitrogen, Carlsbad, Calif., USA), using electroporation with an ECM600 (BTX Instrument Division, Harvard Apparatus, South Natick, Mass., USA) according to the instructions from the supplier (50 F, 186 ⍀ and 1.5 kV).…”

Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain

“…Combining selection/directed evolution with scaffold-based protein libraries provides an excellent route to engineer new protein function. For example, protein libraries based on stable, well-expressed protein scaffolds have been implemented for in vitro selection techniques, such as phage display, ribosome display, and mRNA display (Koide et al 1998;Beste et al 1999;Xu et al 2002;Ciarapica et al 2003;Stumpp et al 2003;Wahlberg et al 2003;Binz et al 2004;Cicortas Gunnarsson et al 2004;Binz and Pluckthun 2005). The utility of such libraries depends on how tolerant the scaffolds are to randomization.…”