A carbohydrate binding module as a diversity-carrying scaffold.
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.
Cicortas Gunnarsson
L
L
Department of Immunotechnology, Lund University, PO Box 7031, S-220 07 Lund, Sweden.
Nordberg Karlsson
E
E
Albrekt
A-S
AS
Andersson
M
M
Holst
O
O
Ohlin
M
M
D000595:Amino Acid Sequence; D001435:Bacteriophages; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D050260:Carbohydrate Metabolism; D002942:Circular Dichroism; D016384:Consensus Sequence; D017124:Conserved Sequence; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000821:virology; D014644:Genetic Variation; D005822:Genetic Vectors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D045245:Rhodothermus / Q000201:enzymology; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014995:Xylosidases