title: High-throughput screening of cohesin mutant libraries on cellulose microarrays.
abstract: The specificity of cohesin-dockerin interactions is critically important for the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome). In order to investigate the origins of the observed specificity, a variety of selected amino acid positions at the cohesin-dockerin interface can be subjected to mutagenesis, and a library of mutants can be constructed. In this chapter, we describe a protein-protein microarray technique based on the high affinity of a carbohydrate-binding module (CBM), attached to mutant cohesins. Using cellulose-coated glass slides, libraries of mutants can be screened for binding to complementary partners. The advantages of this tool are that crude cell lysate can be used without additional purification, and the microarray can be used for screening both large libraries as initial scanning for "positive" plates, and for small libraries, wherein individual colonies are printed on the slide. Since the time-consuming step of purifying proteins can be circumvented, the approach is also appropriate for providing molecular insight into the multicomponent organization of complex cellulosomes.
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Slutzki
authors.forename: Vered
authors.initials: V
authors.lastname: Ruimy
authors.forename: Ely
authors.initials: E
authors.lastname: Morag
authors.forename: Yoav
authors.initials: Y
authors.lastname: Barak
authors.forename: Rachel
authors.initials: R
authors.lastname: Haimovitz
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D044603:Cellulosomes / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004867:Equipment Design; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D009154:Mutation; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D000097722:Cohesins
chemical_list: D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D002482:Cellulose; D002868:Chromosomal Proteins, Non-Histone; D000097722:Cohesins