Affiliation:
1. Section of Molecular and Cellular Biology, University of California, Davis, California 95616
2. Division of Cellular, Molecular and Microbial Biology, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Abstract
ABSTRACT
In cellulosomes produced by
Clostridium
spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex. Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a “designer” cellulosome, or a recombinant cellulosome with a specific function. We report the preparation of
Clostridium cellulovorans
recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units. The full-length EngB containing the dockerin domain was expressed by
Bacillus subtilis
WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with
Escherichia coli
. The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
91 articles.
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