Expression, purification and characterization of a functional carbohydrate-binding module from Streptomyces sp. SirexAA-E

Sungsoo Lim, Shishir Chundawat, Brian G. Fox

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Streptomyces sp. SirexAA-E (ActE) has been identified as a highly cellulolytic actinobacterium capable of deconstructing lignocellulosic biomass. SirexAA-E CAZymes most frequently contain a carbohydrate-binding module from family 2a (CBM2a). The DNA encoding the CBM2a from gene locus SACTE-0237, the most abundantly expressed cellulase from SirexAA-E, was cloned into an Escherichia coli expression vector and expressed as a C-terminal fusion protein to GFP. The GFP-CBM2a fusion protein was purified from insoluble inclusion bodies and refolded. The solubilized protein was separated by size-exclusion chromatography into high molecular weight GFP-CBM2a multimers and monomeric GFP-CBM2a. Only the monomeric CBM2a protein was found to have high relative affinity (partition coefficient of 0.62 ± 0.04 L/g) to cellulose. Binding of monomeric CBM2a prepared in this manner exhibits fully reversible, high affinity binding to cellulose.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalProtein Expression and Purification
Volume98
DOIs
StatePublished - Jun 1 2014
Externally publishedYes

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Streptomyces
Carbohydrates
Cellulose
Proteins
Cellulase
Actinobacteria
Inclusion Bodies
Biomass
Gel Chromatography
Molecular Weight
Escherichia coli
DNA
Genes

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

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abstract = "Streptomyces sp. SirexAA-E (ActE) has been identified as a highly cellulolytic actinobacterium capable of deconstructing lignocellulosic biomass. SirexAA-E CAZymes most frequently contain a carbohydrate-binding module from family 2a (CBM2a). The DNA encoding the CBM2a from gene locus SACTE-0237, the most abundantly expressed cellulase from SirexAA-E, was cloned into an Escherichia coli expression vector and expressed as a C-terminal fusion protein to GFP. The GFP-CBM2a fusion protein was purified from insoluble inclusion bodies and refolded. The solubilized protein was separated by size-exclusion chromatography into high molecular weight GFP-CBM2a multimers and monomeric GFP-CBM2a. Only the monomeric CBM2a protein was found to have high relative affinity (partition coefficient of 0.62 ± 0.04 L/g) to cellulose. Binding of monomeric CBM2a prepared in this manner exhibits fully reversible, high affinity binding to cellulose.",
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