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Establishment of an experimental system allowing immobilization of proteins on the surface of Bacillus subtilis cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
25703 43591 2006 10 PDF Available
Title
Establishment of an experimental system allowing immobilization of proteins on the surface of Bacillus subtilis cells
Abstract

Gram-positive bacteria code for one or more enzymes termed sortases which catalyze the covalent anchoring of substrate proteins on their cell wall. They recognize an amino acid sequence designated sorting motif, present close to the C-terminal end of the substrate proteins, cleave within this motif and catalyze anchoring of the polypeptide chain to the peptide crossbridge linking the peptidoglycan strands in a transpeptidation reaction. Bacillus subtilis has been reported to code for two different sortases but the sorting sequences recognized by them are yet unknown. To be able to immobilize proteins on the surface of B. subtilis cells, we introduced the srtA gene coding for sortase A of Listeria monocytogenes with the known sorting motif (LPXTG) into B. subtilis. L. monocytogenes and B. subtilis share the same peptide crossbridge. Next, we fused the coding region of an α-amylase gene to the C-terminal region of Staphylococcus aureus fibronectin binding protein B containing the sorting motif. Covalent linkage could be proven by treatment of the cells with lysozyme and by immunofluorescence microscopy. Up to 240,000 molecules of α-amylase could be immobilized per cell, 24 times more than previously reported for other bacterial species. To study the influence of the distance between the sorting motif and the C-terminus of α-amylase on the activity of the enzyme, the length of the spacer was varied. It turned out that the highest activity was measured with a spacer length of 123 amino acid residues.

Keywords
Sortase; Cellular chips; Listeria monocytogenes; Fibronectin binding protein B
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Establishment of an experimental system allowing immobilization of proteins on the surface of Bacillus subtilis cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 122, Issue 4, 20 April 2006, Pages 473–482
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us