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Purification of beta-lactamases by affinity chromatography on phenylboronic acid-agarose.

ABSTRACT: Several beta-lactamases, enzymes that play an important part in antibiotic resistance, have been purified by affinity chromatography on boronic acid gels. The procedure is rapid, appears to be selective for beta-lactamases, and allows a one-step purification of large amounts of enzyme from crude cell extracts. We have found the method useful for any beta-lactamase that is inhibited by boronic acids. Two kinds of boronic acid column have been prepared, the more hydrophobic one being reserved for those beta-lactamases that bind boronic acids relatively weakly. beta-Lactamase I from Bacillus cereus, P99 beta-lactamase and K 1 beta-lactamase from Gram-negative bacteria are among the better-known beta-lactamases that have been purified by this method. The procedure has also been used to purify a novel beta-lactamase from Pseudomonas maltophilia in high yield; the enzyme has an exceptionally broad substrate profile and hydrolyses monocyclic beta-lactams such as azthreonam and desthiobenzylpenicillin.

SUBMITTER: Cartwright SJ

PROVIDER: S-EPMC1144066 | biostudies-other | 1984 Jul

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. RESULTS: Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. CONCLUSIONS: Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.

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Purification of beta-lactamases by affinity chromatography on phenylboronic acid-agarose.

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