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Isolation of viral coat protein mutants with altered assembly and aggregation properties

By David S. Peabody and Lina Al-Bitar

Abstract

A method was developed to screen bacteria for synthesis of mutant proteins with altered assembly and solubility properties using bacteriophage MS2 coat protein as a model self-associating protein. Colonies expressing coat protein from a plasmid were covered with an agarose overlay under conditions that caused the lysis of some of the cells in each colony. The proteins thus liberated diffused through the overlay at rates depending on their molecular sizes. After transfer of the proteins to a nitrocellulose membrane, probing with coat protein-specific antiserum revealed spots whose sizes and intensities were related to the aggregation state of coat protein. The method was employed in the isolation of assembly defective mutants and to find soluble variants of an aggregation-prone coat protein mutant

Topics: NAR Methods Online
Publisher: Oxford University Press
Year: 2001
OAI identifier: oai:pubmedcentral.nih.gov:92581
Provided by: PubMed Central
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