Protocol

Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage Lysogens: Lysis of Bacterial Colonies

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

In this protocol, a bacterial lysogen is constructed from a recombinant bacteriophage encoding a fusion protein of interest. The resulting lysogenic colonies are induced to synthesize the fusion protein, which is then isolated in preparation for functional and biochemical analyses.

MATERIALS

E. coli strains Y1090hsdR and Y1089

These strains, which are available from the ATCC (www.atcc.org), are maintained on LB agar plates containing 50 µg/ml ampicillin. The Y1090 and Y1089 strains carry mutations in the lon gene, which encodes an ATP-dependent protease. Fusion proteins expressed in these strains are often more stable than those expressed in strains lacking this mutation. . . . [Full Text of this Article]