Protocol

Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter

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

This protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage T7 promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins.

MATERIALS

1x SDS gel-loading buffer

E. coli strains HMS174(DE3) or BL21(DE3)

Coomassie Brilliant Blue stain or Silver stain

Gene or cDNA fragment of interest

IPTG (1 M)

70 µl is required for each plaque analyzed.

NZCYM agar plates containing 50 µg/ml ampicillin

NZCYM medium containing 50 µg/ml ampicillin

Positive control plasmid (e.g., carrying a bacteriophage T7 promoter that controls expression of a . . . [Full Text of this Article]

METHOD