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The Multifaceted Benefits of Protein Co-expression in Escherichia coli

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Published on: February 5, 2015

Self-processing of a barley subtilase expressed in E. coli.

Stephan Plattner¹, Clemens Gruber², Friedrich Altmann²

¹Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Austria.

Protein Expression and Purification

|June 15, 2014

Summary

Barley protease BAJ93208 undergoes autocatalytic processing in E. coli, removing its inhibitor and tags. Mass spectrometry identified precise N- and C-terminal cleavage sites, crucial for understanding its function.

Keywords:

Proprotein Subtilase Thionin

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Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Barley protease BAJ93208 is a subtilase family serine protease.
Recombinant expression of BAJ93208 in E. coli was performed using a rhamnose-inducible promoter.

Purpose of the Study:

To investigate the processing of barley protease BAJ93208 during recombinant expression in E. coli.
To identify the precise N- and C-terminal cleavage sites of BAJ93208.

Main Methods:

Recombinant expression of wild-type and inactive mutant BAJ93208 in E. coli.
Western blot analysis to detect protein processing.
Mass spectrometry to identify cleavage sites.

Main Results:

Western blot indicated N- and C-terminal processing of BAJ93208.
An inactive mutant confirmed autocatalytic processing.
Mass spectrometry identified four processed forms, with specific N- and C-terminal cleavages removing the inhibitor and strep-tag.
Additional alanine residues were removed at both termini.

Conclusions:

Barley protease BAJ93208 undergoes autocatalytic processing in E. coli.
The processing removes the N-terminal inhibitor and C-terminal strep-tag, impacting detection.
Precise cleavage sites were identified, providing insights into protease maturation.