Protocol

Heterologous Gene Expression in E.coli

Volume 705 of the series Methods in Molecular Biology pp 175-194

Date:

Expression of Recombinant Proteins with Uniform N-Termini

  • Orsolya KirályAffiliated withDepartment of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University
  • , Lan GuanAffiliated withDepartment of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center
  • , Miklós Sahin-TóthAffiliated withDepartment of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University Email author 

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Abstract

Heterologously expressed proteins in Escherichia coli may undergo unwanted N-terminal processing by methionine and proline aminopeptidases. To overcome this problem, we present a system where the gene of interest is cloned as a fusion to a self-splicing mini-intein. This fusion construct is expressed in an engineered E. coli strain from which the pepP gene coding for aminopeptidase P has been deleted. We describe a protocol using human cationic trypsinogen as an example to demonstrate that recombinant proteins produced in this expression system contain homogeneous, unprocessed N-termini.

Key words

Escherichia coli, intein human cationic trypsinogen, in vitro refolding ecotin affinity chromatography, aminopeptidase P