Use of the human hepcidin gene to build a positive-selection vector for periplasmic expression in Escherichia coli

Jérome Haustant; Annesha Sil; Christopher Maillo-Rius; Agnès Hocquellet; Patricia Costaglioli; Bertrand Garbay; Wilfrid Dieryck

doi:10.1016/j.ab.2016.02.002

Use of the human hepcidin gene to build a positive-selection vector for periplasmic expression in Escherichia coli

Anal Biochem. 2016 May 1:500:35-7. doi: 10.1016/j.ab.2016.02.002. Epub 2016 Feb 10.

Authors

Jérome Haustant¹, Annesha Sil¹, Christopher Maillo-Rius¹, Agnès Hocquellet¹, Patricia Costaglioli¹, Bertrand Garbay¹, Wilfrid Dieryck²

Affiliations

¹ Université de Bordeaux, EA 4135, F-33000 Bordeaux, France; Bordeaux-INP, EA4135, F-33000 Bordeaux, France.
² Université de Bordeaux, EA 4135, F-33000 Bordeaux, France; Bordeaux-INP, EA4135, F-33000 Bordeaux, France. Electronic address: [email protected].

PMID: 26873403
DOI: 10.1016/j.ab.2016.02.002

Abstract

Recombinant proteins are often produced in the periplasm of Escherichia coli because this facilitates the purification process. The oxidizing environment favors the formation of disulfide bridges. We showed that the periplasmic expression of the human hormone hepcidin 25 (Hep25) fused to the maltose-binding protein (MBP) resulted in cell death. This toxicity was not observed when MBP-Hep25 accumulated in the bacterial cytoplasm, or when Hep25 was addressed to the periplasm without the MBP tag. We then modified the periplasmic expression vector pMALp2E to create pMALp2EH, a positive-selection vector with Hep25 as counterselection gene.

Keywords: Cloning; Expression vector; Hepcidin; Positive selection.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Base Sequence
Escherichia coli / genetics
Escherichia coli / metabolism*
Genetic Vectors*
Hepcidins / chemistry
Hepcidins / genetics*
Humans
Periplasm / metabolism*

Substances

Hepcidins