Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A sSpAD as a secretion - tagReport as inadecuate




Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A sSpAD as a secretion - tag - Download this document for free, or read online. Document in PDF available to download.

Microbial Cell Factories

, 9:92

First Online: 23 November 2010Received: 26 August 2010Accepted: 23 November 2010

Abstract

BackgroundEscherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.

ResultsWe discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation.

The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 SOD.

ConclusionsIn this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.

Abbreviations usedsSpADStaphylococcus aureus Protein A domain D expressed from a synthetic codon optimized gene

BL21DE3host strain

CCCPcarbonyl cyanide m-chlorophenylhydrazone

DADEhost strain derived from MC4100 ΔtatABCD, ΔtatE

DTTdithiothreitol

EDTAethylene-diamine-tetra-acetic acid

GroELchaperone heat shock protein 60

GFPGreen fluorescent protein

IPTGisopropyl-β-D-thiogalactopyranoside

MalEmaltose binding protein E

PCRpolymerase chain reaction

SDSsodium dodecyl sulphate

SODHuman Superoxide Dismutase 1

TCAtrichloro acetic acid

Electronic supplementary materialThe online version of this article doi:10.1186-1475-2859-9-92 contains supplementary material, which is available to authorized users.

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Author: Thomas Heel - Michael Paal - Rainer Schneider - Bernhard Auer

Source: https://link.springer.com/







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