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

Abstract

Background: Escherichia 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.

Results: We 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).

Conclusions: In 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.

Figure 1

Comparison of the protein solubility with and without sSpAD tag. The solubility of N^proEDDIE-pep6His, SOD and GFPmut3.1 with and without sSpAD tag, expressed under the control of a LacUV5 promoter, was determined under defined cultivation conditions.

Figure 2

Secretion competence of an sSpAD fusion protein. A: The sSpAD tag reacts with most secondary antibodies. Therefore sSpAD fusion proteins were detected on the same membrane as GroEL immuno blots. For the sSpAD immuno blots a secondary antibody was used which reacts against sSpAD. B: Anti GroEL immuno blots of the isolated fractions were performed to confirm that the periplasmic fractions obtained via osmolysis, were not contaminated with cytoplasmic proteins. C: Anti MalE immuno blots were performed to confirm that the isolated fractions correspond with the periplasm as the Maltose Binding Protein E is a periplasmic protein. -I: whole cell sample without induction of recombinant protein expression, +I: whole cell samples with induction of recombinant protein expression, P periplasmic fraction, C: cytoplasmic fraction; CCCP: carbonyl cyanide m-chlorophenylhydrazone, BL21(DE3): host strain, DADE: ΔtatABCDΔtatE host strain derived from MC4100.

Figure 3

Identification of the secretion pathway. In the CM124 strain Sec mediated secretion is inducible via addition of L-arabinose. Incubation times were kept very short to ensure cell viability of the cell culture without functional Sec pathway. Furthermore, low expression levels of the recombinant protein prevent contamination of the subcellular compartments due to overexpression of the target protein. Therefore, the subcellular localization of N^proEDDIE was determined via immuno blot. GroEL immuno blot was performed to control osmolysis mediated cell lysis. -I: whole cell sample without induction of recombinant protein expression, +I: whole cell samples with induction of recombinant protein expression, P: periplasmic fraction, C: cytoplasmic fraction.

Figure 4

IgG column purification of an sSpAD-fusion protein. sSpAD-SOD was purified out of the periplasmic fraction of a shaking flask cultivation in BL21(DE3). P: periplasmic fraction, Fl: flow through, W: washing step, E1-E4: eluted fractions.