Abstract

BackgroundNative as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells.ResultsUpon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua)-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S.) cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase) promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM) studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE) promoter.ConclusionThe mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of otherwise instable proteins in their native conformation. In addition the self assembly properties of the fusion proteins allow their simple purification. Moreover the binding properties of the S-layer part can be used to immobilize the fusion proteins to various surfaces. Arrays of highly ordered and densely structured proteins either immobilized on surfaces or within living cells may be advantageous over the respective soluble variants with respect to stability and their potential interference with cellular metabolism.

Highlights

  • Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry

  • Bacterial cell surface layers (S-layer) as the outermost cell envelope components are a common feature of many bacteria and archaea species

  • Expression of mSbsC-enhanced green fluorescent protein (EGFP) in E. coli The 3207 bp open reading frame (ORF) encoding mSbsC(31–1099) was fused with the 720 bp ORF of EGFP and cloned into pET17b as described in Methods. Both ORFs are separated by a two-aa-linker (Leu-Glu) that was introduced by the non-template-encoded XhoI site (CTCGAG)

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Summary

Introduction

Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. Bacterial cell surface layers (S-layer) as the outermost cell envelope components are a common feature of many bacteria and archaea species (for review see [1,2]). S-layer monomers assemble to two-dimensional highly porous arrays with either oblique, square or hexagonal symmetry. Upon removal of the denaturing agent the isolated S-layer subunits assemble in vitro into regular arrays exhibiting structural features of the authentic cell surface layer. Contrary to the situation in vivo, the in vitro self-assembly process of S-layer proteins in solution can result in double layer sheets or in tubelike structures [7]

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