Vector engineering to improve a staphylococcal surface display system.
A previously developed expression system for surface display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signals from a Staphylococcus hyicus lipase and the cell wall anchoring part of Staphylococcus aureus protein A (SpA) to achieve surface display of expressed recombinant proteins. The system has been successfully used in various applications but the vector has not been considered genetically stable enough to allow protein library display applications, which would be of obvious interest. A new set of vectors, differing in size and devoid of a phage f1 origin of replication, were constructed and evaluated in terms of bacterial growth characteristics and vector stability. Furthermore, surface expression of a model surface protein was monitored by an enzymatic whole-cell assay and flow cytometry. The engineered expression vectors demonstrated dramatically improved stability and growth properties and two of the novel vectors demonstrated retained high surface density of the displayed model protein. The flow cytometry was found to be a powerful tool for observing the surface density of displayed heterologous proteins, and would thus be a rational strategy for monitoring the optimisation of any surface display system. The implications of these improved display vectors for future protein library applications are discussed.
Wernérus
Henrik
H
Department of Biotechnology, SCFAB, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.
Ståhl
Stefan
S
D003124:Colorimetry; D005434:Flow Cytometry / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011995:Recombination, Genetic; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D013210:Staphylococcus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014169:Transformation, Bacterial
D008565:Membrane Proteins; D013205:Staphylococcal Protein A; D008049:Lipase