Vector engineering to improve a staphylococcal surface display system
2002 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 212, no 1, 47-54 p.Article in journal (Refereed) Published
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 toot 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.
Place, publisher, year, edition, pages
2002. Vol. 212, no 1, 47-54 p.
bacterial surface display, fluorescence-activated cell sorting, Staphylococcus aureus protein A, Staphylococcus carnosus, rolling-circle replication, hyicus subsp hyicus, cell-wall, protein-a, combinatorial libraries, biochemical-properties, recombinant proteins, nucleotide-sequence, directed evolution, vaccine delivery
IdentifiersURN: urn:nbn:se:kth:diva-21678ISI: 000176618900008OAI: oai:DiVA.org:kth-21678DiVA: diva2:340376
QC 201005252010-08-102010-08-10Bibliographically approved