title: Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome.
abstract: Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia. ales.berlec@ijs.si
authors.forename: Aleš
authors.initials: A
authors.lastname: Berlec
authors.forename: Petra
authors.initials: P
authors.lastname: Zadravec
authors.forename: Zala
authors.initials: Z
authors.lastname: Jevnikar
authors.forename: Borut
authors.initials: B
authors.lastname: Štrukelj
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D015870:Gene Expression; D006639:Histidine / Q000235:genetics; D013294:Lactococcus lactis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009561:Nisin / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics; D011485:Protein Binding; D020543:Proteome / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012984:Software; D013205:Staphylococcal Protein A / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C471213:His-His-His-His-His-His; D008565:Membrane Proteins; D009842:Oligopeptides; D020543:Proteome; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D009561:Nisin; D006639:Histidine