title: Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli.
abstract: Outer membrane lipoproteins are emerging as key targets for protective immunity to many bacterial pathogens. Heterologous expression of lipoproteins in Escherichia coli does not always result in high level expression of acylated recombinant protein. Thus, these proteins do not take up their correct membrane topology and are lacking the immunostimulatory properties endowed by the lipid. To this end, we have designed a lipoprotein expression vector (pDUMP) that results in the production of fusion proteins containing the E. coli major outer membrane lipoprotein (Lpp) signal sequence, lipoprotein signal peptidase recognition site, and the +2 outer membrane sorting signal at their N termini. To test the ability of pDUMP to express lipoproteins from heterologous hosts, the surface lipoprotein PsaA from the Gram-positive organism Streptococcus pneumoniae and the outer membrane lipoproteins MlpA from the Gram-negative Pasteurella multocida and BlpA from the spirochete Brachyspira hyodysenteriae were cloned into both hexahistidine fusion vectors and pDUMP. High level expression of antigenically active protein from both the hexahistidine fusion vectors and pDUMP resulted in abundant bands of the predicted molecular masses when analyzed by SDS-PAGE. When grown in the presence of 3[H]palmitic acid, proteins encoded by pDUMP were observed to incorporate palmitic acid whilst the hexahistidine fusion proteins did not. Using mass spectrometry and image analysis we determined the efficiency of lipidation between the three clones to vary from 31.7 to 100%. In addition, lipidated, but not hexahistidine, forms of the proteins were presented on the E. coli surface.
authors.affiliation: Department of Microbiology, Bacterial Pathogenesis Research Group, Monash University, Victoria 3800, Australia.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Cullen
authors.forename: Miranda
authors.initials: M
authors.lastname: Lo
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Bulach
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Cordwell
authors.forename: Ben
authors.initials: B
authors.lastname: Adler
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008074:Lipoproteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C097147:mlpA protein, Myxococcus xanthus