Application of a short, disordered N-terminal flagellin segment, a fully functional flagellar type III export signal, to expression of secreted proteins.
Recently, we have demonstrated that the 26-47 segment of Salmonella enterica serovar Typhimurium flagellin is capable of mediating flagellar export. In order to reveal whether other parts of the N-terminal region have any significant influence on secretion, a series of plasmids were constructed containing the lac promoter followed by the 26-47, 2-65, or 2-192 portion of Salmonella flagellin, to which various heterologous proteins of different size were fused (18 constructs overall). Essentially, all three segments could drive protein export; however, the nature of the attached polypeptide also had a significant effect on secretion efficiency. When low export efficiency was observed, it was mainly caused by inclusion body formation. Our data provide strong support for the idea that a short segment within the disordered N-terminal region of axial proteins is recognized by the flagellar type III export machinery. The 26-47 segment of flagellin contains all of the necessary information to direct translocation of attached polypeptide chains. This short (positions 26 to 47) flagellin segment attached to recombinant proteins can be used for secreted protein expression. Certain fusion proteins that are easily degraded within the cells were found to be intact in the medium, implying a potential application of this expression system for proteins with high proteolytic susceptibility.
Dobó
József
J
Institute of Enzymology, BRC, Hungarian Academy of Sciences, Budapest, Hungary.
Varga
János
J
Sajó
Ráchel
R
Végh
Barbara M
BM
Gál
Péter
P
Závodszky
Péter
P
Vonderviszt
Ferenc
F
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D005408:Flagellin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D020409:Molecular Motor Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D005408:Flagellin; D000251:Adenosine Triphosphatases