Expression of proteins using the third domain of the Escherichia coli periplasmic-protein TolA as a fusion partner.
The third domain of the periplasmic protein TolA from Escherichia coli (TolAIII) was used as a fusion partner in the expression of various proteins from bacteria and eukaryotes. TolAIII is small domain, expressed in high yields as a soluble protein in the cytoplasm of E. coli. Proteins were linked to the C-terminus of TolAIII by a short flexible linker containing sites for endopeptidases. Three different vectors were prepared, containing sites for enterokinase, thrombin or factor Xa. Fusion proteins also contain a His(6)-Ser(2) tag at their N-terminus for easier purification. Up to 90 mg fusion protein per liter bacterial culture was obtained using these vectors. Colicin N R-domain was expressed with this system as a fusion and processed further for functional studies. The yield of final pure R-domain was doubled as compared to the direct expression. The system may prove to be useful in the preparation of other peptides and proteins.
Anderluh
Gregor
G
Department of Biology, University of Ljubljana, Vecna pot 111, Ljubljana 1000, Slovenia.
Gökçe
Isa
I
Lakey
Jeremy H
JH
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics*; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019897:Periplasm / Q000737:chemistry*; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; C062093:tolA protein, E coli