Expression of foreign antigens on the surface ofEscherichia coli by fusion to the outer membrane protein TraT
- 52 Downloads
ThetraT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between anEscherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane ofE. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence oftraT, was amplified and obtained by PCR. This sequence was then subcloned downstream of thetac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane ofE. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as anE. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.
Key WordsBacterial display TraT lipoprotein Outer membrane protein Rhodostomin Hepatitis B virus pre-S1 antigen
Unable to display preview. Download preview PDF.
- 2.Adler M, Lazarus RA, Dennis MS, Wagner G. Solution structure of kistrin, a potent platelet aggregation inhibitor and GP IIb-IIIa antagonist. Science 253:445–448;1991.Google Scholar
- 7.Chang HH. Using TraT lipoprotein as a vehicle to express foreign proteins onEscherichia coli surface. PhD, Diss, Taipei; 1996.Google Scholar
- 21.Kay BK, Winter J, McCatferty J. Phage display of peptides and proteins: A laboratory manual. San Diego, Academic Press; 1996.Google Scholar
- 24.Lu Z, Murray KS, Cleave VV, LaVallie ER, Stahl ML, McCoy JM. Expression of thioredoxin random peptide libraries on theEscherichia coli cell surface as functional fusion to flagellin: A system designed for exploring protein-protein interactions. Biotechnology (NY) 13:366–372;1995.CrossRefPubMedGoogle Scholar
- 32.Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467;1979.Google Scholar
- 33.Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science 249:386–390;1990.Google Scholar
- 38.Tindle RW, Croft S, Herd K, Malcolm K, Geczy AF, Stewart T. A vaccine conjugate of ‘ISCAR’ immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants. Clin Exp Immunol 101:265–271;1995.PubMedGoogle Scholar