OVEREXPRESSION OF PROTEIN SEFA DERIVED FROM SALMONELLA ENTERICA SEROV AR ENTERITIDIS IN ESCHERICHIA COLI BL21

Abstract

SEF14 of S Enteritidis fimbriae plays important roles for strong induction ofT-lymphocyte response and protective immunity. The sefA coding for protein SefA, which  omprises thin aggregative fimbriae SEFI4, lacking signal peptide sequence was amplified by PCR and cloned into pCR2.1. The gene of 432 nucleotides was 99.3% homology with the sefA of S. Enteritidis PT4 - the dominant strain caused the major gastroenteritis in humans. Then, the gene was inserted into expression -vector pET32a(+) and transformed into E. coli BL2l Ibr SetA expression. In this recombinant strain, SetA was expressed at very high level under the chimeric form with Trx, S-tag at N terminal, hexahistidine at C terminal then abbreviated to TrxSef. This protein was produced at the highest amount at 30^oC (over 500 mg!1 broth), but hal r or them was inclusion body. The use of low temperature for TrxSef production decreacd the ratio of insoluble TrxSef fraction to total TrxSef. At 22, 25, 30^oC, soluble TrxSef were synthesized at similar amount (over than 200 mg/l broth), thus productivity of insoluble TrxSef increased owing to the significant increase of expression rate. However, at 25°C, the recombinant strain grew reasonably and produced 76% soluhle TrxSetA. IPT(i concentrations did not affect to the cell growth but lightly influenced the TrxSef production.  At the flask scale production, expression rate of TrxScf depended on the cells growth phase. During exponential phase (2 - 3 hours after induction), TrxSef was produced about 60 mg/g dried cells, but when the cell density reached to stationary phase (4 hours after induction), TrxSef was explosively expressed by 2-fold compared to the TrxSef production during exponential phase and then kept nearly stable for I hour. Thus, the largest amount of TrxSef '.vas harvested at hour 5 after induction. This chimeric recombinant protein had high stability with the host proteases. At optimal condition, over 200 mg ofTrxSefper broth correlating approximately 130 mg TrxSef/g dried cells was achieved