, 2000), adenylate kinase 2 of L. donovani (Villa et al., 2003), cysteine protease type A and B (CPA and CPB) genes of L. infantum (Rafati et al., 2003). In practice, it is usually worthwhile to test several different vector/host combinations to obtain the best possible yield of protein in its desired form. Hence,
a number of commercially available strains of E. coli host cells and expression vectors were tested in an attempt to produce LdSSN in the soluble form. Screening of experimental conditions were carried out to obtain high yields of recombinant protein, including growth temperature, medium type and hours of growth after IPTG induction. For maximum overexpression of SSN protein in the soluble fraction, various parameters were standardized viz. E. coli host strains, IPTG concentrations, incubation temperatures before and after induction SP600125 purchase and incubation period after induction. Because the pET-28a-SSN recombinant vector has T7 promoter, various hosts compatible with T7 promoter viz. BL21 (DE3), Rosetta and codon plus cells were attempted for the expression of soluble SSN protein. The maximum solubility of SSN protein was found in BL21 (DE3)
cells as compared with Rosetta and codon plus cells; therefore, further expression of recombinant SSN protein was carried out in BL21 (DE3) cells. IPTG concentrations varying from 0.1 to 1 mM were attempted BMN 673 mw so as to observe the amount of expressed SSN protein. However, no difference in the amount of expressed protein was observed with the above used concentrations. 0.1 mM IPTG concentration the was used for protein induction and overexpression
studies. Addition of >0.1 mM IPTG to the cultures did not lead to further increase in the amount of overexpressed recombinant LdSSN. The level of expression of recombinant LdSSN was tested under various temperatures ranging from 20 to 37 °C. At temperatures 37, 30 and 28 °C, the recombinant SSN protein was expressed at a significant level, but all the expressed protein appeared as inclusion bodies. Reducing the temperature to 20 °C resulted in the expression of the recombinant protein in a soluble form; however, below 20 °C, the solubility of the protein was increased but the total amount of expressed SSN protein was also decreased, and therefore, in further studies, BL21 (DE3) cells were induced at 20 °C with 0.1 mM IPTG. The induction time was varied from 6 to 12 h. The amount of recombinant soluble SSN protein was increased from 6 to 12 h; therefore, in further studies, the induction time was extended to 12 h to obtain the maximum amount of soluble protein. The best suitable parameters selected resulted in nearly 30% of the recombinant protein in the soluble fraction, whereas most of the protein was found in inclusion bodies.