Comparison of success frequencies of expression vectors at each stage of purification. Selected entry clones of 187 ORFs in Bacillus anthracis Ames, Streptococcus pneumonia TIGR4 and Yersinia pestis KIM, were used to prepare expression clones using 5 different Gateway comparable vectors, pHis, pMBP, pSP-MBP, pDsbA and pGST. Target proteins were expressed in 96-well blocks by IPTG induction at OD600 nm = 0.7-0.9 for 20 hours at 20°C. The harvested cell pellets were resuspended in lysis buffer [50 mM Tris-HCl, 100 mM NaCl, 1 mM DTT at pH 7.8 at 4°C] and lysed using PopCulture and lysonase. The recombinant proteins were purified using Ni-NTA agarose column as described in 'Methods and Materials'.