Protein Engineering Protocols - Mycobacteriology research center

270 Campbell-Valois and Michnick21. Alternatively, protein expression could be induced O/N at 30°C.22. Otherwise, if the quantity of clones to analyze was lower, we used Ni-NTA spincolumns. If more protein from fewer independent clones is needed, regular Ni-NTA agarose column purification is the method of choice. In this case, it may beuseful and reasonable to perform the purification under native conditions.23. The described system only works with Millipore large collector and sealing block.It is also possible to replace vacuum steps by centrifugation.24. A gauge is included with the Millipore manifold.25. If required, the flow-through and the filtrate from the diverse washing steps arecollected by placing a fraction collector in the collection chamber, in Subheading3.7., steps 9 and 10.26. In our case, the protein is eluted under denaturing conditions and the sample dilutedsufficiently to reduce the denaturant to a concentration that does not interfere withprotein function. The concentration of the denaturant used in the elution buffershould be fixed according to the stability of the protein studied. Nevertheless, if it isdesired to elute protein in its native condition, Qiagen recommends using a gradientof decreasing concentration of Gdn-HCl. The protein is then eluted with the appropriatebuffer, without denaturant. One could also simply purify the protein undernative conditions (see the Qiagen Expressionist and Ni-NTA spin column handbook).We have improved elution by using diluted glacial acetic acid, in quantitiessufficient to buffer 25 or 50 mM NaOAc to pH 5.0 (see Note 27 concerning pHadjustment after elution). Urea could also be used as denaturant, provided that it willdenature the protein under study at reasonable concentrations. In addition, care mustbe taken because urea in solution equilibrates with cyanate. However, urea does notprecipitate SDS even at high concentrations, thus, facilitating the characterization ofthe protein at individual purification steps by SDS-PAGE electrophoresis.27. DTT is added to the sample after elution because it is not recommended for usewith Ni-NTA. The pH of the eluate can be adjusted to a relevant value by the additionof NaOH or of an appropriate weak base, such as Tris or NaOAc. The quantityof base to be added is determined empirically on larger volumes with a pH meter.28. If the samples are not going to be characterized immediately, we recommendadding 40% glycerol (v/v) and 1 mM NaN 3for storing of samples at –20°C. Theglycerol can then be removed by dialysis or ultrafiltration before characterization.Alternatively, samples without glycerol can be flash frozen in liquid nitrogen andpreserved at –80°C.29. BL21 pREP4 chemiocompetent cells prepared according to QIAEXpressionnistHandbook with TFB buffer (RbCl) are usually more competent, but because weneeded to transform only super-coiled DNA in our protocols, the Inoue methodwas satisfactory (48).AcknowledgmentsWe are grateful to Emil Pai, David Waugh, and Shigekazu Nagata for thecDNAs of h-ras, raf RBD, and the CAD domain’s of cad and icad, respectively.We also want to thank Dimitri Sans for carefully reading this manuscript, Jérôme