Protein Engineering Protocols - Mycobacteriology research center

300 Hecky et al.for the first transition, and of 11.4 kJ/mol for the second transition. It is importantto note that the optimized mutants denature at higher urea concentrationsthan the wt-clone, confirming the stability ranking observed in the thermoactivityassays.4. Notes1. To ensure that growth results can be compared, a preculture is required to provideconsistent starting conditions.2. The error rate of the shuffling process was determined to be 0.83 by analysis of2529 bases.3. Elution of bound the DNA fragment is increased by elongated incubation (15 min)at elevated temperatures, e.g., 50°C.4. No extra primers are added at this point to facilitate mutual priming and shufflingof the isolated DNA fragments.5. Increased Mg 2+ concentrations stabilize noncomplementary basepairs, whereasthe presence of 0.5 mM manganese ions diminishes the template specificity of thepolymerase. Error rates can be controlled over a wide range, for example, by varyingthe number of template molecules, the cycle number, the period of extension,the source and amount of polymerase, the concentrations and ratios of dNTPs, andthe type of dNTP analogs used. The error rate also depends on the nucleotide compositionof the target gene. Thus, the use of an identical protocol may give quitedifferent results in different trials.6. If the enzyme activity is very low in the first round, selection can alternatively beperformed in liquid medium (LB/Cm with different amounts of ampicillin), wherethe selection stringency is lower than on plates.7. The percent of unprocessed protein was quantified from scanned Coomassiestainedgels using the image analysis software, Scion/NIH image.8. If expression is performed at temperatures significantly below 37°C, it is best toalso lower the temperature of the overnight culture to avoid a lag phase when startingthe expression culture.9. The optimal expression temperature needs to be adjusted for every mutant by performinga small-scale growth test at various temperatures.10. Two pH values (7.0 and 7.2) were tested, with equal results.11. 1 M (NH 4)SO 4is desirable to ensure proper binding, but, if protein precipitationis a problem, the concentration of (NH 4)SO 4can be reduced. Because sampleswith wt β-lactamase started to precipitate after addition of 0.5 M (NH 4)SO 4,afinal concentration of only 0.65 M was used. In this case, the native form of theenzyme was in the flow-through, whereas the unprocessed form was retained onthe column.12. Absorbance spectra were measured from 350 to 220 nm, and the absorption at280 nm was corrected for background absorbance by extrapolating the absorbancebetween 320 and 350 nm linearly to 280 nm. Molar extinction coefficients andmolecular masses were calculated according to Gill and Hippel (45).