Random mutant library

The ratio of the residual activity after incubation to the initial activity provides a measure of stability. Because it is a ratio of activities, this stability index automatically corrects for differences in activity due to variation in enzyme expression levels in the random mutant library. The majority of random mutants are less stable than the parent. However, a small number of more stable mutants can be identified provided the variation inherent in the screen is small (Gershenson and Arnold, 2000). 

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

Fig. 14. Distribution of activity and stability in a first generation random mutant library of S41. Ellipse shows one standard deviation of the values obtained for wild-type clones in this assay.

During the evolution of S41, selective pressure was applied to both stability and activity. Stabilized mutants were accepted only if they showed no or little decrease in activity. Wintrode et al. (2000) adopted a different strategy for the evolution of SSII s activity at low temperature. Random mutant libraries of SSII were prepared either by random mutagenesis or in vitro recombination and screened for catalytic activity at 10°C. Libraries were not screened for thermostability, and mutants that showed improved activity at 10°C were selected regardless of changes, positive or negative, in stability. This experiment addressed the question of what happens to thermostability when no selective pressure is applied. 

It is not yet clear whether any rules for thermal adaptation are to be found in the qualitative features of the mutations discovered during directed evolution. The trends seen in Table V (preference for surface substitutions over buried substitutions, conservative over nonconservative, etc.) would most likely be seen in any pool of functional mutants selected from random mutant libraries. 

The sequence of chapters mirrors the steps in a standard directed-evolution experiment. In the beginning, various methods for the creation of molecular diversity are considered. S. Brakmann and B.F. Lindemann (Chapter 2) present protocols for the generation of mutant libraries by random mutagenesis. Two chapters deal with the particularly powerful approach of in-vitro recombination. H. Suenaga, M. Goto, and K. Furukawa (Chapter 3) describe the application of DNA shuffling, and M. Ninkovic (Chapter 4) presents DNA recombination by the S tEP method. 

Generation of Mutant Libraries Using Random Mutagenesis... 

Macken and Perelson studied antibody affinity maturation as a random walk on the random energy landscape (Macken and Perelson, 1989 Macken et al., 1991 Macken and Perelson, 1991). The total number of mutants tried before a positive mutation is discovered T(F) is a measure of the change in the necessary size of the mutant library. The expected value of T, given that F is not a local optimum, is derived as... 

Shafikhani, S., Siegel, R. A., Ferrari, E., and Schellenberger, V. (1997). Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimeri-zation. Biotechniques, 23(2), 304-310. 

Hermes JD, Parekh SM, Blacklow SC, Koster H, Knowles JR, A reliable method for random mutagenesis The generation of mutant libraries using spiked oligodeoxyribonucleotide primers, Gene, 84 143-151, 1989. 

Because of the significant role of TEM-1 P-lactamase and its mutant derivatives in antibiotic resistance, it is of interest to understand how the amino acid sequence of the enzyme establishes its structure and activity. We have determined the tolerance of each residue in TEM-1 P-lactamase to amino acid substitutions to identify those residues that make critical contributions to the structure and activity of the enzyme. The tolerance of each residue was determined by randomizing three to six contiguous codons to create a random library containing all possible amino acid substitutions for the region randomized (Palzicill and Botstein, 1992). Functional random mutants were then selected from the libraries and sequenced to identify permissible substitutions at each position. The sequences for each set of mutants allowed the importance of... 

To determine the identity of allowable substitutions at each residue position, the DNA sequence of an average of 9 functional random mutants from each library were determined. In total, 43 out of the 263 (16%) mutated residues are inferred to be critical for TEM-1 p-lactamase structure and function since only the wild type amino acid is found at these positions among the sequenced mutants. This set of essential residues includes catalytic residues and a number of other amino acids that are buried in the hydrophobic core of the enzyme. A detailed description and analysis of these results has been published elsewhere (Huang et al., 1996). 

The method for random mutagenesis of genes using error-prone polymerase chain reaction (PCR) was adapted from previous reports (34, 35). An error rate of approximately 0.5% should be expected using this protocol. For a single yeast library of random mutants of approximately 105 clones, you should prepare enough reactions to yield 50—80 Llg error-prone amplified insert (between 3 and 8 reactions). 

---