Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2×108 and 6×107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.
Directed evolution is a proven approach to fine tune or modify biomolecules for various applications ranging from research to industry. The process of evolution requires methods that are capable of not only generating genetic diversity but also to distinguish the variants of desired characteristics. One method that is synonymous with directed evolution of proteins is phage display. Here, we present a protocol describing the application of magnetic nanoparticles coupled with a processor to carry out the identification of monoclonal antibodies (mAbs) from a diverse antibody library via phage display. Target antigens are coupled to magnetic nanoparticles as the solid phase for the isolation of the binding mAbs via affinity. A gradual enrichment in clones would result in increasing ELISA readouts with increasing rounds of panning. During monoclonal level analysis, positivity can be deduced with comparison to background and controls. The biopanning process can also be adopted for the directed evolution of enzymes, scaffold proteins or even peptides.
The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.