Construction of “Small-Intelligent” Focused Mutagenesis Libraries using Well-Designed Combinatorial Degenerate Primers

Abstract

Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel small-intelligent strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.

MDC-Analyzer: A Novel Degenerate Primer Design Tool for the Construction of Intelligent Mutagenesis Libraries with Contiguous Sites

“…The temperature program used was: 98C for 3 min, followed by 30 cycles of 10 s at 98C, 45 s at 50C, and 2 min at 72C, with a final incubation at 72C for 10 min. In the case study of library construction using DC-Analyzer methodology, two active-site residues (P135/F136) of HheC were fully randomized according to the procedure as previously described (13). …”

“…In addition to chemical synthesis methodology (11, 12), our previously developed DC-Analyzer shows great potential for constructing a mutagenesis library with one gene per protein (13), where stop codons, rare codons of E. coli , and codon bias are all eliminated in the constructed small-intelligent libraries. Meanwhile, a quite similar approach called 22c-trick using a mixture of three oligonucleotides (two degenerate) with NDT (12 codons) VHG (9 codons) and one TGG primer was also reported (14).…”

Designer protein delivery: From natural to engineered affinity-controlled release systems

“…Combinatorial approaches such as diand tri-nucleotide block precursors (68,69,74) for oligonucleotide synthesis can further reduce or eliminate redundancy but may be prohibitively expensive for some laboratories. Novel combinatorial strategies are being devised to generate both cost-effective and nondegenerate mutagenic primers and have been assisted by computational efforts (75)(76)(77). Investigators should consider the type or scope of mutations incorporated and the potential forms of bias in each approach when developing selection and evolution strategies for a particular application (74).…”

A gain-of-function positive-selection expression plasmid that enables high-efficiency cloning

“…Much ingenuity has gone into designing schemes to minimize redundancy in these libraries, and hence the number of clones that must be screened for effective coverage. For example, if targeting a single codon, sequences containing the degenerate nucleotide triplets NDT, VMA, ATG, and TGG can be mixed at a 12:6:1:1 molar ratio to enable all 20 amino acids to be specified without redundancy (Tang et al 2012). These approaches can make low throughput screening strategies feasible but, in such cases, it is imperative that effort is not expended screening empty vectors, i.e., that every clone examined contains a gene variant.…”