Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering

Abstract

Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and comprehensive, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple protocol.

Effects of Proline Substitutions on the Thermostable LOV Domain from Chloroflexus aggregans

“…Earlier studies focused on the effects of mutating the conserved cysteine, which forms a covalent bond with the flavonoid cofactor during the photocycle, and some random mutations on flavin binding and photochemical reactivity [55,56]. Later studies probed the effects of mutations on other properties, particularly the absorption spectrum [47,50,57,58], photocycle lifetime [57,59], brightness of the cysteine-less variants [9,19,20,60], generation of radicals [15,61,62] and thermal stability [21][22][23]. Many of these mutations were rational, or could be rationalized after initial discovery, thus allowing one to apply the same principles to impart a different LOV domain with the desirable properties.…”

“…We expect that the effects of the proline substitutions may also be transferable to other LOV domains, since the rationales that we employed (consensus design [26][27][28], stabilization by prolines [27,28]) will hold. Interestingly, out of three LOV domain thermal stabilization studies [21][22][23], only one reported the proline substitutions [22], which were, however, not included in the most stable variant with multiple mutations. Thus, testing the effects of proline substitutions may be a complementary approach to recombination, and directed evolution to speed up the discovery of the most stable variants.…”

“…Recombination and directed evolution have been used to develop iLOV and phiLOV proteins, which were brighter and more photostable compared to their natural counterparts [19,20]. Rational engineering has been used to thermostabilize YtvA [21], while plasmid recombineering and directed evolution have been used to thermostabilize iLOV [22,23]. As an alternative to the engineering of mesophilic proteins, different LOV domains from thermophilic microorganisms have been screened for thermo-and photostability [24].…”

Modern methods for laboratory diversification of biomolecules

“…Using an iterative recombineering approach called SIRCAS (stepwise integration of rolling circle amplified segments) [64], the Salmonella typhimurium genome was extensively modified in the largest genomic recoding effort to date, enabling 1557 synonymous leucine substitutions. In addition to whole genome mutagenesis through recombineering, the flexibility of this oligo-based approach was recently demonstrated in strategy that extended its applications to directed protein evolution [65]. As a proof of concept, the authors used plasmid recombineering to subject the 110-residue iLOV protein to saturation mutagenesis with near complete coverage (99.8% of all mutations), uncovering many variants with improved thermostability.…”

Systematic genome engineering approaches to investigate mutational effects and evolutionary processes

“…violacein [38,44,45]) and proteins [46][47][48][49], as well as by genome-wide codon replacement to create a so-called "genomically recoded organism" (GRO) engineered to depend on synthetic amino acids [50][51][52], MAGE has paved the way for previously unimaginable evolutionary innovations.…”