Molecular evolution by staggered extension process (StEP) in vitro recombination

Abstract

We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65 degrees C is 50 times that of wild-type subtilisin E.

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

“…After incubation with saturating concentrations of BODIPY-NT (8)(9)(10)(11)(12)(13), bacteria expressing the largest number of functional receptors correspondingly exhibit the greatest fluorescence, and these cells were collected directly in growth medium and then expanded for a subsequent round. A single selection round, which consisted of library expansion, induced receptor expression, incubation with fluorescent ligand, and FACS to recover the most fluorescent bacterial cells, took approximately 1 day.…”

“…After a third randomization step followed by four more rounds of FACS, the evolved pool was split into two. One half was randomized by epPCR a fourth time and the other half was shuffled, using the staggered extension process (StEP) (12 Fig. S8).…”

“…In principle, selectivity selections could be performed independently of selections for expression level. However, in the case of NTR1, the FACS signal of the WT receptor was so weak with BODIPY-NT (8)(9)(10)(11)(12)(13) alone, that the addition of excess SR 48692 would drop the signal of any positive clones into the background. Thus, the selectivity screen was only used after an initial selection for expression level to ensure that the MFI of the pool was significantly above background (after the fourth epPCR; see Fig.…”

Directed evolution of adeno-associated virus to an infectious respiratory virus

“…For the initial plasmid library, the AAV2 cap gene was amplified via PCR using 5Ј-CATGGGAAAGGTGCCAGACG-3Ј and 5Ј-CGCA-GAGACCAAAGTTCAACTGA-3Ј as forward and reverse primers, respectively and AAV5 cap gene was amplified via PCR using 5Ј-CATGGGAAAGGTGCCA-GACG-3Ј and 5Ј-AAGCGGCCGCAATGGGTTAAAGGGG -3Ј as forward and reverse primers, respectively. DNA shuffling was performed as previously described (7,8), and chimeric cap genes were cloned into pSub2 for rcAAV production (10). For subsequent evolution rounds, error-prone PCR was performed as previously described (10).…”

“…Directed evolution strategies have demonstrated the power of mutagenesis and DNA shuffling methods to investigate and enhance preexisting functions of or generate novel functions in a protein without underlying mechanistic knowledge (7,8). Recent efforts have increasingly demonstrated the impact of these methods to address novel and high impact problems in protein engineering (9)(10)(11); however, few studies have focused efforts on engineering structurally complex protein assemblies (10) or on direct clinical application (11).…”