Library analysis of SCHEMA‐guided protein recombination

Abstract

The computational algorithm SCHEMA was developed to estimate the disruption caused when amino acid residues that interact in the three-dimensional structure of a protein are inherited from different parents upon recombination. To evaluate how well SCHEMA predicts disruption, we have shuffled the distantly-related ␤-lactamases PSE-4 and TEM-1 at 13 sites to create a library of 2 14 (16,384) chimeras and examined which ones retain lactamase function. Sequencing the genes from ampicillin-selected clones revealed that the percentage of functional clones decreased exponentially with increasing calculated disruption (E ‫ס‬ the number of residue-residue contacts that are broken upon recombination). We also found that chimeras with low E have a higher probability of maintaining lactamase function than chimeras with the same effective level of mutation but chosen at random from the library. Thus, the simple distance metric used by SCHEMA to identify interactions and compute E allows one to predict which chimera sequences are most likely to retain their function. This approach can be used to evaluate crossover sites for recombination and to create highly mosaic, folded chimeras.Keywords: Chimera; lactamase; PSE-4; recombination; schema; TEM-1; directed evolution Numerous protein traits can be improved or altered when recombination is coupled with a screening or selection strategy (for review, see Minshull and Stemmer 1999; Arnold 2001a,b). During laboratory evolution, as in nature, recombination promotes the rapid accumulation of beneficial mutations from multiple parents onto a single offspring (Stemmer 1994b;Moore et al. 1997;Crameri et al. 1998). It also explores a part of sequence space that is particularly rich in folded and functional proteins. Recombination plays a key role in natural evolution of proteins through the swapping of well-defined structural domains . Where a domain structure is not obvious, however, how recombination contributes to the evolution and diversification of protein sequence and function is less well understood.Recently we developed an algorithm, called SCHEMA, for predicting which fragments of homologous proteins can be recombined without disturbing the integrity of the structure (Voigt et al. 2002). Based on the 3D structures of the parent proteins, the algorithm identifies pairs of amino acids that are interacting, defined as those residues within a cutoff distance of 4.5 Å, and determines the net number of interactions broken when a chimeric protein inherits portions of its sequence from different parents (defined as E). Because calculating E for all possible crossover combinations is computationally intractable, it is difficult to identify which crossover locations are optimal with respect to their ability to yield folded chimeras. One version of SCHEMA circumvents this computational difficulty by finding compact, con-

On the conservative nature of intragenic recombination

“…Sequencing of the remaining clones revealed an additional eight sequences for a total of 31 unique functional chimeras (see Table 1, which is published as supporting information on the PNAS web site). Although no point mutations were found in the newly characterized chimeras, one of those previously identified as functional has two adjacent amino acid substitutions (28). Sequencing of unselected chimeras showed that nine of 13 (69%) contained frameshifts introduced during oligonucleotide synthesis.…”

“…In a previous study, we recombined PSE-4 and TEM-1 to create a well defined library of chimeras (28) and selected for those that allowed E. coli XL1-Blue to grow on 20 g͞ml ampicillin. Approximately 100 colonies were observed, and sequencing fifty of these clones identified 23 unique functional chimeras.…”

“…To determine the average effects of recombining PSE-4 and TEM-1 over all possible substitution levels, we partitioned all chimeras into bins of substitution levels containing at least one functional chimera. The number of unique synthesized chimeras in each bin sets an upper bound on the denominator of the fraction of functional chimeras; because of the possibilities that frameshifts inactivated some chimeras and that certain fragments were overrepresented because of biases in library construction (28), it is unlikely that this upper bound was reached. The calculated fraction of functional chimeras therefore represents a lower bound on the true fraction functional at each m.…”

“…The gene products from each library were digested with HindIII and SacI, purified by using a Zymo DNA Clean and Concentrator kit (Zymo Research), and ligated into pMon-1A2 as in a previous study (28). E. coli XL1-Blue was transformed with plasmids containing each library as recommended by the manufacturer and plated on three or more nonselective (10 g͞ml kanamycin) and selective (20 g/ml ampicillin͞10 g/ml kanamycin) plates.…”

“…Structurally related polypeptides have been swapped among homologous single-domain proteins to create functional chimeras with substitution levels much higher than in random mutation experiments (24)(25)(26)(27)(28)(29)(30). The more conservative nature of recombination is likely to arise at least in part because the individual amino acid substitutions created by recombination, having proved compatible with a similar structure, are less likely to be incompatible in the homolog structure than substitutions created by mutation.…”