Searching for common sequence patterns among distantly related proteins

We have developed a program Gap Allowing Pattern Explorer (GAPE)to extract amino acid sequence motifs conserved among distantlyrelated proteins. The GAPE program is designed to allow gapsin the sequences. First, this program generates all possibleamino acid patterns comprising up to five amino acids. Sequencescontaining the amino acid residues in the same order as a generatedpattern are selected as subsequences, where the differencesin the distances between two consecutive amino acids are ignored.Next, the motifs are extracted from the subsequences under conditionsin which all four distances between the five amino acids arefixed. At this stage, motifs with gaps in their subsequenceare also found by relaxing one of the four fixed distances.The statistical significance for a motif obtained is calculatedbased on the amino acid composition of the sequences under consideration.When the GAPE program was applied to 59 pyridoxal-phosphaterelatedsequences and 64 ATP (AMP-forming)-related sequences, motifsextracted with a low expectation of occurrence contained someof the amino acid residues chemically proved to be involvedin the ligand recognition.     

Site-directed protein recombination as a shortest-path problem

Protein function can be tuned using laboratory evolution, in which one rapidly searches through a library of proteins for the properties of interest. In site-directed recombination, n crossovers are chosen in an alignment of p parents to define a set of p(n + 1) peptide fragments. These fragments are then assembled combinatorially to create a library of p(n+1) proteins. We have developed a computational algorithm to enrich these libraries in folded proteins while maintaining an appropriate level of diversity for evolution. For a given set of parents, our algorithm selects crossovers that minimize the average energy of the library, subject to constraints on the length of each fragment. This problem is equivalent to finding the shortest path between nodes in a network, for which the global minimum can be found efficiently. Our algorithm has a running time of O(N(3)p(2) + N(2)n) for a protein of length N. Adjusting the constraints on fragment length generates a set of optimized libraries with varying degrees of diversity. By comparing these optima for different sets of parents, we rapidly determine which parents yield the lowest energy libraries.     

Engineering aggregation-resistant proteins by directed evolution

A method was recently described for selecting aggregation-resistant antibody domains. A repertoire of domains displayed on filamentous bacteriophage were heated/cooled and selected for binding to the affinity ligand protein A specific for the folded domains. Here we describe a generalization of the method based on the selection for retained phage infectivity and for the binding of an appended sequence tag, and applicable to any protein displayable in multivalent form on phage.     

In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants

The use of random mutagenesis in concert with protein display technologies to rapidly select high affinity antibody variants is an established methodology. In some cases, DNA recombination has been included in the strategy to enable selection of mutations which act cooperatively to improve antibody function. In this study, the impact of L-Shuffling DNA recombination on the eventual outcome of an in vitro affinity maturation has been experimentally determined. Parallel evolution strategies, with and without a recombination step, were carried out and both methods improved the affinity of an anti-Fas single chain variable fragment (scFv). The recombination step resulted in an increased population of affinity-improved variants. Moreover, the most improved variant, with a 22-fold affinity gain, emerged only from the recombination-based approach. An analysis of mutations preferentially selected in the recombined population demonstrated strong cooperative effects when tested in combination with other mutations but small, or even negative, effects on affinity when tested in isolation. These results underline the ability of combinatorial library approaches to explore very large regions of sequence space to find optimal solutions in antibody evolution studies.     

Natural history as a predictor of protein evolvability

Natural selection generally produces specific and efficient enzymes. In contrast, directed evolution experiments usually produce enzyme variants with broadened substrate specificity or enhanced catalytic promiscuity. Some proteins may be more evolvable than others, but few workers consider this problem when choosing starting points for laboratory evolution. Here, we review the variables associated with enzyme evolvability, namely promiscuity and mutational robustness. We present a qualitative model of adaptive evolution and recommend that protein engineers exploit their knowledge of natural history to identify evolvable wild-type proteins. Three examples of 'generalist' proteins that evolved in the laboratory into 'specialists' are described to illustrate the practical utility of this point.     

A second-generation system for unbiased reading frame selection

Reading frame selection of nucleic acids has important implications for protein engineering and genomics. Current methods are limited because selection of the gene of interest inevitably depends on the solubility of its translated product. Here we report the construction of the pInSALect vector, which provides strict reading frame selection without concomitant selection for protein solubility or folding. This plasmid incorporates the cis-splicing VMA intein sequence from Saccharomyces cerevisiae to facilitate the post-translational self-excision of the protein of interest, thereby eliminating potential aggregation problems. Results from two libraries of chimeric glycinamide ribonucleotide formyltransferases confirm the superior performance of pInSALect over existing reading frame selection systems.     

Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance

Both freezing tolerance and NaCI tolerance are improved whenantifreeze proteins are expressed as fusion proteins with twodomains of staphylococcal protein A (SPA) in Escherichia coli.To characterize these properties further we created a randomlymutated expression library in E.coli, based on the winter flounderantifreeze protein HPLC-8 component gene. Low-fidelity PCR productsof this gene were fused to the spa gene encoding two domainsof the SPA. The library was screened for enhanced NaCl toleranceand four clones were selected. The freezing tolerance of eachof the selected clones was enhanced to varying extents. DNAsequencing of the isolated mutants revealed that the amphiphilicproperties of the native antifreeze protein were essentiallyconserved. Furthermore, by studying the primary sequence ofthe randomly mutated clones, in comparison with the degree offreezing tolerance, we have identified clues which help in understandingthe relationship between salt and freezing tolerance.     

A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T50) of the most stable variant, 4-43D6, is increased by 11-12 degrees C over the wild-type enzymes and the half-life of activity at 53 degrees C is increased approximately 190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.     

Directed evolution of PDZ variants to generate high-affinity detection reagents

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.     

A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries

The in vitro MutaGen procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol beta and pol eta. Three large libraries (>10(5) independent clones) were generated (one with pol beta and two with pol eta). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol beta were 4-7-fold less mutated than those created with pol eta, indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol beta and pol eta provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol eta displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen thus appears as a very useful tool for gene and protein randomisation.     

Digital screening methodology for the directed evolution of transglycosidases

Engineering of glycosidases with efficient transglycosidasesactivity is an alternative to glycosyltransferases or glycosynthasesfor the synthesis of oligosaccharides and glycoconjugates. However,the engineering of transglycosidases by directed evolution methodologiesis hampered by the lack of efficient screening systems for sugar-transferactivity. We report here the development of digital imaging-basedhigh-throughput screening methodology for the directed evolutionof glycosidases into transgalactosidases. Using this methodology,we detected transglycosidase mutants in intact Escherichia colicells by digital imaging monitoring of the activation of non-or low-hydrolytic mutants by an acceptor substrate. We screenedseveral libraries of mutants of β-glycosidase from Thermusthermophilus using this methodology and found variants withup to a 70-fold overall increase in the transglycosidase/hydrolysisactivity ratio. Using natural disaccharide acceptors, thesetransglycosidase mutants were able to synthesise trisaccharides,as a mixture of two regioisomers, with up to 76% yield.     

Probing the structural plasticity of an archaeal primordial cobaltochelatase CbiX(S)

Insertion of metal ions into tetrapyrrole macrocycles is catalyzed by a diverse group of enzymes called chelatases. Structures are known for several chelatases catalyzing metal insertion into protoporphyrin IX or sirohydrochlorin. Despite a lack of significant amino acid sequence similarity, these ferro- and cobaltochelatases share a high degree of structural similarity. Cobaltochelatase CbiK and ferrochelatase HemH are bilobial enzymes with two alpha/beta domains, which were suggested to origin from a common ancestral protein via gene duplication. Small, single-domain chelatases (CbiX(S)) were recently described in archaea and are believed to represent primordial chelatases. Here, we tested the structural plasticity of an archaeal cobaltochelatase CbiX(S) by rearranging its structure with a novel method producing random in-frame deletions, duplications and insertions. A number of functional chelatase variants with insertion of duplicated sequence stretches, encompassing from one to nine secondary structural elements, were obtained. CbiX(S) was found to tolerate large sequence rearrangements in four out of the nine loop regions of the protein, indicating a high degree of structural plasticity. The predicted topologies of two variants (M51 and M518) are strikingly similar to CbiK and HemH, suggesting that we recreated duplication events that are believed to have created the bilobial chelatases.     

Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation

We have developed an efficient optimization technique, 'biased mutation-assembling', for improving protein properties such as thermostability. In this strategy, a mutant library is constructed using the overlap extension polymerase chain reaction technique with DNA fragments from wild-type and phenotypically advantageous mutant genes, in which the number of mutations assembled in the wild-type gene is stochastically controlled by the mixing ratio of the mutant DNA fragments to wild-type fragments. A high mixing ratio results in a mutant composition biased to favor multiple-point mutants. We applied this strategy to improve the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as a case study and found that the proportion of thermostable mutants in a library increased as the mixing ratio was increased. If the proportion of thermostable mutants increases, the screening effort needed to find them should be reduced. Indeed, we isolated a mutant with a 1200-fold longer activity half-life at 60 degrees C than that of wild-type prolyl endopeptidase after screening only 2000 mutants from a library prepared with a high mixing ratio. Our results indicate that an aggressive accumulation of advantageous mutations leads to an increase in the quality of the mutant library and a reduction in the screening effort required to find superior mutants.     

Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase

Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E.     

Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant

One of the main obstacles in employing P450 monooxygenases for preparative chemical syntheses in cell-free systems is their requirement for cofactors such as NAD(P)H. In order to engineer P450 BM3 from Bacillus megaterium for cost-effective process conditions in vitro, a validated medium throughput screening system based on cheap Zn dust as an electron source and Cobalt(III)sepulchrate (Co(III)sep) as a mediator was reported. In the current study, the alternative cofactor system Zn/Co(III)sep was used in a directed evolution experiment to improve the Co(III)sep-mediated electron transfer to P450 BM3. A variant, carrying five mutations (R47F F87A V281G M354S D363H, Table I), P450 BM3 M5 was identified and characterized with respect to its kinetic parameters. P450 BM3 M5 achieved for mediated electron transfer a 2-fold higher k(cat) value and a 3-fold higher catalytic efficiency compared with the starting point mutant P450 BM3 F87A (k(cat): 62 min(-1) compared with 28 min(-1); k(cat)/K(m): 62 microM(-1)min(-1) compared to 19 microM(-1)min(-1)). For obtaining first insights on electron transfer contributions, three reductase-deficient variants were generated. The reductase-deficient mutant of P450 BMP M5 exhibited a catalytic efficiency of 69% and a k(cat) value of 89% of the values obtained for P450 BM3 M5.     

Pairwise iterative superposition of distantly related proteins and assessment of the significance of 3-D structural similarity

A challenge lies in identifying distant protein 3-D structuralsimilarity by rigid-body superposition. The most common measureof structural similarity is r.m.s. distance (r.m.s.d.) betweentopologically equivalent residues, and most automated methodsof protein modelling rely on the assembly of rigid fragmentsfrom known 3-D structures. A fast method of improving the definitionof a common protein fold by superposition, especially for distantrelationships, is described. The definition of topological equivalenceby the standard dynamic programming sequence alignment algorithmis extended by refining the entire structure alignment (notjust those equivalenced residues within a given cut-off distance)and determining whether the alignment can be continued at thetermini. The most appropriate distance-based definition of topologicalequivalence for a given comparison is identified. Despite thefact that hitherto the distant similarity between the globinfold and colicin A has not been recognized directly by rigid-bodysuperposition, this new approach defines more equivalent residueswith a lower r.m.s.d. between them than that obtained by thesuperposition of equivalences identified by a more elaboratemethod. A previous distance metric of 3-D structural similarityderived from rigid-body superposition has been extended to theassessment of superpositions where topological equivalenceshave been determined by methods other than rigid-body ones.     

Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation

Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis is synthesized in a prepro-form (prepro-Tk-subtilisin), secreted in a pro-form (pro-Tk-subtilisin), and matured to an active form (mat-Tk-subtilisin*; a Ca(2+)-bound active form of matured domain of Tk-subtilisin) upon autoprocessing and degradation of the propeptide [Tk-propeptide; propeptide of Tk-subtilisin (Gly1-Leu69)]. Pro-Tk-subtilisin exhibited halo-forming activity only at 80 degrees C, but not at 70 and 60 degrees C, because Tk-propeptide is not effectively degraded by mat-Tk-subtilisin* and forms an inactive complex with mat-Tk-subtilisin* at <80 degrees C. Random mutagenesis in the entire prepro-Tk-subtilisin gene, followed by screening for mutant proteins with halo-forming activity at 70 and 60 degrees C, allowed us to identify single Gly56 --> Ser mutation in the propeptide region responsible for low-temperature adaptation of pro-Tk-subtilisin. SDS-PAGE analyses and mat-Tk-subtilisin* activity assay of pro-G56S-subtilisin indicated more rapid maturation than pro-Tk-subtilisin. The resultant active form was indistinguishable from mat-Tk-subtilisin* in activity and stability, indicating that Gly56 --> Ser mutation does not seriously affect the folding of the mature domain. However, this mutation greatly destabilized the propeptide, making it unstructured in an isolated form. As a result, Tk-propeptide with Gly56 --> Ser mutation (G56S-propeptide) was more susceptible to proteolytic degradation and less effectively inhibited mat-Tk-subtilisin* activity than Tk-propeptide. These results suggest that pro-G56S-subtilisin is more effectively matured than pro-Tk-subtilisin at lower temperatures, because autoprocessed G56S-propeptide is unstructured upon dissociation from mat-Tk-subtilisin* and is therefore effectively degraded by mat-Tk-subtilisin*.     

Selective gene amplification

We describe a system for directed evolution based on in vitro compartmentalisation in which amplification of a gene is coupled to the formation of product by the enzyme it encodes. This approach mimics the process of natural selection; 'fitter' genes--encoding more efficient enzymes--have more 'offspring'. It allows selection for any activity so long as a product-specific ligand (e.g. an antibody) is available.     

Amino acid alphabet size in protein evolution experiments: better to search a small library thoroughly or a large library sparsely?

We compare the results obtained from searching a smaller librarythoroughly versus searching a more diverse, larger library sparsely.We study protein evolution with reduced amino acid alphabets,by simulating directed evolution experiments at three differentalphabet sizes: 20, 5 and 2. We employ a physical model forevolution, the generalized NK model, that has proved successfulin modeling protein evolution, antibody evolution and T-cellselection. We find that antibodies with higher affinity arefound by searching a library with a larger alphabet sparselythan by searching a smaller library thoroughly, even with well-designedreduced libraries. We also find ranked amino acid usage frequenciesin agreement with observations of the CDR-H3 variable regionof human antibodies.     

In vivo selection for the enhancement of Thermotoga maritima exopolygalacturonase activity at neutral pH and low temperature

The aim of this study was to develop an Escherichia coli-based metabolic selection system for the uncovering of new oligogalacturonate-active enzymes. Based on the expression of the specific permease TogMNAB, this system enabled the entry of oligogalacturonates into the cytoplasm of E. coli thus providing a modified strain usable for this purpose. This tool was used for the metabolic selection of Thermotoga maritima exopolygalacturonase (TmGalU) mutants enabling the uptake of sodium trigalacturonate as the sole carbon source by the bacterium. In only one round of error-prone PCR and selection, mutants of TmGalU with a 4-fold increased turnover at pH 7.0 and 2-fold more active at 37 degrees C than wild-type enzyme were isolated. These results show the versatility of this strain for the evolution of oligogalacturonate-active enzymes.