A Method for the Selection of Catalytic Activity Using Phage Display and Proximity Coupling

362

372

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication. P olynucleotide polymerases occupy a central role in the maintenance, transmission, and expression of genetic information (1). They also have enabled core technologies of molecular biology like sequencing, PCR, site-directed mutagenesis, and cDNA cloning. However, polymerases available from nature are often not optimally suited for these applications and attempts have been made to tailor polymerase function by using either design or selection strategies. Structural studies have greatly advanced our understanding of polymerase function (2-4) and together with insights gained from site-directed mutagenesis have allowed the rational design of some polymerase variants with improved properties. Among these are polymerases with improved dideoxynucleotide incorporation for cycle-sequencing (5) or increased processivity (6). Other ''designer'' polymerases include truncation variants (7,8), some of which show improved thermostability and fidelity, although at the cost of reduced processivity. Despite these advances, our ability to engineer designer polymerases for specific applications remains limited.Repertoire selection methods have proven to be an effective means to obtain biopolymers with desired properties. Polymerases have been selected successfully for activity by phage display (9) and by complementation of a DNA polymerase I-deficient Escherichia coli strain (10). Careful screening of the complementing polymerase mutants yielded polymerases with a range of interesting properties such as alt...

mentioning

confidence: 99%

Directed evolution of polymerase function by compartmentalized self-replication

Ghadessy

Ong

Holliger

2001

362

372

“…For example, with water-in-oil emulsion technology (14), Holliger and coworkers (15) evolved DNA polymerases that were either more thermally stable or more resistant to an inhibitor. Winters and coworkers (16) demonstrated that phage pIII-displayed DNA polymerases could be isolated based on their activity-dependent modification of an attached substrate. However, the substrate was attached to the major phage coat protein, pVIII, raising the concern of cross-reactivity between a polymerase on one phage and a substrate attached to another phage.…”

mentioning

confidence: 99%

Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

Xia

Chen

Sera

et al. 2002

151

118

The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template͞primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wildtype enzyme incorporates dNTP substrates.

“…Only opal stop codon-containing clones were selected from the library, whereas, in their absence, almost exclusively chimeric gene fusions leading to a frameshift between the barnase and p3 genes were selected (unpublished data). Phage was prepared by using the helper phage KM13 (27), which encodes a trypsin-sensitive p3 mutant, to reduce contributions to infectivity from phage that do not display the fusion protein (28).…”

Section: Methodsmentioning

confidence: 99%

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

Riechmann

Winter

2000

Self Cite

It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and͞or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a ␤-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the proteaseresistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.