Acinetobacter baylyi ADP1 is naturally competent and proficient at homologous recombination, so it can be transformed without restriction digests or ligation reactions. Expression vectors for this system, however, are not yet widely available. Here we describe the construction and characterization of inducible expression vectors that replicate as plasmids in A. baylyi or integrate into a nonessential part of its chromosome. These tools will facilitate the engineering of genes and genomes in this promising model organism.
Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase. Chimeric primers containing plasmid sequence at the 5′ ends and insert sequence at the 3′ ends were used to PCR-amplify insertion sequences of various sizes, namely the genes for GFP (gfp), β-D-glucuronidase (gusA), and β-galactosidase (lacZ), as well as the entire luxABCDE operon. These inserts were employed as mega-primers in a second PCR with a circular plasmid template. The original plasmid templates were then destroyed in restriction digests with DpnI, and the overlap extension PCR products were used to transform competent Escherichia coli cells. Phusion DNA polymerase was used for the amplification and fusion reactions, so both reactions were easy to monitor and optimize.
The Calvin Cycle is the primary conduit for the fixation of carbon dioxide into the biosphere; ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the rate-limiting fixation step. Our goal is to direct the evolution of RuBisCO variants with improved kinetic and biophysical properties. The Calvin Cycle was partially reconstructed in Escherichia coli; the engineered strain requires the Synechococcus PCC6301 RuBisCO for growth in minimal media supplemented with a pentose. We randomly mutated the gene encoding the large subunit of RuBisCO (rbcL), co-expressed the resulting library with the small subunit (rbcS) and the Synechococcus PCC7492 phosphoribulokinase (prkA), and selected hypermorphic variants. The RuBisCO variants that evolved during three rounds of random mutagenesis and selection were over-expressed, and exhibited 5-fold improvement in specific activity relative to the wild-type enzyme. These results demonstrate a new strategy for the artificial selection of RuBisCO and other non-native metabolic enzymes.
Plasmodium vivax and P. falciparum cause malaria, so proteins essential for their survival in vivo are potential anti-malarial drug targets. Adenosine deaminases (ADA) catalyze the irreversible conversion of adenosine into inosine, and play a critical role in the purine salvage pathways of Plasmodia and their mammalian hosts. Currently, the number of selective inhibitors of Plasmodium ADAs is limited. One potent and widely used inhibitor of the human ADA (hADA), erythro-9-(2-hydroxy-3-nonly)adenine (EHNA), is a very weak inhibitor (Ki = 120uM) of P. falciparum ADA (pfADA). EHNA-like compounds are thus excluded from consideration as potential inhibitors of Plasmodium ADA in general. However, EHNA activity in P. vivax ADA (pvADA) has not been reported. Here we applied computational molecular modeling to identify the mechanisms of the ligand recognition unique for P. vivax and P. falciparum ADA. Based on the computational studies, we performed molecular biology experiments to show that EHNA is at least 60-fold more potent against pvADA (Ki = 1.9uM) than against pfADA. The D172A pvADA mutant is bound even more tightly (Ki = 0.9uM). These results improve our understanding of the mechanisms of ADA ligand recognition and species-selectivity, and facilitate the rational design of novel EHNA-based ADA inhibitors as anti-malarial drugs. To demonstrate a practical application of our findings we have computationally predicted a novel potential inhibitor of pvADA selective versus the human ADA.
The prevalence of paralogous enzymes implies that novel catalytic functions can evolve on preexisting protein scaffolds. The weak secondary activities of proteins, which reflect catalytic promiscuity and substrate ambiguity, are plausible starting points for this evolutionary process. In this study, we observed the emergence of a new enzyme from the ASKA collection of Escherichia coli open reading frames (ORFs). The over-expression of (His)6-tagged glutamine phosphoribosylpyrophosphate amidotransferase (PurF) unexpectedly rescued a ΔtrpF E. coli strain from starvation on minimal media. The wild-type PurF and TrpF enzymes are unrelated in sequence, tertiary structure or catalytic mechanism. The promiscuous phosphoribosylanthranilate isomerase (PRAI) activity of the ASKA PurF variant apparently stems from a pre-existing affinity for phosphoribosylated substrates. The relative fitness of the (His)6-PurF/ΔtrpF strain was improved 4.8-fold to nearly wild-type levels by random mutagenesis of purF and genetic selection. The evolved and ancestral PurF proteins were purified and reacted with phosphoribosylanthranilate in vitro. The best evolvant (kcat/KM = 0.3 s−1.M−1) was ~25-fold more efficient than its ancestor, but >107–fold less efficient than the wild-type PRAI. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms.
Our objective is to produce a protein biosensor (or molecular switch) that is specifically activated in solution by a monoclonal antibody. Many effector-dependent enzymes have evolved in nature, but the introduction of a novel regulatory mechanism into a normally unregulated enzyme poses a difficult design problem. We used site-saturation mutagenesis and screening to generate antibody-activated variants of the reporter enzyme beta-glucuronidase (GUS). The specific activity of the purified epitope-tagged GUS variant was increased by up to ~500-fold by the addition an equimolar concentration of a monoclonal antibody. This molecular switch is modular in design, so it can easily be re-engineered for the detection of other peptide-specific antibodies. Such antibody-activated reporters could someday enable point-of-care serological assays for the rapid detection of infectious diseases.
Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli β-galactosidase (BGAL) variants with enhanced β-fucosidase activity (tenfold increase in kcat/KM in reactions with the novel para-nitrophenyl-β-d-fucopyranoside substrate; 39-fold decrease in reactivity with the “native” para-nitrophenyl-β-d-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of β-fucosidases that are significantly more active (180-fold increase in kcat/KM in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Site-saturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway.
The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including β-galactosidase, alkaline phosphatase, and β-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be “reprogrammed” for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.
Our goal is to understand how enzymes adapt to utilize novel substrates. We and others have shown that directed evolution tends to generate enzyme variants with broadened substrate specificity. Broad-specificity enzymes are generally deleterious to living cells, so this observed trend might be an artifact of the most commonly employed high throughput screens. Here, we demonstrate a more natural and effective screening strategy for directed evolution. The gene encoding model enzyme HIV protease was randomly mutated, and the resulting library was expressed in Escherichia coli cells to eliminate cytotoxic broad-specificity variants. The surviving variants were screened for clones with activity against a reporter enzyme. The wild-type human immunodeficiency virus type I protease (HIV PR) is cytotoxic and exhibits no detectable activity in reactions with beta-galactosidase (BGAL). In contrast, the selected variants were nontoxic and exhibited greater activity and specificity against BGAL than did the wild-type HIV PR in reactions with any substrate. A single round of whole gene random mutagenesis and conventional high-throughput screening does not usually effect complete inversions of substrate specificity. This suggests that a combination of positive and purifying selection engenders more rapid adaptation than positive selection alone.
Our long-term goal is to direct the evolution of novel protease variants. To this end we have engineered a new type of protease-activated reporter enzyme. Many protease-activated enzymes evolved in nature, but the introduction of novel regulatory mechanisms into normally unregulated enzymes poses a difficult design challenge. Random Elongation Mutagenesis [1] was used to fuse the p6 peptide, which is recognized and cleaved by HIV protease, and twelve random sequence amino acids to the C-termini of beta-glucuronidase (GUS) and alkaline phosphatase (AP). The resulting GUS- p6-(NNN)12 and AP-p6-(NNN)12 libraries were expressed in E. coli and screened for clones that were inactivated by the C-terminal extension (tail). The inactivated clones were co-expressed with HIV protease, and those that were re-activated were isolated. The AP and GUS activities of the most responsive clones were each >3.5-fold higher when co-expressed with HIV protease, and this activation is correlated with in vivo proteolysis. It should be possible to generalize this strategy to different reporter enzymes, different target proteases, and perhaps to other types of protein-modifying enzymes.