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At least 721 records · Page 40Linked to original sources
PubMed2006

Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria.

An agar plate freezing assay is described based on the incorporation of fluorescein dye in agar medium. Upon addition of fluorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes.

Agar↗
PubMed2005

A generic system for the Escherichia coli cell-surface display of lipolytic enzymes.

EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.

Amino Acid Sequence↗
PubMed2004

Process implementation aspects for biocatalytic hydrocarbon oxyfunctionalization.

Oxidoreductases catalyze a large variety of regio-, stereo-, and chemoselective hydrocarbon oxyfunctionalizations, reactions, which are important in industrial organic synthesis but difficult to achieve by chemical means. This review summarizes process implementation aspects for the in vivo application of the especially versatile enzyme class of oxygenases, capable of specifically introducing oxygen from molecular oxygen into a large range of organic molecules. Critical issues such as reaching high enzyme activity and specificity, product degradation, cofactor recycling, reactant toxicity, and substrate and oxygen mass transfer can be overcome by biochemical process engineering and biocatalyst engineering. Both strategies provide a growing toolset to facilitate process implementation, optimization, and scale-up. Major advances were achieved via heterologous overexpression of oxygenase genes, directed evolution, metabolic engineering, and in situ product removal. Process examples from industry and academia show that the combined use of different concepts enables efficient oxygenase-based whole-cell catalysis of various commercially interesting reactions such as the biosynthesis of chiral compounds, the specific oxyfunctionalization of complex molecules, and also the synthesis of medium-priced chemicals. Better understanding of the cell metabolism and future developments in both biocatalyst and bioprocess engineering are expected to promote the implementation of many and various industrial biooxidation processes.

Biotechnology↗
PubMed2006

Micro-colony array based high throughput platform for enzyme library screening.

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.

Calibration↗
PubMed2006

Highly efficient ribosome display selection by use of purified components for in vitro translation.

Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. The ribosome display process exploits cell-free translation to achieve coupling of phenotype and genotype by the production of stabilised ribosome complexes in which translated proteins and their encoding mRNA remain attached to the ribosome. Current ribosome display systems that are well proven, by the evolution of high affinity antibodies and the optimisation of defined protein characteristics, use an Escherichia coli cell extract for in vitro translation and display of an mRNA library. Recently, a cell-free translation system has been produced by combining recombinant E. coli protein factors with purified 70S ribosomes. We have applied this development in cell-free translation technology to ribosome display by using the reconstituted system to generate stabilised ribosome complexes for selection. We show that higher cDNA yields are recovered from ribosome display selections when using a reconstituted translation system and the degree of improvement seen is selection specific. These effects are likely to reflect higher mRNA and protein stability and potentially other advantages that may include protein specific improvements in expression. Reconstituted translation systems therefore enable a highly efficient, robust and accessible prokaryotic ribosome display technology.

Amino Acid Sequence↗
PubMed2004

Phage integrases: biology and applications.

Phage integrases are enzymes that mediate unidirectional site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB. Integrases may be grouped into two major families, the tyrosine recombinases and the serine recombinases, based on their mode of catalysis. Tyrosine family integrases, such as lambda integrase, utilize a catalytic tyrosine to mediate strand cleavage, tend to recognize longer attP sequences, and require other proteins encoded by the phage or the host bacteria. Phage integrases from the serine family are larger, use a catalytic serine for strand cleavage, recognize shorter attP sequences, and do not require host cofactors. Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale. These properties give phage integrases growing importance for the genetic manipulation of living eukaryotic cells, especially those with large genomes such as mammals and most plants, for which there are few tools for precise manipulation of the genome. Integrases of the serine family have been shown to work efficiently in mammalian cells, mediating efficient integration at introduced att sites or native sequences that have partial identity to att sites. This reaction has applications in areas such as gene therapy, construction of transgenic organisms, and manipulation of cell lines. Directed evolution can be used to increase further the affinity of an integrase for a particular native sequence, opening up additional applications for genomic modification.

Animals↗
PubMed2003

Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system.

The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering. To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates. In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized. There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme. A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs. Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases. Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.

Base Sequence↗
PubMed2004

Recombination of hybrid target sites by binary combinations of Flp variants: mutations that foster interprotomer collaboration and enlarge substrate tolerance.

Strategies of directed evolution and combinatorial mutagenesis applied to the Flp site-specific recombinase have yielded recombination systems that utilize bi-specific hybrid target sites. A hybrid site is assembled from two half-sites, each harboring a distinct binding specificity. Satisfying the two specificities by a binary combination of Flp variants, while necessary, may not be sufficient to elicit recombination. We have identified amino acid substitutions that foster interprotomer collaboration between partner Flp variants to potentiate strand exchange in hybrid sites. One such substitution, A35T, acts specifically in cis with one of the two partners of a variant pair, Flp(K82M) and Flp(A35T, R281V). The same A35T mutation is also present within a group of mutations that rescue a Flp variant, Flp(Y60S), that is defective in establishing monomer-monomer interactions on the native Flp target site. Strikingly, these mutations are localized to peptide regions involved in interdomain and interprotomer interactions within the recombination complex. The same group of mutations, when transferred to the context of wild-type Flp, can relax its specificity to include non-native target sites. The hybrid Flp systems described here mimic the naturally occurring XerC/XerD recombination system that utilizes two recombinases with distinct DNA binding specificities. The ability to overcome the constraints of binding site symmetry in Flp recombination has important implications in the targeted manipulations of genomes.

Base Sequence↗
PubMed2004

Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase.

Variation in gene sequences generated by directed evolution approaches often does not assure a minimalist design for obtaining a desired property in proteins. While screening for enhanced thermostability, structural information was utilized in selecting mutations that are generated by error-prone PCR. By this approach we have increased the half-life of denaturation by 300-fold compared to the wild-type Bacillus subtilis lipase through three point mutations generated by only two cycles of error-prone PCR. At lower temperatures the activity parameters of the thermostable mutants are unaltered. High-resolution crystal structures of the mutants show subtle changes, which include stacking of tyrosine residues, peptide plane flipping and a better anchoring of the terminus, that challenge rational design and explain the structural basis for enhanced thermostability. The approach may offer an efficient and minimalist solution for the enhancement of a desired property of a protein.

Bacillus subtilis↗
PubMed2005

Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization.

We describe here the selection of ultra-potent anti-respiratory syncytial virus (RSV) antibodies for preventing RSV infection. A large number of antibody variants derived from Synagis (palivizumab), an anti-RSV monoclonal antibody that targets RSV F protein, were generated by a directed evolution approach that allowed convenient manipulation of the binding kinetics. Palivizumab variants with about 100-fold slower dissociation rates or with fivefold faster association rates were identified and tested for their ability to neutralize virus in a microneutralization assay. Our data reveal a major differential effect of the association and dissociation rates on the RSV neutralization, particularly for intact antibodies wherein the association rate plays the predominant role. Furthermore, we found that antibody binding valence also plays a critical role in mediating the viral neutralization through a mechanism that is likely unrelated to antibody size or binding avidity. We applied an iterative mutagenesis approach, and thereafter were able to identify palivizumab Fab variants with up to 1500-fold improvement and palivizumab IgG variants with up to 44-fold improvement in the ability to neutralize RSV. These anti-RSV antibodies likely will offer great clinical potential for RSV immunoprophylaxis. In addition, our findings provide insights into engineering potent antibody therapeutics for other disease targets.

Amino Acid Sequence↗
PubMed2005

Novel strategy for protein exploration: high-throughput screening assisted with fuzzy neural network.

To engineer proteins with desirable characteristics from a naturally occurring protein, high-throughput screening (HTS) combined with directed evolutional approach is the essential technology. However, most HTS techniques are simple positive screenings. The information obtained from the positive candidates is used only as results but rarely as clues for understanding the structural rules, which may explain the protein activity. In here, we have attempted to establish a novel strategy for exploring functional proteins associated with computational analysis. As a model case, we explored lipases with inverted enantioselectivity for a substrate p-nitrophenyl 3-phenylbutyrate from the wild-type lipase of Burkhorderia cepacia KWI-56, which is originally selective for (S)-configuration of the substrate. Data from our previous work on (R)-enantioselective lipase screening were applied to fuzzy neural network (FNN), bioinformatic algorithm, to extract guidelines for screening and engineering processes to be followed. FNN has an advantageous feature of extracting hidden rules that lie between sequences of variants and their enzyme activity to gain high prediction accuracy. Without any prior knowledge, FNN predicted a rule indicating that "size at position L167," among four positions (L17, F119, L167, and L266) in the substrate binding core region, is the most influential factor for obtaining lipase with inverted (R)-enantioselectivity. Based on the guidelines obtained, newly engineered novel variants, which were not found in the actual screening, were experimentally proven to gain high (R)-enantioselectivity by engineering the size at position L167. We also designed and assayed two novel variants, namely FIGV (L17F, F119I, L167G, and L266V) and FFGI (L17F, L167G, and L266I), which were compatible with the guideline obtained from FNN analysis, and confirmed that these designed lipases could acquire high inverted enantioselectivity. The results have shown that with the aid of bioinformatic analysis, high-throughput screening can expand its potential for exploring vast combinatorial sequence spaces of proteins.

Fuzzy Logic↗
PubMed2006

Role of solvation barriers in protein kinetic stability.

The stability of several protein systems of interest has been shown to have a kinetic basis. Besides the obvious biotechnological implications, the general interest of understanding protein kinetic stability is emphasized by the fact that some emerging molecular approaches to the inhibition of amyloidogenesis focus on the increase of the kinetic stability of protein native states. Lipases are among the most important industrial enzymes. Here, we have studied the thermal denaturation of the wild-type form, four single-mutant variants and two highly stable, multiple-mutant variants of lipase from Thermomyces lanuginosa. In all cases, thermal denaturation was irreversible, kinetically controlled and conformed to the two-state irreversible model. This result supports that the novel molecular-dynamics-focused, directed-evolution approach involved in the preparation of the highly stable variants is successful likely because it addresses kinetic stability and, in particular, because heated molecular dynamics simulations possibly identify regions of disrupted native interactions in the transition state for irreversible denaturation. Furthermore, we find very large mutation effects on activation enthalpy and entropy, which were not accompanied by similarly large changes in kinetic urea m-value. From this we are led to conclude that these mutation effects are associated to some structural feature of the transition state for the irreversible denaturation process that is not linked to large changes in solvent accessibility. Recent computational studies have suggested the existence of solvation/desolvation barriers in at least some protein folding/unfolding processes. We thus propose that a solvation barrier (arising from the asynchrony between breaking of internal contacts and water penetration) may contribute to the kinetic stability of lipase from T. lanuginosa (and, possibly, to the kinetic stability of other proteins as well).

Ascomycota↗
PubMed2004

T cell receptors: affinities, cross-reactivities, and a conformer model.

Based on findings with the T cell receptor from mouse CTL clone 2C, and other TCRs, we propose a model that could account for degeneracy of T cell recognition. This "conformer model" holds that a single TCR exists in multiple conformations that are in equilibrium. The model is consistent with (1) the characterization of multiple ligands that bind to the 2C TCR, and other TCRs, (2) the binding properties and predicted structural features of various 2C TCR mutants, including higher affinity variants isolated by directed evolution, and (3) the three-dimensional structures of TCRs and antibodies, with emphasis on the conformational diversity exhibited by proteins with the same primary amino acid sequence. We propose that the advantages of multiple conformers (e.g. ability of a single T cell to recognize many different ligands) outweigh the disadvantages (e.g. lower TCR affinity and possibly T cell sensitivity; detrimental cross-reactivity with structurally unrelated self-ligands).

Animals↗
PubMed2021

Novel human liver-tropic AAV variants define transferable domains that markedly enhance the human tropism of AAV7 and AAV8.

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. Here, we report the bioengineering of a set of next-generation AAV vectors, named AAV-SYDs (where "SYD" stands for Sydney, Australia), with increased human hepato-tropism in a liver xenograft mouse model repopulated with primary human hepatocytes. We followed a two-step process that staggered directed evolution and domain-swapping approaches. Using DNA-family shuffling, we first mapped key AAV capsid regions responsible for efficient human hepatocyte transduction in vivo. Focusing on these regions, we next applied domain-swapping strategies to identify and study key capsid residues that enhance primary human hepatocyte uptake and transgene expression. Our findings underscore the potential of AAV-SYDs as liver gene therapy vectors and provide insights into the mechanism responsible for their enhanced transduction profile.

AAV↗
PubMed2004

Horseradish peroxidase: a modern view of a classic enzyme.

Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism, cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed.

Animals↗
PubMed2004

The interaction of proteins with solid surfaces.

The interaction of proteins with solid surfaces is a fundamental phenomenon with implications for nanotechnology, biomaterials and biotechnological processes. Kinetic and thermodynamic studies have long indicated that significant conformational changes may occur as a protein encounters a surface; new techniques are measuring and modeling these changes. Combinatorial and directed evolution techniques have created new peptide sequences that bind specifically to solid surfaces, similar to the natural proteins that regulate crystal growth. Modeling efforts capture kinetics and thermodynamics on the colloidal scale, but detailed treatments of atomic structure are still in development and face the usual challenges of protein modeling. Opportunities abound for fundamental discovery, as well as breakthroughs in biomaterials, biotechnology and nanotechnology.

Adsorption↗
PubMed2005

Detoxification of organophosphate nerve agents by bacterial phosphotriesterase.

Organophosphates have been widely used as insecticides and chemical warfare agents. The health risks associated with these agents have necessitated the need for better detoxification and bioremediation tools. Bacterial enzymes capable of hydrolyzing the lethal organophosphate nerve agents are of special interest. Phosphotriesterase (PTE) isolated from the soil bacteria Pseudomonas diminuta displays a significant rate enhancement and substrate promiscuity for the hydrolysis of organophosphate triesters. Directed evolution and rational redesign of the active site of PTE have led to the identification of new variants with enhanced catalytic efficiency and stereoselectivity toward the hydrolysis of organophosphate neurotoxins. PTE has been utilized to protect against organophosphate poisoning in vivo. Biotechnological applications of PTE for detection and decontamination of insecticides and chemical warfare agents are developing into useful tools. In this review, the catalytic properties and potential applications of this remarkable enzyme are discussed.

Catalysis↗
PubMed2005

Improving enzyme properties: when are closer mutations better?

Study of mutations that improve enzyme properties reveals that in many, but not all, cases closer mutations are more effective than distant ones. For enantioselectivity, substrate selectivity and new catalytic activity (catalytic promiscuity) closer mutations improved enzymes more effectively than distant ones. However, both close and distant mutations can improve activity, thermal stability and also probably stability toward organic solvents. Typical random mutagenesis methods, such as error-prone PCR, create greater numbers of distant mutations than close mutations because enzymes contain more amino acids distant from the active site than close to the active site. This suggests that instead of mutating the entire enzyme, focusing mutations near the substrate-binding site might dramatically increase the success rate in many directed evolution experiments.

Animals↗