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Biomedical subjects

F H Arnold

Publications and source records attributed to F H Arnold.

At least 19 recordsLinked to original sources
PubMed1999

Directed evolution of biocatalysts.

Directed evolution is being used increasingly in academic and industrial laboratories to modify and improve important biocatalysts. Significant advances during this period of review include compartmentalization of genes and the in vitro translation apparatus in emulsions, as well as several impressive demonstrations of catalyst improvement. Shuffling of homologous genes offers a new way to utilize natural diversity in the evolution of novel catalysts.

Catalysis↗
PubMed1999

Directed evolution converts subtilisin E into a functional equivalent of thermitase.

We used directed evolution to convert Bacillus subtilis subtilisin E into an enzyme functionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris. Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5, whose half-life at 83 degrees C (3.5 min) and temperature optimum for activity (Topt, 76 degrees C) are identical with those of thermitase. The Topt of the evolved enzyme is 17 degrees C higher and its half-life at 65 degrees C is >200 times that of wild-type subtilisin E. In addition, 5-3H5 is more active towards the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-type at all temperatures from 10 to 90 degrees C. Thermitase differs from subtilisin E at 157 amino acid positions. However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable. The eight substitutions, which include known stabilizing mutations (N218S, N76D) and also several not previously reported, are distributed over the surface of the enzyme. Only two (N218S, N181D) are found in thermitase. Directed evolution provides a powerful tool to unveil mechanisms of thermal adaptation and is an effective and efficient approach to increasing thermostability without compromising enzyme activity.

Amino Acids↗
PubMed1998

Directed evolution of a thermostable esterase.

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14 degreesC increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

Amino Acid Substitution↗
PubMed1998

Random-priming in vitro recombination: an effective tool for directed evolution.

A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences is reported. The method involves priming template polynucleotide(s) with random-sequence primers and extending to generate a pool of short DNA fragments which contain a controllable level of point mutations. The fragments are reassembled during cycles of denaturation, annealing and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. Screening or selecting the expressed gene products leads to new variants with improved functions, as demonstrated by the recombination of genes encoding different thermostable subtilisins in order to obtain enzymes more stable than either parent.

Bacillus subtilis↗
PubMed1998

Combinatorial protein design by in vitro recombination.

DNA recombination is a powerful engine for the creation of new phenotypes. Recently, methods for in vitro DNA recombination (DNA shuffling) have been developed and applied to the evolution of novel molecules in the laboratory. An exciting new development is the shuffling of homologous genes to create diversity for directed evolution.

DNA↗
PubMed1998

Molecular evolution by staggered extension process (StEP) in vitro recombination.

We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65 degrees C is 50 times that of wild-type subtilisin E.

Bacillus subtilis↗
PubMed1997

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences.

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.

Carboxylic Ester Hydrolases↗
PubMed1997

Functional and nonfunctional mutations distinguished by random recombination of homologous genes.

We describe a convenient method for distinguishing functional from nonfunctional or deleterious mutations in homologous genes. High fidelity in vitro gene recombination ("DNA shuffling") coupled with sequence analysis of a small sampling of the shuffled library exhibiting the evolved behavior allows identification of those mutations responsible for the behavior in a background of neutral and deleterious mutations. Functional mutations are expected to occur in 100% of the sequenced screened sample; neutral mutations are found in 50% on average, and deleterious mutations do not appear at all. When used to analyze 10 mutations in a laboratory-evolved gene encoding a thermostable subtilisin E, this method rapidly identified the two responsible for the observed protease thermostability; the remaining eight were neutral with respect to thermostability, within the precision of the screening assay. A similar approach, coupled with selection for growth and survival of the host organism, could be used to distinguish adaptive from neutral mutations.

Calorimetry, Differential Scanning↗
PubMed1997

Langmuir monolayer characterization of metal chelating lipids for protein targeting to membranes.

Targeting and organization of proteins on lipid membranes led to applications in both biological and materials sciences. Coordination of membrane-bound metal ions by surface histidine residues provides a general method for targeting of proteins to membrane surfaces. Here we report the Langmuir monolayer properties of a new class of metal-chelating lipids. The lipids utilize the metal chelator iminodiacetate (IDA) as the hydrophilic headgroup, allowing display of divalent transition metal ions on the aqueous side of the membrane. Changes in surface pressure-molecular area isotherms were used to observe metal binding, and an association constant for Cu2+ binding to the IDA lipids of 10(7-8) M-1 was estimated. The ability to control binding site density is important for many applications. The IDA lipid was found to be miscible with both distearoylphosphocholine (DSPC) and 1-stearoyl-2-oleoyl-phosphocholine (SOPC) at most compositions and surface pressures.

Binding Sites↗
PubMed1997

Optimization of DNA shuffling for high fidelity recombination.

A convenient 'DNA shuffling' protocol for random recombination of homologous genes in vitro with a very low rate of associated point mutagenesis (0.05%) is described. In addition, the mutagenesis rate can be controlled over a wide range by the inclusion of Mn2+or Mg2+during DNase I digestion, by choice of DNA polymerase used during gene reassembly as well as how the genes are prepared for shuffling (PCR amplification versus restriction enzyme digestion of plasmid DNA). These protocols should be useful for in vitro protein evolution, for DNA based computing and for structure-function studies of evolutionarily related genes.

Biological Evolution↗
PubMed1997

Optimizing industrial enzymes by directed evolution.

Enzymes can be tailored for optimal performance in industrial applications by directing their evolution in vitro. This approach is particularly attractive for engineering industrial enzymes. We have created an efficient para-nitrobenzyl esterase over six generations of random point mutagenesis and recombination coupled with screening for improved variants. The best clones identified after four generations of sequential random mutagenesis and two generations of random recombination display more than 150 times the p-nitrobenzyl esterase activity of wild type towards loracarbef-p-nitrobenzyl ester in 15% dimethylformamide. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (< 2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.

Carboxylic Ester Hydrolases↗
PubMed1997

Directed evolution of enzyme catalysts.

Directed enzyme evolution has emerged in the past few years as a powerful alternative to rational approaches for engineering biocatalysts. Prerequisites for successful directed evolution are functional expression in a suitable microbial host, a rapid screen for the desired feature(s) and a well-thought-out working strategy for navigating protein landscapes. The rapidly growing body of literature on enzyme evolution in vitro includes techniques for creating and searching combinatorial enzyme libraries, as well as several successful examples of different evolutionary strategies being used.

Biotechnology↗
PubMed1997

Combinatorial protein design: strategies for screening protein libraries.

Powerful strategies for screening protein libraries further strengthen the arguments for applying 'irrational' approaches to understanding and designing new proteins. Developments during the past year include the application of functional complementation and automation to reduce screening loads, as well as the use of computerized data acquisition to characterize whole protein libraries rather than just selected individuals.

Automation↗
PubMed1997

A glucose-sensing polymer.

We have prepared a robust polymer that can be used to measure glucose concentrations in complex biological media. At alkaline pH, the metal-complexing polymer binds glucose and instantly releases protons in proportion to the glucose concentration over a clinically relevant range (0 to 25 mM). The inexpensive polymer is sufficiently selective to provide an easily measurable response to glucose in porcine plasma. The polymer's ability to function at nonphysiological pH (at which the buffer capacity of biological samples is small) makes it possible to design simple and inexpensive sensing devices based on measurement of changes in proton concentration.

Animals↗
PubMed1996

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

Bacterial Proteins↗