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PubMed2003

mRNA display: ligand discovery, interaction analysis and beyond.

In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides.

Adenosine Triphosphate↗
PubMed1999

Incremental truncation as a strategy in the engineering of novel biocatalysts.

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.

Amino Acid Motifs↗
PubMed2003

Cloning, over-expression, purification, and characterisation of N-acetylneuraminate synthase from Streptococcus agalactiae.

N-acetylneuraminate synthase (NeuAc-synthase; E.C. 4.1.3.19) is one of the two enzymes responsible for sialic acid (N-acetylneuraminic acid) synthesis in bacteria. Potential genes encoding NeuAc synthase in Streptococcus agalactiae and Bacillus subtilis were identified from a BLAST search of the EMBL/GenBank/DDBJ database using the E. coli neuB gene sequence as a probe and the genes cloned and expressed at high level in Escherichia coli. The neuB gene of S. agalactiae was shown to encode an active NeuAc synthase, whereas the spsE gene product from B. subtilis did not have this activity. Expression of the native S. agalactiae neuB gene product enzyme in E. coli resulted in a product that was prone to proteolysis during purification so the protein was tagged with a hexa-histidine tag at its N-terminus and the enzyme was rapidly purified to homogeneity by ammonium sulphate fractionation and Ni-chelating affinity chromatography in two steps. Measurement of the subunit molecular mass by electrospray ionisation mass spectrometry (M(r) = 38, 987 +/- 3) and of the native molecular mass by gel filtration chromatography (M(r) = 78,000) clearly demonstrated that the enzyme is dimeric. The effects of EDTA, temperature, and pH on the activity of the S. agalactiae NeuAc synthase were examined. Enzyme activity was maximal at pH 7 and was dependent on the presence of metal ions such as Mg(2+), Mn(2+) or Co(2+). The purified enzyme was inhibited by the reagent phenylglyoxal and the substrates N-acetyl mannosamine or phosphoenol pyruvate afforded protection against this inhibition, suggesting that one or more arginine residues are involved in substrate recognition and binding. The ease of expression and the properties of the enzyme should now permit a thorough study of the specificity of the enzyme and provide the prerequisites for attempts to alter this specificity by directed evolution for the production of novel sialic acid analogues.

Ammonium Sulfate↗
PubMed2001

Receptor-mediated gene transfer by phage-display vectors: applications in functional genomics and gene therapy.

Recent studies have demonstrated targeted gene-delivery to mammalian cells using modified phage-display vectors. Specificity is determined by the choice of the genetically displayed targeting ligand. Without targeting, phage particles have virtually no tropism for mammalian cells. Thus, novel ligands can be selected from phage libraries by their ability to deliver a reporter gene to targeted cells. Together with advances in cDNA display technologies, these findings offer new opportunities for the use of phage-display technology in functional genomics. In addition, targeted phage particles have potential as alternative gene therapy vectors that can be further improved using directed evolution.

Journal Article↗
PubMed2002

Incorporation of non-natural amino acids into proteins.

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.

Acylation↗
PubMed1999

Extremozymes.

Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. From recent work, major approaches to extending the range of applications of extremozymes have emerged. Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications. Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.

Bacteria↗
PubMed2001

Metabolic engineering as an integrating platform for strain development.

Integration of the analytical framework and experimental tools of metabolic engineering with emerging technologies such as DNA microarrays and directed evolution stands to dramatically improve the approaches by which strain improvement and biocatalyst design are pursued in the future. Progress in genomics and applied molecular biology, together with increasing emphasis on renewable resource utilization for chemical production, has advanced metabolic engineering to the forefront of biotechnological interest.

Industrial Microbiology↗
PubMed1999

Commodity scale production of sugars from starches.

The production of sugars from starch sources is an industry that exists in its present form due to the application of industrial enzymology to solve process related problems. As the industry matures, the demand for more efficient enzymes leading to higher quality products and lower production costs for the starch processor has increased. Researchers are now finding or tailoring enzymes for specific operational needs of the processor using a combination of tools such as protein engineering, directed evolution and improved accessing of natural diversity.

Aspergillus↗
PubMed2004

Polarimetric assay for the medium-throughput determination of alpha-amino acid racemase activity.

A polarimetric assay has been developed for the identification of alpha-amino acid racemase activity. The setup consists of a microcuvette polarimeter (40 microL volume) connected to a pipetting robot for microtiter plates, a pump, and data processing. It could be demonstrated for a glutamate racemase from Lactobacillus fermentii, expressed in Escherichia coli, serving as model enzyme, that its activity can be determined from the time-dependent change of the optical rotation using l-glutamate as substrate. Thus, the specific activity was determined to 111.4 mdeg/min which corresponds to 45.7 micromol/min per mg purified enzyme. Moreover, a protocol was developed that allows the measurement of racemase activity from 96-well microtiter plates using purified enzymes. Thus, the method described can be used to determine racemase activity in an automatic manner. It should be also applicable for the screening of enzyme libraries created by directed evolution.

Amino Acid Isomerases↗
PubMed2002

Crystal structure of D-hydantoinase from Bacillus stearothermophilus: insight into the stereochemistry of enantioselectivity.

Industrial production of antibiotics, such as semisynthetic penicillins and cephalosporins, requires optically pure D-p-hydroxylphenylglycine and its derivatives as important side-chain precursors. To produce optically pure D-amino acids, microbial D-hydantoinase (E.C. 3.5.2.2) is used for stereospecific hydrolysis of chemically synthesized cyclic hydantoins. We report the apo-crystal structure of D-hydantoinase from B. stearothermophilus SD1 at 3.0 A resolution. The structure has a classic TIM barrel fold. Despite an undetectable similarity in sequence, D-hydantoinase shares a striking structural similarity with the recently solved structure of dihydroorotase. A structural comparison of hydantoinase with dihydroorotase revealed that the catalytic chemistry is conserved, while the substrate recognition is not. This structure provides insight into the stereochemistry of enantioselectivity in hydrolysis and illustrates how the enzyme recognizes stereospecific exocyclic substituents and hydrolyzes hydantoins. It should also provide a rationale for further directed evolution of this enzyme for hydrolysis of new hydantoins with novel exocyclic substituents.

Amidohydrolases↗
PubMedDate not supplied

Imaging polarimetry for high throughput chiral screening.

Imaging polarimetry was demonstrated as a highly parallel method of determining optical rotation of biochemical samples. The imaging polarimeter utilized a bright, uniform light source wavelength-filtered to near the sodium D line, a sample array flanked by inlet and analyzing polarizers, and a CCD camera fitted with an equal-perspective telecentric lens. The prototype apparatus was demonstrated to have an optical resolution better than 0.08 degrees. The potential for high throughput screening was demonstrated by imaging chiral solutions in 1536-well microtiter plates and by real-time monitoring of 30 simultaneous chiral enzymatic reactions. Improvements in polarizer and CCD technology may broadly expand the technique's applicability to fields such as directed evolution and combinatorial chemistry, where screening throughput is currently limiting for chiral applications.

Algorithms↗
PubMed2002

Structural chemistry of a green fluorescent protein Zn biosensor.

We designed a green fluorescent protein mutant (BFPms1) that preferentially binds Zn(II) (enhancing fluorescence intensity) and Cu(II) (quenching fluorescence) directly to a chromophore ligand that resembles a dipyrrole unit of a porphyrin. Crystallographic structure determination of apo, Zn(II)-bound, and Cu(II)-bound BFPms1 to better than 1.5 A resolution allowed us to refine metal centers without geometric restraints, to calculate experimental standard uncertainty errors for bond lengths and angles, and to model thermal displacement parameters anisotropically. The BFPms1 Zn(II) site (KD = 50 muM) displays distorted trigonal bipyrimidal geometry, with Zn(II) binding to Glu222, to a water molecule, and tridentate to the chromophore ligand. In contrast, the BFPms1 Cu(II) site (KD = 24 muM) exhibits square planar geometry similar to metalated porphyrins, with Cu(II) binding to the chromophore chelate and Glu222. The apo structure reveals a large electropositive region near the designed metal insertion channel, suggesting a basis for the measured metal cation binding kinetics. The preorganized tridentate ligand is accommodated in both coordination geometries by a 0.4 A difference between the Zn and Cu positions and by distinct rearrangements of Glu222. The highly accurate metal ligand bond lengths reveal different protonation states for the same oxygen bound to Zn vs Cu, with implications for the observed metal ion specificity. Crystallographic anisotropic thermal factor analysis validates metal ion rigidification of the chromophore in enhancement of fluorescence intensity upon Zn(II) binding. Thus, our high-resolution structures reveal how structure-based design has effectively linked selective metal binding to changes in fluorescent properties. Furthermore, this protein Zn(II) biosensor provides a prototype suitable for further optimization by directed evolution to generate metalloprotein variants with desirable physical or biochemical properties.

Biosensing Techniques↗
PubMed2005

Paralog-selective ligands for bcl-2 proteins.

There is considerable current interest in molecules that bind intra- or extracellular protein surfaces and inhibit protein-protein interactions. Previously we have reported that miniature proteins based on pancreatic-fold polypeptides can recognize even shallow alpha-helix binding clefts with high affinity and selectivity against unrelated proteins. One such miniature protein, PPBH3-1, binds the anti-apoptotic protein paralogs Bcl-2 and Bcl-XL with nanomolar affinity and a DeltaDeltaG = 1.2 kcal.mol-1 preference for Bcl-XL. Here we describe the directed evolution of PPBH3-1 into two new miniature proteins, PPBH3-5 and PPBH3-6, whose paralog specificity is reversed relative to PPBH3-1. PPBH3-5 and PPBH3-6 bind Bcl-2 with nanomolar affinity and a DeltaDeltaG = 0.9-1.3 kcal.mol-1 preference for Bcl-2 over Bcl-XL. Experiments with Bcl-XL variants suggest that PPBH3-5 and PPBH3-6 achieve high paralog specificity by exploiting subtle structural or electrostatic differences in the Bcl-2 and Bcl-XL molecular landscapes. PPBH3-5 and PPBH3-6 may have unique applications as early examples of nonnatural ligands that interact selectively with Bcl-2 proteins.

Amino Acid Sequence↗
PubMed2006

A minimalist approach toward protein recognition by epitope transfer from functionally evolved beta-sheet surfaces.

New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small beta-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the beta-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of beta-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, beta-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 A was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the beta-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 A linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action.

Amino Acid Sequence↗
PubMed2006

Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents.

Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.

Acetylcholinesterase↗
PubMed2003

Chromosomal characteristics of rDNA in European grayling Thymallus thymallus (Salmonidae).

The chromosomal characteristics, locations and variations of two classes of ribosomal DNA (5S and 18S) were studied in European grayling karyotype (Thymallus thymallus, Salmonidae). Major rDNA sites as revealed by sequential CMA3/Ag staining and confirmed by in situ hybridization with a 18S rDNA probe were situated in two loci and were found to be polymorphic in size and displaying several distinct forms. The 5S rDNA was located by PRINS on three pairs of subtelocentric chromosomes, additional minor signal was present at the centromere of one metacentric element. 5S sites were not associated with NORs. The dosage compensation mechanism was proposed as an explanation of high frequency of lethal rDNA-deleted forms of the NOR-bearing chromosomes. Double variable pattern in the number and location of NORs supported the bi-directional evolution of salmonid rDNA loci.

Animals↗
PubMed2016

Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.

No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.

Animals↗