An Engineered Protease that Cleaves Specifically after Sulfated Tyrosine

The bacterial protease OmpT has been engineered to cleave after sulfotyrosine residues in peptide substrates but not after phosphotyrosine (see scheme). A selection/counterselection screen was used to identify OmpT variants with the desired specificity and high levels of overall catalytic activity

In vitro scanning saturation mutagenesis of proteins

The present invention combines PCR.TM. mutagenesis with in vitro transcription/translation and ELISA for the rapid generation and characterization of protein mutants. The PCR.TM. products are used directly as the template for the in vitro transcription/translation reactions and because no cloning steps are required, the in vitro saturation mutagenesis of one residue can be completed in duplicate within a week by a single investigator. This high throughput enables the saturation mutagenesis of numerous residues of interest, a process that can be described as in vitro scanning saturation mutagenesis. Compositions and methods of use of such a process are described herein.Board of Regents, University of Texas Syste

Antibodies with increased affinities for anthrax antigens

The invention overcomes the deficiencies of the prior art by providing antibody compositions having improved affinities for Bacillus anthracis antigens. The compositions have important thereapeutic and diagnostic applications, including treatment or detection of infection by Bacillus anthracis.Board of Regents, University of Texas Syste

Antibodies with increased affinities for anthrax antigens

The invention overcomes the deficiencies of the prior art by providing antibody compositions having improved affinities for Bacillus anthracis antigens. The compositions have important thereapeutic and diagnostic applications, including treatment or detection of infection by Bacillus anthracis.Board of Regents, University of Texas Syste

Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression.

For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries

Highly active and selective endopeptidases with programmed substrate specificities

A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (kcat/KM > 104 M?1 s?1). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including Glu?Arg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic

Subtle Recognition of 14-Base Pair DNA Sequences via Threading Polyintercalation

ABSTRACT: Small molecules that bind DNA in a sequence-specific manner could act as antibiotic, antiviral, or anticancer agents because of their potential ability to manipulate gene expression. Our laboratory has developed threading poly-intercalators based on 1,4,5,8-naphthalene diimide (NDI) units connected in a head-to-tail fashion by flexible peptide linkers. Previously, a threading tetraintercalator composed of alternating minor−major−minor groove-binding modules was shown to bind specifically to a 14 bp DNA sequence with a dissociation half-life of 16 days [Holman, G. G., et al. (2011) Nat. Chem. 3, 875−881]. Herein are described new NDI-based tetraintercalators with a different major groove-binding module and a reversed N to C directionality of one of the minor groove-binding modules. DNase I footprinting and kinetic analyses revealed that these new tetraintercalators are able to discriminate, by as much as 30-fold, 14 bp DNA binding sites that differ by 1 or 2 bp. Relative affinities were found to correlate strongly with dissociation rates, while overall C2 symmetry in the DNA-binding molecule appeared to contribute to enhanced association rates

Phosphoramidite derivatives of texaphyrins

The invention is directed to a method of incorporating expanded porphyrins, and particularly of incorporating a sapphyrin or a texaphyrin, before, during, or after chemical synthesis of an oligomer to form an expanded porphyrin-oligonucleotide conjugate, and particularly a sapphyrin- or texaphyrin-oligonucleotide conjugate. This method includes reacting derivatized nucleotides and a sapphyrin or a texaphyrin in a desired order in an automated or manual DNA synthesizer having a solid support to form a sapphyrin- or a texaphyrin-oligonucleotide conjugate.Board of Regents, University of Texas Syste

Isolation of binding proteins with high affinity to ligands

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides via “display-less” library screening. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in soluble form in the periplasmic space of gram negative bacteria, such as Escherichia coli, and are mixed with a labeled ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the concentration of the labeled ligand bound to the binding protein is increased and allows the cells to be isolated from the rest of the library. Where fluorescent labeling of the target ligand is used, cells may be isolated by fluorescence activated cell sorting (FACS). The approach is more rapid than prior art methods and avoids problems associated with the surface-expression of ligand fusion proteins employed with phage display.Board of Regents, University of Texas Syste

Method for separating molecules

The present invention provides various novel covalently modified sapphyrin derivatives and conjugates; polymers including sapphyrin or derivatives thereof; and chromatographic supports including sapphyrins and other expanded porphyrins and derivatives thereof. Disclosed are water soluble sapphyrins, including polyhydroxysapphyrins and sapphyrin-sugar derivatives; sapphyrin-metal chelating conjugates; sapphyrin nucleobase conjugates; oligosapphyrins and polysapphyrins, including sapphyrin dimers, trimers, oligomers and higher polymers; and polymer supported expanded porphyrin compositions, including advantageous rubyrin- and sapphyrin-based chromatography columns and electrophoretic supports. Sapphyrin oligomers and polymers and polymer supported expanded porphyrins, such as, e.g., glass and silica expanded porphyrin constructs, are disclosed which include both repeating units of sapphyrin derivatives alone and which include other units, for example, nucleobases, sapphyrin-nucleobase conjugates and long chain alkyl groups.Board of Regents, University of Texas Syste