Mapping of polyglutamylation in tubulins using nanoLC-ESI-MS/MS.

Tubulin polyglutamylation is a polymeric modification that extends from the carboxyl-terminus of tubulins. Molecular description of amino acids and their branching polyglutamyls is a hallmark of tubulin in microtubules. There are different chemical approaches for detecting these polymeric structures, mostly reported prior to development of nESI peptide analysis.

Solid-phase enzyme catalysis of DNA end repair and 3' A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA.

The use of next-generation sequencing (NGS) has been instrumental in advancing biological research and clinical diagnostics. To fully utilize the power of NGS, complete, uniform coverage of the entire genome is required. In this study, we identified the primary sources of bias observed in sequence coverage across AT-rich regions of the human genome with existing amplification-free DNA library preparation methods.

Enhancing Multistep DNA Processing by Solid-Phase Enzyme Catalysis on Polyethylene Glycol Coated Beads.

Covalent immobilization of enzymes on solid supports provides an alternative approach to homogeneous biocatalysis by adding the benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized (IM) enzymes generally suffer from reduced activity compared to their soluble counterparts. The nature and hydrophobicity of the supporting material surface can introduce enzyme conformational change, spatial confinement, and limited substrate accessibility, all of which will result in loss of the immobilized enzyme activity.

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D.

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E.

Genetically encoded fragment-based discovery of glycopeptide ligands for carbohydrate-binding proteins.

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD.

Biochemical characterization of a Naegleria TET-like oxygenase and its application in single molecule sequencing of 5-methylcytosine.

Modified DNA bases in mammalian genomes, such as 5-methylcytosine ((5m)C) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic conversion of (5m)C to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like (5m)C oxygenase (NgTET1) from Naegleria gruberi, a single-celled protist evolutionarily distant from vertebrates.

Multivalent site-specific phage modification enhances the binding affinity of receptor ligands.

High-throughput screening of combinatorial chemical libraries is a powerful approach for identifying targeted molecules. The display of combinatorial peptide libraries on the surface of bacteriophages offers a rapid, economical way to screen billions of peptides for specific binding properties and has impacted fields ranging from cancer to vaccine development. As a modification to this approach, we have previously created a system that enables site-specific insertion of selenocysteine (Sec) residues into peptides displayed pentavalently on M13 phage as pIII coat protein fusions.

Chemical posttranslational modification of phage-displayed peptides.

Phage-displayed peptide library has fueled the discovery of novel ligands for diverse targets. A new type of phage libraries that displays not only linear and disulfide-constrained cyclic peptides but moieties that cannot be encoded genetically or incorporated easily by bacterial genetic machinery has emerged recently. Chemical posttranslational modification of phage library is one of the simplest approaches to encode nonnatural moieties.

Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries.

A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.

Expanding the genetic code of Escherichia coli with phosphoserine.

O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNA(Sep)), its cognate Sep-tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNA(Sep) binding.

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes.

Photocatalysts: splitting water with viruses.

A bacteriophage can be used as a template for assembling catalytic nanostructures for the light-driven oxidation of water molecules.

The appearance of pyrrolysine in tRNAHis guanylyltransferase by neutral evolution.

tRNA(His) guanylyltransferase (Thg1) post-transcriptionally adds a G (position -1) to the 5'-terminus of tRNA(His). The Methanosarcina acetivorans Thg1 (MaThg1) gene contains an in-frame TAG (amber) codon. Although a UAG codon typically directs translation termination, its presence in Methanosarcina mRNA may lead to pyrrolysine (Pyl) incorporation achieved by Pyl-tRNA(Pyl), the product of pyrrolysyl-tRNA synthetase.

RNA polymerases.

This unit describes DNA-dependent, RNA-dependent, and template-independent RNA polymerases. DNA-dependent RNA polymerases include the related bacteriophage T7, T3, and SP6 polymerases, the most commonly used RNA polymerases for in vitro transcription reactions. Reaction conditions to produce preparative quantities of transcribed RNA and labeled RNA probes are covered, as are the major applications of these reactions.

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide.

Single M13 bacteriophage tethering and stretching.

The ability to present biomolecules on the highly organized structure of M13 filamentous bacteriophage is a unique advantage. Where previously this viral template was shown to direct the orientation and nucleation of nanocrystals and materials, here we apply it in the context of single-molecule (SM) biophysics. Genetically engineered constructs were used to display different reactive species at each of the filament ends and along the major capsid, and the resulting hetero-functional particles were shown to consistently tether microscopic beads in solution.

Revised Escherichia coli selenocysteine insertion requirements determined by in vivo screening of combinatorial libraries of SECIS variants.

To investigate the stringency of the Escherichia coli selenocysteine insertion sequence (SECIS) requirements, libraries of SECIS variants were screened via a novel method in which suppression of the selenocysteine (Sec) opal codon was coupled to bacteriophage plaque formation. The SECIS variant libraries were designed with a mostly paired lower stem, so that randomization could be focused on the upper stem and loop regions. We identified 19 functional non-native SECIS sequences that violated the expected pairing requirements for the SECIS upper stem.

Creation of an artificial bifunctional intein by grafting a homing endonuclease into a mini-intein.

The majority of inteins are comprised of a protein splicing domain and a homing endonuclease domain. Experimental evidence has demonstrated that the splicing domain and the endonuclease domain in a bifunctional intein are largely independent of each other with respect to both structure and activity. Here, an artificial bifunctional intein has been created through the insertion of an existing homing endonuclease into a mini-intein that is naturally lacking this functionality.

Selection for mutants improving expression of an anti-MAP kinase monoclonal antibody by filamentous phage display.

It has recently been reported that single amino acid residues can strongly influence the expression of recombinant antibody fragments in Escherichia coli. Prediction of these critical positions can be difficult even with prior knowledge of the primary sequence and the three-dimensional folded structure of the antibody. To circumvent this, a Fab phage display library containing random point mutations was generated from a hybridoma specific for activated p44/p42 mitogen-activated protein (MAP) kinases.