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Biomedical subjects

Todd A Naumann

Publications and source records attributed to Todd A Naumann.

6 recordsLinked to original sources
PubMed2005

Engineering an affinity tag for genetically encoded cyclic peptides.

Peptide expression libraries are valuable probes of cellular function. SICLOPPS technology merges the principal advantages of both genetic methods and small-molecule approaches in yielding superior library sizes of operationally stable, structurally well-defined entities with an established biological and medicinal record. Here, we describe development, application, and the first-generation library implementation of an expressed affinity tag for a library of cyclic peptides. A tripeptide streptavidin-binding motif (HPQ) proved to be compatible with presentation from a backbone cyclized template. A resulting peptide was employed as a sensitive indicator of peptide splicing, expression, and recovery as well as an affinity tag for one-step purification. Specific recognition of the tag by streptavidin was also critical for an analysis of intein mutants. Finally, the initially identified probe was used as a template for design of a streptavidin-responsive cyclic peptide library.

Amino Acid Motifs↗
PubMed2003

Chromosomal deletion formation system based on Tn5 double transposition: use for making minimal genomes and essential gene analysis.

In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes.

Chromosome Deletion↗
PubMed2002

Production of combinatorial libraries of fused genes by sequential transposition reactions.

The use of in vivo and in vitro transposition reactions to perform non-combinatorial manipulation of DNAs in molecular biology is widespread. In this work we describe a technique that utilizes two sequential, directed transposition reactions in order to carry out combinatorial DNA manipulations. The methodology relies on the use of two different mutant Tn5 transposase proteins that have different transposon end recognition specificities. We demonstrate that the technique can be used to create large libraries of random fusions between two genes. These transpositional fusions are defined by insertion of a 32 bp linker sequence. We applied the technique to a model system, chloramphenicol acetyl transferase, to create functional fusions from N- and C-terminally truncated, non-functional genes. Comparative structural analysis suggests that both sides of the linker are inserted into disordered regions in functional proteins.

Base Sequence↗
PubMed2002

Tn5 transposase active site mutants.

Tn5 transposase (Tnp) is a 53.3-kDa protein that is encoded by and facilitates movement of transposon Tn5. Tnp monomers contain a single active site that is responsible for catalyzing a series of four DNA breaking/joining reactions at one transposon end. Based on primary sequence homology and protein structural information, we designed and constructed a series of plasmids that encode for Tnps containing active site mutations. Following Tnp expression and purification, the active site mutants were tested for their ability to form protein-DNA complexes and perform each of the four catalytic steps in the transposition pathway in vitro. The results demonstrate that Asp-97, Asp-188, and Glu-326, visible in the active site of Tn5 crystal structures, are absolutely required for all catalytic steps. Mutations within a series of amino acid residues that are conserved in the IS4 family of transposases and retroviral integrases also impair Tnp catalytic activity. Mutations at either Tyr-319 or Arg-322 reduce both hairpin resolution and strand transfer activity within protein-DNA complexes. Mutations at Lys-333 reduce the ability of Tnps to form protein-DNA complexes, whereas mutations at the less strongly conserved Lys-330 have less of an effect on both synaptic complex formation and catalytic activity.

Arginine↗
PubMed2002

Tn5 transposase with an altered specificity for transposon ends.

Tn5 is a composite bacterial transposon that encodes a protein, transposase (Tnp), required for movement of the transposon. The initial step in the transposition pathway involves specific binding of Tnp to 19-bp end recognition sequences. Tn5 contains two different specific end sequences, termed outside end (OE) and inside end (IE). In Escherichia coli, IE is methylated by Dam methylase (IE(ME)). This methylation greatly inhibits recognition by Tnp and greatly reduces the ability of transposase to facilitate movement of IE defined transposons. Through use of a combinatorial random mutagenesis technique (DNA shuffling), we have isolated an IE(ME)-specific hyperactive form of Tnp, Tnp sC7v.2.0, that is able to promote high levels of transposition of IE(ME) defined transposons in vivo and in vitro while functioning at wild-type levels with OE transposons. This protein contains a critical glutamate-to-valine mutation at amino acid 58 that is responsible for this change in end specificity.

DNA Transposable Elements↗