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Marcus D Hughes

Publications and source records attributed to Marcus D Hughes.

6 recordsLinked to original sources
PubMed2005

Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins.

We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using 'MAX' randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40,000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.

Binding Sites↗
PubMed2003

Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.

Cell Division↗
PubMed2003

Removing the redundancy from randomised gene libraries.

Amino acid substitution plays a vital role in both the molecular engineering of proteins and analysis of structure-activity relationships. High-throughput substitution is achieved by codon randomisation, which generates a library of mutants (a randomised gene library) in a single experiment. For full randomisation, key codons are typically replaced with NNN (64 sequences) or NN(G)(CorT) (32 sequences). This obligates cloning of redundant codons alongside those required to encode the 20 amino acids. As the number of randomised codons increases, there is therefore a progressive loss of randomisation efficiency; the number of genes required per protein rises exponentially. The redundant codons cause amino acids to be represented unevenly; for example, methionine is encoded just once within NNN, whilst arginine is encoded six times. Finally, the organisation of the genetic code makes it impossible to encode functional subsets of amino acids (e.g. polar residues only) in a single experiment. Here, we present a novel solution to randomisation where genetic redundancy is eliminated; the number of different genes equals the number of encoded proteins, regardless of codon number. There is no inherent amino acid bias and any required subset of amino acids may be encoded in one experiment. This generic approach should be widely applicable in studies involving randomisation of proteins.

Amino Acid Substitution↗
PubMed2003

Fluorescent microplate-based analysis of protein-DNA interactions. II: Immobilized DNA.

A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.

Adsorption↗