Cloning and characterisation of LEA1-EM genes in the resurrection plant, Xerophyta humilis

Includes abstract.Includes bibliographical references (leaves 75-86).The presence and expressIon patterns of orthologues of LEA group 1 genes has been characterised in the resurrection plant, Xerophyta humilis. The group I LEAs (Em I and Em6) were first identified as proteins that were abundantly and specifically expressed during the desiccation and germination phase of angiosperm seed development. The group I LEA genes are characterised by the presence of one or more tandemly repeated 20-amino acid motifs that are particularly rich in Gly residues

High-Throughput Sequencing Enhanced Phage Display Identifies Peptides That Bind Mycobacteria

Bacterial cell wall components have been previously used as infection biomarkers detectable by antibodies. However, it is possible that the surface of the Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), also possesses molecules which might be non-antigenic. This makes the probing of biomarkers on the surface of M. tb cell wall difficult using antibodies. Here we demonstrate the use of phage display technology to identify peptides that bind to mycobacteria. We identified these clones using both random clone picking and high throughput sequencing. We demonstrate that random clone picking does not necessarily identify highly enriched clones. We further showed that the clone displaying the CPLHARLPC peptide which was identified by Illumina sequencing as the most enriched, binds better to mycobacteria than three clones selected by random picking. Using surface plasmon resonance, we showed that chemically synthesised CPLHARLPC peptide binds to a 15 KDa peptide from M.tb H37Rv whole cell lysates. These observations demonstrate that phage display technology combined with high-throughput sequencing is a powerful tool to identify peptides that can be used for investigating potential non-antigenic biomarkers for TB and other bacterial infections

Sequence enrichment profiles after high-throughput sequencing of the phage displayed libraries using Illumina technology.

<p>(A) Number of unique peptides observed in the different rounds of biopanning (B) Frequency of the selected peptides at each round of biopanning (C) Manhattan plot showing peptide sequence enrichment (GWAS) results for round 3 and 5 of biopanning.</p

Binding of the selected phage clones to mycobacteria species.

<p>(A) <i>M. tb</i> (Δleu/Δpan), (B) <i>M. smegmatis</i>. A two-tailed, unpaired t-test was used to analyse significance (**<i>p</i><0.01 and *<i>p</i><0.05). Error bars represent standard deviations of the arithmetic means of the normalised optical densities at 405 nm performed in triplicates.</p