Phage display biopanning identifies the translation initiation and elongation factors (IF1α-3 and eIF-3) as components of Hsp70–peptide complexes in breast tumour cells

Abstract

The heat shock protein, HSP70, is over-expressed in many tumours and acts at the crossroads of key intracellular processes in its role as a molecular chaperone. HSP70 associates with a vast array of peptides, some of which are antigenic and can mount adaptive immune responses against the tumour from which they are derived. The pool of peptides associated with HSP70 represents a unique barcode of protein metabolism in tumour cells. With a view to identifying unique protein targets that may be developed as tumour biomarkers, we used purified HSP70 and its associated peptide pool (HSP70-peptide complexes, HSP70-PCs) from different human breast tumour cell lines as targets for phage display biopanning. Our results show that HSP70-PCs from each cell line interact with unique sets of peptides within the phage display library. One of the peptides, termed IST, enriched in the biopanning process, was used in a 'pull-down' assay to identify the original protein from which the HSP70-associated peptides may have been derived. The eukaryotic translation initiation factor 3 (eIF-3), a member of the elongation factor EF1α family, and the HSP GRP78, were pulled down by the IST peptide. All of these proteins are known to be upregulated in cancer cells. Immunohistochemical staining of tumour tissue microarrays showed that the peptide co-localised with HSP70 in breast tumour tissue. The data indicate that the reservoir of peptides associated with HSP70 can act as a unique indicator of cellular protein activity and a novel source of potential tumour biomarkers.

In situproduction of humanβdefensin-3 in lager yeasts provides bactericidal activity against beer-spoiling bacteria under fermentation conditions

“…The samples along with 0·25 μ g synthetic HBD3 were separated on 15% polyacrylamide‐SDS gels (Sambrook and Russell ; Siebke et al . ), and the proteins were electrotransferred to membranes and processed as previously described (Siebke et al . ), except that polyvinylidene difluoride (PVDF, Immobilon‐P Millipore, Billerica, MA, USA) membrane was used according to manufacturer's recommendations.…”

“…), and the proteins were electrotransferred to membranes and processed as previously described (Siebke et al . ), except that polyvinylidene difluoride (PVDF, Immobilon‐P Millipore, Billerica, MA, USA) membrane was used according to manufacturer's recommendations. The membranes were blocked in 1% BSA in PBS‐Tween (2–12 h) and then incubated (2–12 h) with biotinylated anti‐HBD3 polyclonal antibody (detection antibody, 0·25 μ g ml −1 Peprotech) in PBS‐BSA‐DB.…”

Molecular Mimics of the Tumour Antigen MUC1

“…The basic outline of biopanning procedure was described earlier [17], [25]. Approximately 10 11 –10 12 phage particles from the relevant phage display library were used for each biopanning experiment.…”

“…The Ph.D. C7C library was used for the first step biopannings while both the Ph.D. C7C and the Ph.D-12 libraries (New England Biolabs Inc., MA) were employed in the reverse biopannings. The basic outline of biopanning procedure was described earlier [17] , [25] . Approximately 10 11 –10 12 phage particles from the relevant phage display library were used for each biopanning experiment.…”

Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library

“…Peptides can selectively bind to targets in tumor tissue and can be isolated. Phage display has successfully selected peptides that are specific for tumor cells, such as breast tumor cells , bladder tumor cells , colon tumor cells , prostate tumor cells , and renal tumor cells .…”