Abstract
Purpose: To screen an hTfR affinity peptide and investigate its activity in vitro.Methods: hTfR affinity phage clones were screened from 7-mer phage display library, and their binding ability evaluated by enzyme-linked immunosorbent assay (ELISA). A competitive assay was performed to discover the peptide BP9 (BP9) binding site on the cells. The inhibitory effect of BP9 on the cells was determined using thiazolyl blue (MTT) assay. EGFP-BP9 fusion protein was expressed in E. coli, and its binding and localization on cells were determined by fluorescence microscopy and confocal microscopy, respectively.Results: After three rounds of panning, recovery efficiency was 48-fold higher than that of the first round. The peptide BP9 sharing 2 identical amino acids to Tf showed high-affinity to hTfR, and possessed strong proliferation inhibition ratio on different tumour cells of 70 % (HepG2 cells)/77 % (SMMC-7221 cells) at a concentration of 0.1 mM, and 85 % (HepG2 cells)/81 % (SMMC-7221 cells) at a concentration of 0.001 mM for 48 h. The recombinant protein EGFP–BP9 could bind to tumour cells andgain entry via the endocytic pathway.Conclusion: BP9 can bind to TfR and inhibit the proliferation of the tumour cells over-expressing TfR. The DNA sequence coding for BP9 was able to target the macromolecule to combine with TfR. BP9 may possess potential applications in cancer therapy.Keywords: Peptide, hTfR, Transferrin receptor, Phage display technology, Enhanced green fluorescence protein, Target, Cancer cells
Highlights
Phage display is a selection technique in which a library of peptide or protein variants is expressed on the outside of a phage virion, while the genetic material encoding each variant resides on the inside [1]
In order to further research whether the BP9 peptide could mediate other macromolecules to combine with transferrin receptor (TfR), the DNA sequence coding for peptide BP9 were fused with enhanced green fluorescence protein (EGFP) by PCR and inserted into expressing vector pET-22b
These results show that the EGFP-BP9 fusion protein can combine with TfR on HepG2 cells and SMMC-7221 cells, and that BP9 has the ability to mediate other molecules to bind to TfR
Summary
Phage display is a selection technique in which a library of peptide or protein variants is expressed on the outside of a phage virion, while the genetic material encoding each variant resides on the inside [1]. A number of peptides have been identified by phage display for targeting different tumors and cell types [2,3,4]. The Ph.D.-7TM Phage Display Peptide Library kit (New England Biolabs, Beverly, MA, USA) was used to screen specific peptides binding to hTfR. Cells were inoculated on 24-well tissue culture plates overnight They were washed and incubated in serum-free medium for 3 h and preincubated with 1 % (w/v) BSA to block nonspecific binding at room temperature for 1 h. The synthetic peptide (0.00001 mM, 0.0001 mM, 0.001 mM, 0.01 mM, 0.1 mM) was diluted in PBS and incubated with the cells at 37 °C for 3 h, incubated with 1×1011 pfu/well of phage clones at 37 °C for 2 h. His-EGFP-BP9 fusion proteins (8 μM) were added to the culture medium and incubated at 37 °C for the indicated time. The differences were considered to be significant at p < 0.05
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