Screening and identification of a renal carcinoma specific peptide from a phage display peptide library

Tu Xiangan, Zhuang Jintao, Wang Wenwei, Zhao Liang, Zhao Liangyun, Zhao Jiquan, Deng Chunhua, Qiu Shaopeng, Zhang, Zhang Yuanyuan

Description

Specific peptide ligands to cell surface receptors have been extensively used in tumor research and clinical applications. Phage display technology is a powerful tool for the isolation of cell-specific peptide ligands. To screen and identify novel markers for renal cell carcinoma, we evaluated a peptide that had been identified by phage display technology. Methods A renal carcinoma cell line A498 and a normal renal cell line HK-2 were used to carry out subtractive screening in vitro with a phage display peptide library. After three rounds of panning, there was an obvious enrichment for the phages specifically binding to the A498 cells, and the output/input ratio of phages increased about 100 fold. A group of peptides capable of binding specifically to the renal carcinoma cells were obtained, and the affinity of these peptides to the targeting cells and tissues was studied. Results Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, the Phage ZT-2 and synthetic peptide ZT-2 were shown to specifically bind to the tumor cell surfaces of A498 and incision specimens, but not to normal renal tissue samples. Conclusion A peptide ZT-2, which binds specifically to the renal carcinoma cell line A498 was selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of renal carcinoma or targeted drug delivery in chemotherapy.

Sujets

• Renal cell carcinoma
• Phage display
• Peptide
• Targeting

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 11
• Langue: English

Extrait

Tu et al. Journal of Experimental & Clinical Cancer Research 2011, 30:105
http://www.jeccr.com/content/30/1/105
RESEARCH Open Access
Screening and identification of a renal carcinoma
specific peptide from a phage display peptide
library
1*† 1† 1 1 1 1 1Xiangan Tu , Jintao Zhuang , Wenwei Wang , Liang Zhao , Liangyun Zhao , Jiquan Zhao , Chunhua Deng ,
1 2*Shaopeng Qiu and Yuanyuan Zhang
Abstract
Background: Specific peptide ligands to cell surface receptors have been extensively used in tumor research and
clinical applications. Phage display technology is a powerful tool for the isolation of cell-specific peptide ligands. To
screen and identify novel markers for renal cell carcinoma, we evaluated a peptide that had been identified by
phage display technology.
Methods: A renal carcinoma cell line A498 and a normal renal cell line HK-2 were used to carry out subtractive
screening in vitro with a phage display peptide library. After three rounds of panning, there was an obvious
enrichment for the phages specifically binding to the A498 cells, and the output/input ratio of phages increased
about 100 fold. A group of peptides capable of binding specifically to the renal carcinoma cells were obtained,
and the affinity of these to the targeting cells and tissues was studied.
Results: Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and
immunofluorescence, the Phage ZT-2 and synthetic peptide ZT-2 were shown to specifically bind to the tumor cell
surfaces of A498 and incision specimens, but not to normal renal tissue samples.
Conclusion: A peptide ZT-2, which binds specifically to the renal carcinoma cell line A498 was selected from
phage display libraries. Therefore, it provides a potential tool for early diagnosis of renal carcinoma or
targeted drug delivery in chemotherapy.
Keywords: Renal cell carcinoma, Phage display, Peptide, Targeting
Introduction However, metastatic disease recurs in a third of these
Renal cell carcinoma (RCC) accounts for 3% of all adult patients. The patients with metastatic RCC have a poor
malignancies and is the most lethal urological cancer. It prognosis with a median survival time of 1 to 2 years [3].
accounted more than 57, 000 new cases and 13, 000 can- Detection of RCC in early stages helps increase the life
cer-related deaths in the United States in 2009[1]. In expectancy of the patient [4]. Two diagnosis methods,
hisChina around 23, 000 new patients with RCC are diag- topathology and image procedures (computed tomography
nosed each year, and the incidence is increasing rapidly scan, ultrasonography, or magnetic resonance imaging)
due to the aging population [2]. Approximately 60% of provide increase the early detection of the RCC.
Histopatients have clinically localized disease at presentation, pathologically, although several promising biomarkers
with the majority undergoing curative nephrectomy. such as Carbonic anhydrase IX, B7-H1 and P53 for RCC
have been under investigation, none currently have been
validated or are inroutine use [5,6]. Therefore, some novel* Correspondence: [e-mail]; [e-mail]
† Contributed equally molecular markers must be screened and identified for
1Department of Urology, The First Affiliated Hospital, Sun Yat-sen University,
improving early diagnosis and prognosis ofRCC.
Guangzhou 510700, Guangdong, PR China
2 Phage display is a molecular diversity technology thatWake Forest Institute for Regenerative Medicine, Wake Forest University
Health Sciences, Winston-Salem, NC, 27157, USA allows the presentation of large peptide and protein
Full list of author information is available at the end of the article
© 2011 Tu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.Tu et al. Journal of Experimental & Clinical Cancer Research 2011, 30:105 Page 2 of 6
http://www.jeccr.com/content/30/1/105
libraries on the surface of filamentous phage. Phage dis- repertoire of 12-mer peptide sequences that express
ranplay libraries permit the selection of peptides and pro- dom twelve-amino-acid sequences. Extensively
sequenteins, including antibodies, with high affinity and cing the naive library has revealed a wide diversity of
specificity for all targets. An important distinctive mark sequences with no obvious positional biases.
+
of this technology is the direct link that exists between The E. coli host strain ER2738 (a robust F strain with
the experimental phenotype and its encapsulated geno- a rapid growth rate) (New England Biolabs) was used
type. Phage display technology is a powerful tool for the for M13 phage propagation. The A498 and HK-2 cells
selection of cell-specific peptide ligands at present [7]. were cultured in DMEM supplemented with penicillin,
Some laboratories have applied this technology to isolate streptomycin, and 10% fetal bovine serum. Cells were
peptide ligands with good affinity and specificity for a harvested when subconfluent, and the total number of
variety of cell types. The specific ligands isolated from cells was counted using a hemocytometer.
phage libraries can be used in diagnostic probe,
therapeutic target validation, and drug design and vaccine In Vitro Panning
development [8-10]. A498 cells were taken as the target cells, and HK-2 as
In the present study, we identified a specific novel the absorber cells for a whole-cell subtractive screening
peptide that bound to the cell surface of renal carci- from a phage display 12-peptide library. Cells were
culnoma cell line A498 generated in this laboratory by turedinDMEMwith10%FCSat37°Cinahumidified
using in vitro phage-displayed random peptide libraries. atmosphere containing 5% CO . HK-2 cells were washed2
Our results demonstrate that this biopanning strategy with PBS and kept in serum-free DMEM for 1 h before
can be used to identify tumor-specific targeting peptides. blocking with 3 mL blocking buffer (BF, PBS + 5% BSA)
11One of our selected peptides, ZT-2 was most effective in for 10 min at 37°C. Approximately 2 × 10 pfu phages
targeting cells and tissues, indicating its potential for use were added and mixed gently with the blocked HK-2 for
in early diagnosis and targeted therapy of RCC. 1 h at 37°C. Cells were then pelleted by centrifuging at
1000 rpm (80 g)for5min.HK-2andphagesboundto
Materials these cells were removed by centrifugation. Those
Renal carcinoma line A498 and a normal renal cell line phages in the supernatant were incubated with the
BFHK-2 were obtained from Medical Academy of China blocked A498 cells for 1 h at 37°C before cells were
pel(Beijing, PR China). Fetal calf serum (FCS) and Dulbecco’s leted again. After that, the pelleted cells were washed
modified eagle’s medium (DMEM) were purchased from twice with 0.1% TBST (50 mM Tris-HCl, pH 7.5, 150
Gibco(Invitrogen,Carlsbad,USA).PhageDNAsequen- mM NaCl, 0.1% Tween-20) to remove unbound phage
cing was performed by Shanghai Sangon Corp (Shanghai, particles. A498 cells and bound phages were both
incuPR China). Peptide ZT-2 (QQPPMHLMSYAG) and a bated with the E. coli host strain ER2738. Then, the
nonspecific control peptide (EAFSILQWPFAH) were phages were rescued by infection with bacteria while the
synthesized and labeled with fluorescein isothiocyanate cells died. The phage titer was subsequently evaluated
(FITC) by Shanghai Bioengineering Ltd. Mass analysis of by a blue plaque-forming assay on agar plates containing
the peptides was confirmed by a matrix-assisted laser des- tetracycline. Finally, a portion of purified phage
preparaorption/ionization time-of-flight mass spectrometry, and tion was used as the input phage for the next round of
all peptides were > 90% pure as determined by reverse- in vitro selection.
11phase HPLC. Peptide stock solutions were prepared in For each round of selection, more than 1.5 × 10 pfu
PBS (pH 7.4). Horseradish peroxidase-conjugated sheep of collected phages were used. The panning intensity
anti-rabbit antibody and rabbit anti-M13 bacteriophage was increased by prolonging the phage incubation
perantibody were purchased from Pharmacia (Peapack, NJ, iod with HK-2 for 1.25 h or 1.5 h, shortening the phage
USA). Trizol reagents were purchased from Gibco BRL incubation with A498 for 45 min and 30 min in the
sec(Gaithersburg, MD, USA) and the reverse transcriptase ond and third rounds individually, and increasing
washpolymerase chain reaction (RT-PCR)system kits werepur- ing with TBST for 4 times and 6 times in the second
chased from Promega (Madison, WI, USA). and third round individually.
ThePh.D.-12phagedisplaypeptidelibrarykit(New
England Biolabs, Beverly, MA, USA) was used to screen Sequence Analysis of Selected Phages and Peptide
specific peptides binding to A498 cells. The phage dis- Synthesis
play library contains random peptides constructed at the After three rounds of in vitro panning, 60 blue plaques
N terminus of the minor coat protein (cpIII) of the M13 were randomly selected and their sequences were
ana13
phage. The titer of the library is 2.3 × 10 pfu (plaque- lyzed with an ABI Automatic DNA Analyzer (Shanghai
forming units). The library contains a mixture of 3.1 × Sangon Corp)