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Novel Recombinant Traceable C-Met Antagonist-Avimer Antibody Mimetic Obtained by Bacterial Expression Analysis

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Novel Recombinant Traceable C-Met Antagonist-Avimer Antibody Mimetic Obtained by Bacterial Expression Analysis Original Article Novel Recombinant Traceable c-Met Antagonist-Avimer Antibody Mimetic Obtained by Bacterial Expression Analysis Bahram Baghban Kohnehrouz 1, Afsaneh Talischian 2, Alireza Dehnad 3, and Shahnoush Nayeri 4 1. Department of Plant Breeding & Biotechnology, University of Tabriz, Tabriz, Iran 2. Department of Biological Sciences, Higher Education Institute of Rab-Rashid, Tabriz, Iran 3. Department of Biotechnology, East Azerbaijan Research and Education Center Agricultural and Natural Resources, AREEO, Tabriz, Higher Education Institute of Rab-Rashid, Tabriz, Iran 4. Department of Biotechnology, Shahid Beheshti University, Tehran, Iran Abstract Background: Avimers are originally types of artificial proteins with multiple binding sites for specific binding to certain antigens. Various radioisotopes and nanoparticles link these molecules, which are widely used in early detection in tissue imaging, treat- ment and study on carcinogenesis. Among these, c-Met antagonist avimer (C426 avi- mer), with ability to bind the c-Met receptor of tyrosine kinase (RTK) is an attractive candidate for targeted cancer therapy. In this study, a novel traceable C426 avimer gene was designed and introduced by adding the 12nt tracer binding site encoded four specific amino acid residues at the C-terminal region of C426 avimer coding se- quence. Methods: The 282 bp DNA sequence encoded 94aa avimer protein was synthesized and sub-cloned into prokaryotic pET26b expression vector. The expression of the ma- ture peptide encoding the traceable avimer molecule was carried out in Escherichia coli strain BL21 using IPTG (Isopropyl β-D-1-thiogalactopyranoside) induction process. The expression level of the 11 kDa traceable avimer was studied by SDS-PAGE, west- * Corresponding author: Bahram Baghban Kohnehrouz, ern blot and ELISA analysis. Ph.D., Department of Plant Results: Docking analysis of C426 avimer protein and its ligand c-Met showed that Breeding & Biotechnology, the traceability related changes happened at the best conformation and optimal en- University of Tabriz, Tabriz, Iran ergy. The SDS-PAGE, western blotting and ELISA analysis results demonstrated that Tel: +98 41 33392031 the expression of the 11 kDa C426 avimer molecule was detectable without any deg- Fax: +98 41 33536003 radation compared with the control group. E-mail: [email protected] Conclusion: Concerning the consequences of this work, this new approach can be Received: 18 Nov 2016 widely used in the medical field and provide an opportunity to evaluate the affinity Accepted: 13 Mar 2017 and traceability features. Avicenna J Med Biotech 2018; 10(1): 9-14 Keywords: Antibody avidity, E. coli strain BL21, Enzyme-linked immunosorbent assay, Molecular docking analysis Introduction Recently, a class of antibody mimetics molecules ing domains is a powerful approach to increase their including avimers is developed as the non-immuno- affinity and specificity 4. These molecules have several globulin affinity proteins through protein engineering. advantageous compared to common antibodies includ- These molecules are artificial proteins with multiple ing small size, high stability, strong binding affinity to binding sites that can bind to the specific molecule like the target molecule, specificity and high resistance antigens. Avimer proteins are composed of two or against heat denaturation and enzymatic degradation 5,6. more peptide sequences with the length of about 30- In addition, the avimer molecules can be produced 35aa, which are connected to each other by linker pep- based on bacterial expression systems 7. The develop- tides. The main origin of individual peptide sequences ment of these kinds of molecules has provided a strong is the A domain of various membrane receptors 1. Do- background for production of a variety of avimeric mains of this family bind over 100 different known tar- drugs depends on their target cells for clinical uses, gets, including small molecules, proteins and viruses treatment of diseases like Crohn's, autoimmune and 2,3. The avidity generated by combining multiple bind- various cancers including leukemia and carcinoma. Copyright © 2018, Avicenna Journal of Medical Biotechnology. All rights reserved. Vol. 10, No. 1, January-March 2018 9 Novel Recombinant Traceable Avimer According to the reports, a number of avimer pro- vali-dated the modelled protein. The physio-chemical teins can bind to the turmeric targets including C426 properties of the traceable avimer including the molec- avimer protein, which has an ability to bind the c-Met ular weight, theoretical pI, amino acid composition, receptor. C-Met is a Receptor Tyrosine Kinase (RTK) atomic composition, extinction coefficient, estimated which is activated upon binding to its only known lig- half-life, instability index, aliphatic index, and grand and, Hepatocyte Growth Factor (HGF). Deregulated average of hydropathy (GRAVY) were computed by expression/activation of cMet is found in many neo- ProtParam tool (http://web.expasy.org/cgi-bin/pro- plasms severity and poor prognosis makes this RTK an tparam). attractive candidate for targeted anticancer therapy. Identification of C426 avimer protein binding site Avimer molecules along with various radioisotopes Positions of the amino acids present in binding and nanoparticles can be used as biomarkers in tissue site of the protein and tertiary structure representa- imaging too. The imaging of tissue using modern mo- tion of the binding site were determined by FTsite lecular techniques and nuclear medicine has important server from Boston University, which is freely availa- roles in the early detection of cancer, treatment and 8 ble online at http://ftsite.bu.edu/. study of disease process . Hence, the sequence of a novel avimer gene encod- Molecular cloning of C426 avimer ing the traceable c-Met antagonist avimer protein was The deduced nucleotide sequence of avimer mole- designed and introduced. The expression of the mature cule was synthesized and cloned into pGH cloning peptide of traceable c-Met antagonist avimer molecule vector by GENEray Co. (Shanghai, China). The pET- was reported in Escherichia coli (E. coli) BL21-Cod- 26b(+) expression vector with kanamycin resistance onPlus (DE3)-RIL. It should be noted that this is the gene was used for cloning and expression purpose. The first report introducing the traceable c-Met antagonist DNA fragment of C426 avimer was excised from pGH avimer, which is provided as an opportunity to evaluate cloning vector using NcoI and HindIII restriction en- the affinity and traceability features of antibody mime- zymes and was recovered by gel purification kit (Bio- tics at future researches of anticancer therapy. neer, South Korea; cat. no. k-3035) according to the manufacturer’s instructions. Then, it was sub-cloned Materials and Methods into linear pET-26b(+) cloning vector by T4 DNA lig- C426 avimer design ase (Vivantis, USA). The E. coli strain DH5α compe- The original amino acid sequence of C426 avimer tent cells were prepared by TSS reagent and recombi- (PMID: 16299519) was retrieved from GenBank at nant plasmid was transformed using heat shock meth- NCBI (www.ncbi.nlm.nih.gov). The amino acid se- od. For screening, the transformed cells were plated on quence of C426 avimer was converted into the deduced LB-agar medium containing kanamycin antibiotic (50 nucleotide sequence using EMBOSS Backtranseq tool. µg/ml). Recombinant plasmid was purified using the For prokaryotic expression of the avimer molecule, the plasmid extraction kit (Bioneer, South Korea; cat. no. appropriate codons were adjusted with codon usage k-3112). The size of insert was validated by restriction table of E. coli and the deduced amino acids sequence digestion analysis with NcoI and HindIII and electro- was checked out using Expasy translator tool (www. phoresis on 1.5% agarose gel. The precision of the ORF region was verified by sequencing analysis (GENE- web.expasy.org/translate). Then, the 12nt tracer bind- ray, China). ing site encoding four specific amino acid residues (glycine, glycine, glycine, cysteine) was added to the Sequencing and bioinformatics analysis 3'end of coding sequence. The cutting restriction sites The sequencing result of pET containing avimer for nucleotide sequence of C426 avimer were deter- coding sequence was checked out by BLASTn tool at mined using Webcutter (toolrna.lundberg.gu.se/cut- NCBI (https://blast.ncbi.nlm.nih.gov) for the correct ter2). The non-cutting restriction sites including NcoI/ positioning of C426 avimer ORF region in the pET- NdeI and HindIII (arbitrarily defined) were added at 26b (+). the 5' and 3' ends of the fragment for sub-cloning into The expression of the C426 avimer protein the prokaryotic expression vector, respectively. For prokaryotic gene expression study, the pET26- The 3D structure of C426 avimer using homology model- avimer plasmid DNA was transformed into E. coli ling strains BL21 competent cells using heat shock method. The protein secondary structure of designed and The positive colonies were screened out by LB-agar original avimer protein was compared by SWISS- containing kanamycin antibiotic (50 µg/ml). Followed MODEL server (http://swissmodel.expasy.org). The by the overnight LB-broth containing kanamycin (50 three-dimensional structure of the designed avimer µg/ml) culture of the positive colony, it was treated at protein and its receptor were checked out by ClusPro 37°C for 4 hr under 400 μl IPTG (10 mM) treatment in 2.0 automated web-based program (https://cluspro.bu.
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