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 * Corresponding author: Bahram Baghban Kohnehrouz, Ph.D., Department of Plant Breeding & Biotechnology, University of Tabriz, Tabriz, Iran Tel: +98 41 33392031 Fax: +98 41 33536003 E-mail: Received: 18 Nov 2016 Accepted: 13 Mar 2017 sites for specific binding to certain antigens. Various radioisotopes and nanoparticles link these molecules, which are widely used in early detection in tissue imaging, treatment and study on carcinogenesis. Among these, c-Met antagonist avimer (C426 avimer), 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 sequence. Methods: The 282 bp DNA sequence encoded 94aa avimer protein was synthesized and sub-cloned into prokaryotic pET26b expression vector. The expression of the mature 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, western blot and ELISA analysis. Results: Docking analysis of C426 avimer protein and its ligand c-Met showed that the traceability related changes happened at the best conformation and optimal energy. The SDS-PAGE, western blotting and ELISA analysis results demonstrated that the expression of the 11 kDa C426 avimer molecule was detectable without any degradation compared with the control group. Conclusion: Concerning the consequences of this work, this new approach can be widely used in the medical field and provide an opportunity to evaluate the affinity 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 including avimers is developed as the non-immunoglobulin affinity proteins through protein engineering. These molecules are artificial proteins with multiple binding sites that can bind to the specific molecule like antigens. Avimer proteins are composed of two or more peptide sequences with the length of about 3035aa, which are connected to each other by linker peptides. The main origin of individual peptide sequences is the A domain of various membrane receptors 1. Domains of this family bind over 100 different known targets, including small molecules, proteins and viruses 2,3 . The avidity generated by combining multiple bind- ing domains is a powerful approach to increase their affinity and specificity 4. These molecules have several advantageous compared to common antibodies including small size, high stability, strong binding affinity to the target molecule, specificity and high resistance against heat denaturation and enzymatic degradation 5,6. In addition, the avimer molecules can be produced based on bacterial expression systems 7. The development of these kinds of molecules has provided a strong background for production of a variety of avimeric drugs depends on their target cells for clinical uses, treatment of diseases like Crohn's, autoimmune and 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 proteins can bind to the turmeric targets including C426 avimer protein, which has an ability to bind the c-Met receptor. C-Met is a Receptor Tyrosine Kinase (RTK) which is activated upon binding to its only known ligand, Hepatocyte Growth Factor (HGF). Deregulated expression/activation of cMet is found in many neoplasms severity and poor prognosis makes this RTK an attractive candidate for targeted anticancer therapy. Avimer molecules along with various radioisotopes and nanoparticles can be used as biomarkers in tissue imaging too. The imaging of tissue using modern molecular techniques and nuclear medicine has important roles in the early detection of cancer, treatment and study of disease process 8. Hence, the sequence of a novel avimer gene encoding the traceable c-Met antagonist avimer protein was designed and introduced. The expression of the mature peptide of traceable c-Met antagonist avimer molecule was reported in Escherichia coli (E. coli) BL21-CodonPlus (DE3)-RIL. It should be noted that this is the first report introducing the traceable c-Met antagonist avimer, which is provided as an opportunity to evaluate the affinity and traceability features of antibody mimetics at future researches of anticancer therapy. Materials and Methods C426 avimer design The original amino acid sequence of C426 avimer (PMID: 16299519) was retrieved from GenBank at NCBI (www.ncbi.nlm.nih.gov). The amino acid sequence of C426 avimer was converted into the deduced nucleotide sequence using EMBOSS Backtranseq tool. For prokaryotic expression of the avimer molecule, the appropriate codons were adjusted with codon usage table of E. coli and the deduced amino acids sequence was checked out using Expasy translator tool (www. web.expasy.org/translate). Then, the 12nt tracer binding site encoding four specific amino acid residues (glycine, glycine, glycine, cysteine) was added to the 3'end of coding sequence. The cutting restriction sites for nucleotide sequence of C426 avimer were determined using Webcutter (toolrna.lundberg.gu.se/cutter2). The non-cutting restriction sites including NcoI/ NdeI and HindIII (arbitrarily defined) were added at the 5' and 3' ends of the fragment for sub-cloning into the prokaryotic expression vector, respectively. The 3D structure of C426 avimer using homology modelling The protein secondary structure of designed and original avimer protein was compared by SWISSMODEL server (http://swissmodel.expasy.org). The three-dimensional structure of the designed avimer protein and its receptor were checked out by ClusPro 2.0 automated web-based program (https://cluspro.bu. edu/home.php). Ramachandran Plot gener (...truncated)