Straightforward and Cost-Effective Production of RADA-16I Peptide in Escherichia coli

Document Type : Research Paper


1 PhD Candidate, Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

2 Associate Professor, Department of Nanobiotechnology, Tarbiat Modares University, Tehran, Iran

3 Professor, Faculty of Biological Sciences, Tarbiat Modares University Tehran, Iran



Background: RADA16I represents one of promising hydrogel forming peptides. Several implementations of RADA16I hydrogels have proven successful in the field of regenerative medicine and tissue engineering. However, RADA16I peptides used in various studies utilize synthetic peptides and so far, only two research articles have been published on RADA16I peptide recombinant production. Moreover, previous studies utilized non- or less routine expression and purification methods to produce RADA16I peptide recombinantly.
Objectives: The main goal was to produce the self-assembling peptide, RADA16I, in Escherichia coli by exploiting routine and widely used vectors and purification methods, in shake flask.
Material and Methods: RADA16I coding sequence was inserted in pET31b+, and the construct was transformed into E. coli. Purified fusion constructs were purified using Nickel Sepharose. RADA16I unimers were released using CNBr cleavage. CD and FTIR spectroscopy were used to study recombinant RADA16I’s confirmation. TEM was used to confirm fibril formation of recombinant RADA16I. Furthermore, MTT assay was implemented to assess cytocompatibility of recombinant RADA16I.
Results: The biochemical, biophysical and structural analysis proved the ability of the recombinant RADA16I to form self-assembling peptide nanofibers. Furthermore, the nanofibers exhibited no cytotoxicity and retained their cell adhesive activity.
Conclusions: We successfully produced RADA16I in acceptable levels and established a basis for future investigation for the production of RADA16I under fermentation conditions.


Main Subjects

1.           Langer R, Vacanti JP. Tissue engineering. Science. 1993;260(5110):920-926. doi: 10.1126/science.8493529 pmid: 8493529
2.           Stupp SI, Zha RH, Palmer LC, Cui H, Bitton R. Self-assembly of biomolecular soft matter. Faraday Discuss. 2013;166:9-30. doi: 10.1039/c3fd00120b pmid: 24611266
3.           Zhang S, Holmes TC, DiPersio CM, Hynes RO, Su X, Rich A. Self-complementary oligopeptide matrices support mammalian cell attachment. Biomaterials. 1995;16(18):1385-1393. doi: 10.1016/0142-9612(95)96874-Y pmid: 8590765
4.           Zhang S, Lockshin C, Cook R, Rich A. Unusually stable beta-sheet formation in an ionic self-complementary oligopeptide. Biopolymers. 1994;34(5):663-672. doi: 10.1002/bip.360340508 pmid: 8003624
5.           Zhang S, Gelain F, Zhao X. Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin Cancer Biol. 2005;15(5):413-420. doi: 10.1016/j.semcancer.2005.05.007 pmid: 16061392
6.           Zhang S, Holmes T, Lockshin C, Rich A. Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc Natl Acad Sci U S A. 1993;90(8):3334-3338. doi: 10.1073/pnas.90.8.3334 pmid: 7682699
7.           Aramvash A, Seyedkarimi MS. All-atom molecular dynamics study of four RADA 16-I peptides: the effects of salts on cluster formation. J Cluster Sci. 2015;26(2):631-643. doi: 10.1007/s10876-014-0836-8
8.           Yokoi H, Kinoshita T, Zhang S. Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc Natl Acad Sci U S A. 2005;102(24):8414-8419. doi: 10.1073/pnas.0407843102 pmid: 15939888
9.           Arosio P, Owczarz M, Wu H, Butte A, Morbidelli M. End-to-end self-assembly of RADA 16-I nanofibrils in aqueous solutions. Biophys J. 2012;102(7):1617-1626. doi: 10.1016/j.bpj.2012.03.012 pmid: 22500762
10.        Zhang H, Luo H, Zhao X. Mechanistic study of self-assembling peptide rada16-i in formation of nanofibers and hydrogels. J Nanotechnol Eng Med. 2010;1(1):011007. doi: 10.1115/1.4000301
11.        Cormier AR, Pang X, Zimmerman MI, Zhou HX, Paravastu AK. Molecular structure of RADA16-I designer self-assembling peptide nanofibers. ACS Nano. 2013;7(9):7562-7572. doi: 10.1021/nn401562f pmid: 23977885
12.        Nagarkar RP, Schneider JP. Synthesis and primary characterization of self-assembled peptide-based hydrogels. Methods Mol Biol. 2008;474:61-77. doi: 10.1007/978-1-59745-480-3_5 pmid: 19031061
13.        Ye Z, Zhang H, Luo H, Wang S, Zhou Q, Du X, et al. Temperature and pH effects on biophysical and morphological properties of self-assembling peptide RADA16-I. J Pept Sci. 2008;14(2):152-162. doi: 10.1002/psc.988 pmid: 18196533
14.        Holmes TC, de Lacalle S, Su X, Liu G, Rich A, Zhang S. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proc Natl Acad Sci U S A. 2000;97(12):6728-6733. doi: 10.1073/pnas.97.12.6728 pmid: 10841570
15.        Semino CE, Merok JR, Crane GG, Panagiotakos G, Zhang S. Functional differentiation of hepatocyte-like spheroid structures from putative liver progenitor cells in three-dimensional peptide scaffolds. Differentiation. 2003;71(4-5):262-270. doi: 10.1046/j.1432-0436.2003.7104503.x pmid: 12823227
16.        Semino CE, Kasahara J, Hayashi Y, Zhang S. Entrapment of migrating hippocampal neural cells in three-dimensional peptide nanofiber scaffold. Tissue Eng. 2004;10(3-4):643-655. doi: 10.1089/107632704323061997 pmid: 15165480
17.        Narmoneva DA, Oni O, Sieminski AL, Zhang S, Gertler JP, Kamm RD, et al. Self-assembling short oligopeptides and the promotion of angiogenesis. Biomaterials. 2005;26(23):4837-4846. doi: 10.1016/j.biomaterials.2005.01.005 pmid: 15763263
18.        Ellis-Behnke RG, Liang YX, Tay DK, Kau PW, Schneider GE, Zhang S, et al. Nano hemostat solution: immediate hemostasis at the nanoscale. Nanomedicine. 2006;2(4):207-215. doi: 10.1016/j.nano.2006.08.001 pmid: 17292144
19.        Guo J, Su H, Zeng Y, Liang YX, Wong WM, Ellis-Behnke RG, et al. Reknitting the injured spinal cord by self-assembling peptide nanofiber scaffold. Nanomedicine. 2007;3(4):311-321. doi: 10.1016/j.nano.2007.09.003 pmid: 17964861
20.        Guo J, Leung KK, Su H, Yuan Q, Wang L, Chu TH, et al. Self-assembling peptide nanofiber scaffold promotes the reconstruction of acutely injured brain. Nanomedicine. 2009;5(3):345-351. doi: 10.1016/j.nano.2008.12.001 pmid: 19268273
21.        Kisiday J, Jin M, Kurz B, Hung H, Semino C, Zhang S, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair. Proc Natl Acad Sci U S A. 2002;99(15):9996-10001. doi: 10.1073/pnas.142309999 pmid: 12119393
22.        Kuliopulos A, Walsh CT. Production, purification, and cleavage of tandem repeats of recombinant peptides. J Am Chem Soc. 1994;116(11):4599-4607. doi: 10.1021/ja00090a008
23.        Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990;96(1):23-28. pmid: 2265755
24.        Paradís‐Bas M, Tulla‐Puche J, Zompra AA, Albericio F. RADA‐16: A Tough Peptide–Strategies for Synthesis and Purification. Eur J Organ Chem. 2013;2013(26):5871-5878. doi: 10.1002/ejoc.201300612
25.        Reed DC, Barnard GC, Anderson EB, Klein LT, Gerngross TU. Production and purification of self-assembling peptides in Ralstonia eutropha. Protein Expr Purif. 2006;46(2):179-188. doi: 10.1016/j.pep.2005.08.023 pmid: 16249097
26.        Mie M, Oomuro M, Kobatake E. Hydrogel scaffolds composed of genetically synthesized self-assembling peptides for three-dimensional cell culture. Polymer J. 2013;45(5):504. doi: 10.1038/pj.2012.216
27.        Nevskaya NA, Chirgadze YN. Infrared spectra and resonance interactions of amide-I and II vibration of alpha-helix. Biopolymers. 1976;15(4):637-648. doi: 10.1002/bip.1976.360150404 pmid: 1252599
28.        Sarroukh R, Goormaghtigh E, Ruysschaert JM, Raussens V. ATR-FTIR: a "rejuvenated" tool to investigate amyloid proteins. Biochim Biophys Acta. 2013;1828(10):2328-2338. doi: 10.1016/j.bbamem.2013.04.012 pmid: 23746423