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

Abstract

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.

Bioengineering and Biotechnology Approaches in Cardiovascular Regenerative Medicine, Volume II

“…The self-assembled peptides have excellent performances, such as programmable, good viscoelasticity, good biocompatibility, low immunogenicity and low cost (Kondo, Nagasaka et al, 2014;Rad-Malekshahi, Lempsink et al, 2016;Gao, Xu et al, 2017). Moreover, without the help of traditional hemostatic methods, such as pressure, cautery, vasoconstriction, coagulation and cross-linking agent, it can achieve complete and rapid hemostasis in the fields of the brain, spinal cord, liver, femoral artery and skin wounds (Fouani, Nikkhah et al, 2019).…”

Advances in the development and optimization strategies of the hemostatic biomaterials

“…The self-assembled peptides have excellent performances, such as programmable, good viscoelasticity, good biocompatibility, low immunogenicity and low cost ( Kondo, Nagasaka et al, 2014 ; Rad-Malekshahi, Lempsink et al, 2016 ; Gao, Xu et al, 2017 ). Moreover, without the help of traditional hemostatic methods, such as pressure, cautery, vasoconstriction, coagulation and cross-linking agent, it can achieve complete and rapid hemostasis in the fields of the brain, spinal cord, liver, femoral artery and skin wounds ( Fouani, Nikkhah et al, 2019 ).…”