January 2021
E-poster with recorded talk at online conference of Greek Society of Biochemistry and Molecular Biology
Self-assembling “recombinamers” destined for ECM scaffold for cartilage regeneration
Paraskevas Lamprou1, Eleni Papachristou1, Aglaia Mantsou1, Eleftherios Andriotis2, Rigini Papi1 and Theodora Choli-Papadopoulou1
1 Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, Greece
2 Laboratory of Pharmaceutical Technology, School of Pharmacy, Aristotle University of Thessaloniki, Greece
Self-assembling proteins may form entangled fibrous networks that can be used as scaffolds for bottom-up biotechnological applications, e.g. tethering various cell types or even single molecules for tissue engineering purposes. “Recombinamer” polypeptides have been designed by genetic engineering techniques, comprising highly repeated tandem building blocks inspired by human elastin peptides, (VPGVG)x, silk fibroin and mussel “glue” protein, biotin-binding motifs and a high number of lysines for cross-linking between polypeptide chains. These “recombinamer” polypeptides were also designed to carry biochemical cues for cell adhesion as well as heparin-binding domains which are important components of the ECM. To create each monomer, partially complementary oligonucleotides have been designed so that they function as templates during a single PCR. The monomer of each building block was cloned in an expression vector, and in a next step, the combining of the building blocks was achieved by a method of recursive directional ligation using specifically designed non-palindromic restriction sites. In this way, the number of the tandem repeats and their location could be adjusted. Each “recombinamer” polypeptide was purified by affinity chromatography in order to obtain it in large quantity. The presence of elastomeric domains and high crystallinity silk-like domains, the cross-linking of the side chains of lysines and the addition of porogenic salts facilitated the formation of a porous scaffold. The scaffold seemed to be able to promote the adhesion of eukaryotic cells, and it also gave a hint that it is able to promote the differentiation of stem cells. Another important finding was that the scaffold offered protection to mucin, a glycoprotein of gastrointestinal tract, against metabolites of anaerobic bacteria which are implicated in cartilage degeneration. Our final aim is the fabrication of a scaffold that can promote cartilage regeneration.
This research has been co‐financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH – CREATE – INNOVATE (project code: T1EDK-04567).
November 2019
Poster at 70th Conference of Greek Society of Biochemistry and Molecular Biology, Athens, Greece
Production of self-assembling “recombinamer” proteins destined for bio-active artificial nanonetworks formation
Paraskevas Lamprou1, Aglaea Mantsou1 and Theodora Choli-Papadopoulou1
1 Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, Greece
Self-assembling proteins may form entangled fibrous networks that can be used as scaffolds for bottom-up biotechnological applications, e.g. tethering various cell types or even single molecules for tissue engineering purposes. A “recombinamer” polypeptide has been designed by genetic engineering techniques, comprising highly repeated tandem building blocks inspired by human elastin peptides, (VPGVG)x, silk fibroin and mussel “glue” protein, biotin-binding motifs and a high number of lysines for cross-linking between polypeptide chains. To create each monomer, partially complementary oligonucleotides have been designed so that they function as templates during a single PCR. The monomer of each building block was cloned in an expression vector, and in a next step, the combining of the building blocks was achieved by a method of recursive directional ligation using specifically designed non-palindromic restriction sites. In this way, the number of the tandem repeats and their location could be adjusted. Efforts are currently being made to find the optimal conditions of purification in order to obtain that protein biomaterial in large quantities. Initially, inspired by the intrinsically hydrophobic nature of elastin-like fusions, we tested a set of organic solvent extraction procedures for rapid recovery of pure protein. Additionally, we make attempts to purify the protein of our interest by affinity chromatography under denaturing conditions followed by other chromatography methods, such as cation-exchange chromatography. Our aim is the fabrication of a 3D scaffold for tethering of biotinylated proteins, e.g. growth factors, for chrondrogenic purposes. Due to the presence of elastomeric domains and high crystallinity silk-like domains, the formation of a micelle-like scaffold will be initiated, while cross-linking of the side chains of lysines is expected to facilitate the formation of a hydrogel.
Paraskevas Lamprou thanks General Secretariat for Research and Technology for financial support (NSRF 2014-2020 action “Research-Create-Innovate”)
November 2018
Poster at 69th Conference of Greek Society of Biochemistry and Molecular Biology, Larissa, Greece
ON THE SYNTHESIS AND STRUCTURAL CHARACTERISATION OF ELASTIN-LIKE RECOMBINAMER HYDROGEL SCAFFOLDS FOR TISSUE REGENERATION
Ioannis Riziotis1, Paraskevas Lamprou1, Aglaia Mantsou1, Theodora Choli-Papadopoulou1
1 Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, Greece
Elastin-like polypeptides (ELPs) are applied in a broad range of biomedical fields, such as drug delivery and tissue engineering. These genetically engineered peptides are designed to mimic the endogenous elastin and are characterised by a repeating (VPGXG)n motif, where X could be any guest residue except for proline. Due to the periodic occurrence of cis-proline residues, such peptides exhibit a partially ordered behaviour, undergoing reversible transitions from a disordered state to a β-spiral secondary structure. This is known as Inverse Transition Cycle (ITC) and occurs under a specific temperature, related to the peptide primary structure. Incorporation of lysine residues in low frequency, provides the ability of chemical crosslinking, thus forming a nanostructured hydrogel of good mechanical properties, able to function as biomaterial for tissue scaffolding.
Ioannis Riziotis and Paraskevas Lamprou thank General Secretariat for Research and Technology for financial support (NSRF 2014-2020 action “Research-Create-Innovate”)