Functional biomaterials Results.html

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Functional biomaterials

Results
We designed and prepared different polypeptides. The fusion proteins shown on Figure 5 were constructed to additionally demonstrate the principle of simple production with high yields in bacteria using KSI as fusion partner of proteins (Polypeptide manufacturing), especially relevant in this case because some of these proteins contain antimicrobial peptide. Next, these fusion proteins will be further used to confirm that potential biomaterial forming polypeptides have antimicrobial potency (when containing antimicrobial peptide LL-37) and capability of cell differentiation stimulation (when containing nerve growth differentiation factor).

The prepared fusion proteins were composed of at least one elastin-like segment, represented by four or more elastin-like repeats (in our case one elastin-like repeat was the motif VPGVG which was repeated 10 times which all together represented elastin-like segment), coiled-coil segment (s) and functional domain (s). The polypeptide ELST-ELST-ELST-NGF contained just three repeats of elastin-like hydrophobic domain (VPGVG)10 (Urry et al., 1974) for formation of cell growth material and mouse nerve growth factor (NGF). This polypeptide could also be used for incorporation of NGF in matrix consisting of other polypeptides that also contain hydrophobic elastin-like segments.  Figure 1: Schematic presentation of the designed and prepared fusion proteins. KSI-ketosteroid isomerase; DP-dipeptide Asp-Pro; LL-37-antimicrobial peptide LL-37; ELST- (VPGVG)10; GCN-coiled-coil segment GCN4-p1 (I-L) forming parallel homodimer; P1- designed coiled-coil forming parallel heterodimer with P2; P2- designed coiled-coil forming parallel heterodimer with P1; NGF-nerve growth factor (mouse); RADA-self-assembling peptide (RADA)5. All fusion proteins also contained KSI-DP on N-terminus. Ketosteroid isomerase (KSI) causes the production of proteins in form of insoluble and protease resistant inclusion bodies and dipeptide aspartate-proline (DP) enables cleavage in acidic environment (as demonstrated in Polypeptide manufacturing).

Coiled-coil segments used in these fusion proteins were either designed, so that they form heterodimers with each other (p1, P2), or modified naturally occurring GCN4-p1 peptide (GCN4-p1 I-L) (Harbury et al., 1993) which forms homodimers was used.

Polypeptides were also fused with one or two functional domains: NGF (mouse nerve growth factor), stimulating differentiation of neurons and/or cathelicidin LL-37 which has antimicrobial activity against bacteria, fungi, and viral pathogens and promotes wound healing.

Cloning and expressing fusion proteins for functional biomaterial production
Coding sequences for the above fusion proteins were cloned into appropriate vector. The fusion proteins were then expressed in E.coli BL21 (DE3) pLysS cells in form of inclusion bodies (IB). Next, the inclusion bodies were washed with lysis buffer, 2M urea and MQ water, dissolved in denaturant GdnHCl and dialyzed against MQ.

The expression of the LL37-elst-GCN-elst-P2 and ELST-ELST-ELST-NGF and their presence mainly in inclusion bodies was confirmed using SDS PAGE (Figure 2, 3). The presence of the fusion protein LL37-elst-GCN-elst-P2 was additionally confirmed with Western blot analysis (Figure 2).  Figure 2: Samples of LL37-elst-GCN-elst-P2 soluble fractions and inclusion bodies on SDS-PAGE (left; 1-standard, 2-soluble fraction, 3-IB) and LL37-elst-GCN-elst-P2 inclusion bodies on Western blot (right; 1-standard, 2-IB). Molecular weight of the polypeptide LL37-elast-GCN-elast-P2 is 23 kDa. After production in E. coli BL21(DE3) polypeptide was mainly found in inclusion bodies (insoluble fraction of cell lysate) as could be seen on the picture above (left, 3). Some of the product was also in soluble fraction of cell lysate (above left, 2). Presence of the polypeptide was also confirmed with Western blot analysis (above right, 2).  Figure 3: Samples of ELST-ELST-ELST-NGF inclusion bodies on SDS-PAGE (left; 1-standard, 2-IB). Molecular weight of the polypeptide is 39,6 kDa. Protein was produced in E. coli BL21(DE3). SDS-PAGE analysis showed that protein was packed in inclusion bodies (above, 2). We designed and confirmed the expression of several functional polypeptides containing coiled-coil segments and elastin-like segments. At the moment of freezing the wiki the biological function of produced functional polypeptides has not been tested yet as we directed, as already mentioned, our energy efforts on the subprojects with more defined assembly of polypeptide nanostructures.

For the continuation of research in the direction of biomaterials we plan to test in the first place the efficiency of providing immobilized growth factors, such as NGF. We expect to achieve the best efficiency by using constructs that contain more than three coiled coil segments, which will allow tighter interconnections of polypeptides. Other interesting directions include usage of coiled-coil-forming segments which are capable of interacting with coiled-coil segments of the existing polypeptide material. The presented approach therefore allows almost endless number of combinations and it will be interesting to see the function of several growth factors. This strategy allows attachment of functional protein domains to the polypeptide material after the initial assembly and potentially targeting within the organism. Regulated assembly/disassembly furthermore represents additional improvements. The new polypeptide would find its use not only for culturing cells, tissues and organs in vitro but could also be used for medical treatment in vivo, for instance for replacement of a damaged portion of ligament, tendon, blood vessel etc. or for construction of elastin-like prostheses, e.g. tubes for blood vessel replacement and sheets for wound and burn healing. }}