Phase 1: BACTERIAL CELLULOSE
Cellulose was the most common biopolymer in the world. The primary form in which the material is found is lignocellulotic in trees, however there are other sources such as bacterial cellulose [1].This was first described by Luis Pasteur in the previous century and reported for the first time its use in a Philippine dessert called coconut cream, however, it was not until 1886 when it was reported as a type of cellulose in an acetic fermentation, after being observed as a floating film in a culture medium[2]. In recent years, several studies have been carried out on the usefulness of bacterial cellulose due to its high degree of purity and its simpler structure than that obtained from plants, in addition to the speed of polymer formation, reducing costs and environmental impact in the purification process to eliminate the lignin and other impurities of the material to be applied in the industries[3].
Bacterial cellulose has been used mainly in the paper industry, in the food for the realization of various desserts and strong dishes and as a material for garment development, due to its great flexibility, it is also impregnated with several nanoparticles to give antimicrobial, antifungal capacities[4]. Its ability to be combined with other proteins gives it the advantage to create new polymers with other desired properties such as bioplastics and drug administrators when combined with therapeutic proteins[5]. The existing biocompatibility between bacterial cellulose and human cells has led to the use of the polymer as a matrix for the regeneration of organs and tissues such as cartilage and skin[6].
References
1. Ummatyotin, S., & Manuspiya, H. (2014). A critical review on cellulose: From fundamental to an approach on sensor technology . Renewable and Sustainable Energy Reviews, 402-409.
2. Iguchi, M., Yamanaka, S., & Budhiono, A. (2000). Review bacterial cellulose-a masterpiece of nature's art . Journal of material science, 261-270.
3. Foresti, L., Vazquez, A., & Boury, B. (2016). Appiation of bacterial cellulose as precusor of carbon and composites with metal oxide, metal sulfide and metal nanoparticles. Carbohydrate polymers.
4. MAneerung, T., Tokura, S., Rujiracanit, & R. (2007). Impregnation of silver nanoparticles into bacterial cellulose for antimicrobial wound dressing. Carbohydrate polymers, 43-51.
5. Helenius, C., Backhdal, H., Bodin, A., Nannmark, U., Gatenholm, P., Risberg, & B. (2005). In vivo biocompatibility of bacterial cellulose. Wiley InterScience, 431-438.
6. Backdahl, H., Helenius, G., Bodin, A., Naanmmark, U., Johansson, R., Risberg, B., & Gatenholm, P. (2006). Mechanical properties of bacterial cellulose and interactions with smooth muscle cells. Biomaterials, 2141-2149.
Phase 2: FUSION PROTEIN CBD-ELP-BMP2
LASTIN-LIKE POLYPEPTIDES
Elastin-like polypeptides (ELP) are genetically encodable artificial biopolymers. They are elastomeric proteins formed by a repetitive pentapeptide of Val-Pro-Gly-Xaa-Gly sequence, Xaa can be any amino acid except proline. [1].
ELPs are thermostable biopolymers whose properties vary depending on the temperature, pH or ionic strength. They can pass from a soluble state to an insoluble one and reversibly depending on their transition temperature (Tt) [2], at temperatures lower than the Tt ELPs are soluble, but insoluble when the temperature exceeds the Tt. This property is maintained even when they are fused with other proteins and has been used in protein purification. The amino acid residues that contain groups susceptible to ionization result in a polymer with a Tt regulated by changes in pH, in addition, the substitution of the Xaa residue allows ELP to be designed with a desired Tt[3].
In biomedicine, ELPs have applications in the specific drug delivery, in tissue engineering and regenerative medicine. It has been possible to selectively transport antineoplastic drugs to pathologically changed tissues, allowing the polymer-drug conjugates to accumulate in the vicinity of a tumour, showing a lower toxicity compared to free-running drugs. [1].
In regenerative medicine, ELPs have been used as scaffolds in tissue regeneration, and have shown promising results in treatments for articular cartilage damage, where a hydrogel made of ELP is used, in which it effectively contributed to the production of a cartilage matrix. Other studies show that ELPs conjugated with polymers such as polyacrylic acid and polyethyleneimine can strongly influence the aggregation, morphology and differentiated function of hepatocytes in vitro, showing the ability to use ELP in the regeneration of liver tissue [1]. In addition, ELPs have shown promising results to be used in the engineering of ocular surface tissues, and in vascular grafts [4].
References
1. KOWALCZYK, Tomasz, et al. Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers. World Journal of Microbiology and Biotechnology, 2014, vol. 30, no 8, p. 2141-2152.
2. PARK, Ji-Eun; WON, Jong-In. Thermal behaviors of elastin-like polypeptides (ELPs) according to their physical properties and environmental conditions. Biotechnology and Bioprocess Engineering, 2009, vol. 14, no 5, p. 662.
3. MCMILLAN, R. Andrew; CONTICELLO, Vincent P. Synthesis and characterization of elastin-mimetic protein gels derived from a well-defined polypeptide precursor. Macromolecules, 2000, vol. 33, no 13, p. 4809-4821.
4. MARTÍNEZ-OSORIO, Hernán, et al. Genetically engineered elastin-like polymer as a substratum to culture cells from the ocular surface. Current eye research, 2009, vol. 34, no 1, p. 48-56.
SUPER FOLDER GREEN FLUORESCENT PROTEIN
More complete variants of GFP are used as fusion markers and protein
expression reporters, but fused proteins can reduce the yield, yield, and
fluorescence of these GFPs.[1] They
perform the process properly, when expressed alone or when it is fused to
well-folded proteins; In addition, the resistance of GFP is dependent on the
chemistry and thermal denaturation. In this project we will use a GFP
super-folder, which is a variation of the green fluorescent protein (GFP).
Frequently, wild-type GFP is misfolded when expressed in E. coli and when
expressed as fusions with other proteins. Unlike this one, the GFP super-folder
contains 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T[2],
giving it a better tolerance to circular permutation, greater resistance to
chemical denaturing[3] and better folding kinetics. Therefore, it
can be folded correctly even though the fused protein is not well folded. In
2006 it was evidenced through X-ray crystallographic structural analysis, the
presence of a network of five-member ion pairs in the GFP superfolder,
based on its S30R mutation; and thus improving its folding compared to the GFP
reporter.
BONE MORPHOGENETIC PROTEIN
II
The discovery
of BMPs by Urist in 1965 has been a breakthrough in
research that has been shown that the protein is able to stimulate bone
production. Due to these properties, this protein is currently used in various
fields such as Traumatology, Tissue Engineering and orthopedic surgery in which
recombinant human BMP2 (rhBMP2) is used. The implantation of BMP2 in a collagen
sponge induces the formation of new bone and can be used as a treatment for
certain bone defects[4].
Oral surgery
has benefited in particular with the commercialization of this protein, since
the use of BMP2 in absorbable collagen sponges has significantly reduced the
costs of the interventions and the pain suffered by patients with degenerative
disease of the lumbar discotheques.
