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].

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].

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.

CELLULOSE-BINDING DOMAINS

Cellulose, the most abundant biopolymer and biorenewable compound Earth, is a recalcitrant polysaccharide^[1]. Cellulolytic organisms are capable of degrading cellulose which involves excretion of endo- and exo-glucanases as well as glucosidases. Structurally, these enzymes are modular, consisting of a catalytic domain and cellulose-binding domain (CBD), as well as possible ancillary domains^[2]. Because of the modules play generally their respective role independently, the CBD has been studied to improve the cellulose degradation as well as to bind other functional proteins. It has been foun that CBD can be found at the N-terminal or at the C-terminal region of these enzymes^[4].

In order to evaluate an N-terminal and a C-terminal CBD we chose the domain of Clostridium thermocellum cellulosome-scaffolding protein A (cipA) and the domain of Cellulomonas fimi exoglucanase (Cex). We used the CBDcipA because the high affinity among other CBDs reported by the Imperail College London team (2014).

The modules are joined by linkers that are variable in terms of length and amino acid composition. The length ranges from a few to up to 150 amino acids whereas the sequences are rich in proline or/and hydroxyamino acids^[3]. Because of the synergistic activity between the catalytic and cellulose-binding domain is dependent of the length and/or linker sequence, we have used their respective endogenous linkers^[2].

References

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Iguchi, M., Yamanaka, S., & Budhiono, A. (2000). Review bacterial cellulose-a masterpiece of nature's art . Journal of material science, 261-270.

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.

MAneerung, T., Tokura, S., Rujiracanit, & R. (2007). Impregnation of silver nanoparticles into bacterial cellulose for antimicrobial wound dressing. Carbohydrate polymers, 43-51.

Helenius, C., Backhdal, H., Bodin, A., Nannmark, U., Gatenholm, P., Risberg, & B. (2005). In vivo biocompatibility of bacterial cellulose. Wiley InterScience, 431-438.

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.

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.

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.

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.

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.

Guan, H., Gurau, G. & Rogers, R. (2012). Ionic liquid processing of cellulose. Chemical Society Reviews. Issue 4, 2012

Poon, D., Withers, Stephen., and McIntosh, L. (2006). Direct demonstration of the flexibility of the glycosylated proline-threonine linker in the Cellulomonas fimi Xylanase Cex through NMR spectroscopic analysis. The Journal of Biological Chemistry 282(3):2091-100.

Gilkes, N., Henrissat, B., Kilburn, D., Miller, R. & Warren, R. (1991). Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiology Reviews. 55, 303–315

Zhang, M., Wu, Sheng-Cheng., Zhou, W. & Xu, B. (2012). Imaging and Measuring Single-Molecule Interaction between a Carbohydrate-Binding Module and Natural Plant Cell Wall Cellulose. The Journal of Physical Chemistry 116, 9949−9956

Schaerli, Y., Munteanu, A., Gili, M., Cotterell, J., Sharpe, J., & Isalan, M. (2014). A unified design space of synthetic stripe-forming networks. Nature communications, 5, 4905.

Pédelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature biotechnology, 24(1), 79.

Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S., & Vale, R. D. (2014). A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell, 159(3), 635-646.

Francisca Pulido, J. G. (10 de 12 de 2013). Actualidad Médica . Obtenido de BMP-2 in Traumatology. Advances in Tissue Engineering: https://www.actualidadmedica.es/archivo/2013/790/rev01.html