Expression and Characterization of Fusion Proteins Containing Amelogenin and Silk

Abstract

Research to improve or to discover new biomaterials is vital for human health and a driving force for innovation. Previous studies have reported the mechanical properties of silk proteins and the self-assembly behaviour of amelogenin. Both proteins have successfully been used in regenerative medicine and are interesting for biomaterial applications.

This study reports a novel insight into the characteristics of six recombinant proteins with different silk sequences fused to the C-terminal of amelogenin. Properties such as structural composition, self-assembly behaviour, and biological effect have been studied by performing various techniques like DLS, FT-IR, MS as well as in vitro cell proliferation and morphology studies on dried protein films. Attention was explicitly brought on two constructs, called 156 and 165, as they were observed to self-assemble into large insoluble aggregates at physiological pH.

Construct 165 was observed to have a temperature-dependent aggregation and a lower dissolution tendency at 37°C compared to wildtype amelogenin. This property is believed to be caused by hydrophobic interactions increasing with temperature. Construct 156 contained a mutation in its gene sequence which led to the addition of a peptide with a propensity to form β-sheets. The β-sheets were detected and believed to be the reason for the self-assembly behaviour of the construct. A much-reduced dissolution tendency was observed compared to wildtype amelogenin, suggesting that the added peptide also had a stabilizing effect.

Murine fibroblastic cells were successfully cultured on dried films of construct 156 and 165, suggesting they could be suitable for biological applications. Inconclusive results were acquired whether the cell attachment could be enhanced with construct 108, constituting of amelogenin fused with an RGD-motif. Additional research is needed to determine whether these findings are reproducible and to outline the characteristics of the recombinant proteins in detail.

Popular Abstract

With applications ranging from tissue regeneration, drug-delivery systems, and implants to restore lost mechanical functions, biomaterials play an integral role in medicine today and are essential for human health. Innovative bioengineers and scientists develop new advancements as a response to medical problems or new opportunities

Amelogenin is a protein vital in human enamel formation. It possesses the ability to self- assemble under certain conditions, an attractive property as it could be utilized as the foundation in biological nanomaterials. Silks also hold remarkable mechanical properties, being both structured yet flexible at the same time due to different domains in its protein structure. Combing the two proteins into one would potentially make a fusion protein with useful features originating from both.

Incorporation of silk gene sequences to either or both the N-terminus or the C-terminus of amelogenin allows for many different combinations and fusion proteins. This study aimed to explore the properties of a hand-full of these fusion proteins to investigate whether they potentially could be utilized as building-blocks in future biomaterials. Indeed, this study is merely the first step in the long and challenging process that is required to create a new biomaterial, yet it is very stimulating! The previously unexplored nature of these fusion proteins means that any finding would be novel.

Numerous characterization studies were carried out on six amelogenin-silk constructs to investigate properties such as structural composition, self-assembly behaviour, and biological effect. Attention was explicitly brought on two constructs, called constructs 156 and construct 165 as it was discovered that they held various intriguing features.

Construct 156 contained a silk sequence derived from silkworm and showed multiple promising properties. One of which was the formation of large and stable aggregates with significantly reduced dissolution tendencies compared to nanoparticles formed by wildtype amelogenin. The structure of the construct was analysed by two separate methods both confirming the presence of β-sheets, a structural segment responsible for the stability of silk proteins. The engineered gene sequence of the construct was other than intended and had led to a part of the vector being used as a template during protein synthesis. Following the silk sequence, a peptide of approximately 2.7 kDa with a propensity to form β-sheet structures had been added to the construct. It was likely the cause of the observed features rather than the silk sequences itself. Nonetheless, great historical inventions have been the product of unintended occurrences, and so it was decided to continue exploring the construct. Cell cultivation studies showed that fibroblastic cells were capable of proliferating when cultured on the construct and that the number of viable cells could be enhanced by incorporating a specific cell-binding protein.

For construct 165, also with silk sequence derived from the silkworm, the presence of β-sheets could not be confirmed; however, a strong tendency to aggregate was observed at 37°C. Whether this was the result of hydrophobic interactions that increased with a raised temperature, it suggested that aggregation can be controlled with temperature. The construct was also shown to be more stable at 37°C than wildtype amelogenin indicating improved durability when used at physiological settings. Similarly to cell cultivation studies on construct 156, fibroblastic cells were able to proliferate on construct 165. This speaks for both constructs to be possible candidates for future biomaterials.

• author: Majtorp, Lovisa
• supervisor: Johan Svensson Bonde; Cedric Dicko
• organization: Pure and Applied Biochemistry; Computational Chemistry
• alternative title: Uttryck och karaktärisering av fusionsproteiner bestående av amelogenin och silke
• course: KBKM05 20201
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering; Biology and Life Sciences
• keywords: Amelogenin, Silk, Biomaterial, Protein, Expression, Characterization, Applied biochemistry, Tillämpad biokemi
• language: English
• id: 9010196
• date added to LUP: 2020-05-26 07:28:49
• date last changed: 2020-05-26 07:28:49