Biologically Inspired Engineering for Protein Stabilisation
Creators: name: Caliskan, Huseyin Burak
Publisher: name: Apollo - University of Cambridge Repository
Year: 2020
Subjects: vaccine
protein stabilisation
thermal degradation
bioinspiration
C-type lectin
cold-chain
global health
thermostability

description: Every year millions of lives are saved by vaccination and millions more could be saved with more efficient vaccination coverage. Thermostability of vaccines is one of the major reasons why many children who need vaccines most do not get all the shots they need. Vaccines need to be kept below 8°C throughout manufacturing, delivering and shipping which requires a cold-chain. This involves carrying vaccines in a cool box for days even weeks to small villages or islands with no electricity or any other infrastructure. One of the grand challenges in global health is to produce vaccines that do not require refrigeration. Here, a biologically inspired vaccine stabilisation method is proposed. The method is based on the fact that proteins entrapped in the fossil avian eggshell crystals are well preserved up to thousands of years in Africa where high-temperature vaccine stability is needed. The persistence of the ancient intra-crystalline proteins suggests that protein incorporation into the inorganic host could retard protein degradation for long periods of time without refrigeration. In the present work, biomimetic protein incorporation in calcium carbonate was investigated to produce intra-crystalline heat-stable vaccines. Intra-crystalline protein persistence in the fossil record has been shown for ostrich eggshell proteins and the most durable proteins are known to belong to the C-type lectin-like protein family. It is thought that at least one C-type lectin-like protein is found in the eggshells of every species. The most well known C-type lectin-like protein is the OC-17 from chicken eggshell. OC-17 and other C-type lectins have the highest concentration in eggshells compared to other eggshell proteins which suggests that they could lead understanding of efficient protein incorporation in calcium carbonate crystals. Apart from ostrich and chicken, C-type lectins from other species have not been studied in detail. For this reason, eggshells from 14 species were studied in the present work. The aim of the investigation is to quantify the effect of the organic matter on the eggshells which could allow inferring protein incorporation efficiency of different species. The mechanical properties of the eggshells were studied with nanoindentation. This allows probing the differences in the elastic modulus and hardness of eggshells which are affected by the intra-crystalline protein content. It was found that the elastic modulus differs among species, which is lowest at around 10 GPa for Bali myna and highest at around 60 GPa for ostrich. Similarly, the hardness changes from around 1 GPa for Bali myna to around 3 GPa for rhea. The chemical analysis was conducted with IR spectroscopy. The deviations in the absorption peaks of eggshells compared to pure calcium carbonate allows probing the extent of the amorphous structure of the eggshells. In addition, the comparison of the full-width at half maximum values of vibration modes in each spectrum provides information of the crystal order of the eggshells. Comparison of mechanical and chemical analyses of different species offers insights on the protein content of different eggshells which could lead identification of the most efficient C-type lectins in terms of protein incorporation ability. Because of the importance of eggshell C-type lectins for the vaccine preservation method proposed in the present work, the OC-17 was studied here in detail. First, a purification method was developed to extract OC-17 from chicken eggshell. Liquid chromatography was used to extract OC-17, OC-23 and lysozyme. Then, lysozyme was removed from solution using a lysozyme binding protein. In addition, OC-17 was cloned to synthesize a recombinant C-type lectin-like eggshell protein in bacteria for the first time. Growth conditions were optimized and OC-17 expression was verified with anti-Histag antibody. Lastly, the model protein incorporation into synthetic calcium carbonate was studied. Because the incorporated and reconstituted protein structure is of utmost importance, the secondary and tertiary protein structures of incorporated proteins were analyzed with circular dichroism and intrinsic tryptophan fluorescence spectroscopy. The effect of both the reconstitution and incorporation were studied independently as intra-crystalline proteins could denature during the interaction with the reconstitution buffer or during the incorporation process. It was shown that the model proteins BSA, lysozyme and diphtheria anti-toxin could be reconstituted using EDTA without structural change. The incorporation was found efficient for BSA only with an efficiency of %84 in terms of total amount of protein incorporated into the crystals. The secondary structure of BSA was shown to be stable during reconstitution and incorporation. A structural change in the tertiary structure was observed in BSA. Possible improvements to incorporate a target protein as a fusion construct using the OC-17 or using the ostrich intra-crystalline peptides as ’incorporation tags’ were discussed in order to move towards a real-world application of biomimetic vaccine stabilisation.