Immobilizing antimicrobial substances onto biocompatible materials is an important approach for the design of novel, functionalized medical devices. By choosing antimicrobial substances from innate immune systems, the risk for development of resistance in pathogenic microbes is lower than if conventional antibiotics are used. Combining natural antimicrobial peptides and bactericidal enzymes with strong and elastic spider silk through recombinant protein technology would enable large-scale production of materials that could serve as functionalized wound dressings. Herein, fusion proteins with the engineered spider silk sequence 4RepCT and five different antimicrobial substances were constructed using two different strategies. In the first, the fusion proteins had a His-tag as well as a solubility-enhancing domain N-terminally to the antimicrobial agent during expression. The tags were cleaved and separated from the target protein during the purification process. The other approach provided a His-tag but no additional solubility domain. The antimicrobial agents included in the work were a charge engineered enzyme and four antimicrobial peptides herein called Peptide A, Peptide B, Peptide C and Peptide D. Four out of five fusion proteins could be expressed in Escherichia coli without exhibiting noticeable toxicity to the host. However, most target proteins were found in the non-soluble fraction. For D-4RepCT, neither soluble nor non-soluble proteins were identified. An operating strategy for expression and purification of antimicrobial spider silk proteins was developed, where the construct system providing the solubility-enhancing domain N-terminally to the antimicrobial sequence, and long time expression at low temperatures is a promising approach. The fusion proteins A-4RepCT and C-4RepCT could be produced in adequate amounts, and they proved to possess the ability to assemble into stable fibers. When incubating solutions of Escherichia coli on the functionalized silk material A-4RepCT, it showed to decrease the number of living bacteria in solution, in contrary to wild-type 4RepCT on which bacteria continued to proliferate. Initial studies of the viability of bacteria adhered to the surface of the functionalized spider silk are so far inconclusive. A larger sample size, complementary experiments and methodology optimization is needed for a proper assessment of antibacterial properties. However, preliminary results for the development of antimicrobial spider silk are positive, and the approach elaborated in this work is believed to be applicable for the construction of functional spider silk with a wide range of natural antimicrobial agents for future wound healing applications.

The market for drugs and treatments using biological products is rapidly growing. This is mainly due to the great potential that biological products have in comparison to traditional drugs. Biological products are composed of bigger molecules such as proteins and antibodies, and therefore allow a much more specific treatment. With the complexity of the molecules comes complexity in the upscaling of manufacturing. Start-up companies tend to lack knowledge of this process; consequently, outsourcing is often a requirement for the companies to grow. Outsourcing can be both risky and costly and the companies could benefit from having the skills in-house instead. This report provides information about needs, obstacles and driving forces regarding the future market for biological medicine and also outsourcing’s effect on the market. Desktop research was used to provide necessary information about the market and biological medicines, and also to find companies, research groups and investors that we mapped according to parameters, such as location and what field of medicine they engage in. Through phone and email contact, we gained further understanding about the actors we targeted. Desktop research and interviews were used to identify needs, obstacles and driving forces regarding the development of biological medicine. The result gives insight into where in Sweden the different candidates operate and it tells what pursuits and holdbacks they are facing when developing biological medicines. The major driving force is providing patients efficient treatments but also the opportunity to capitalise in a novel and successful type of industry. The major obstacle is first and foremost the great amount of money needed. Also, companies fail to grasp the many complex and time consuming steps in developing biological medicines. The interviews also showed that outsourcing is practically inevitable, especially for small companies, due to the difficulties of concentrating the competence in-house. In conclusion, the report shows that the benefits of biological medicine creates great driving forces for its development and that Sweden makes up a good climate for this. It also enlightens the importance of test beds to ensure that the future development relies on competence.