Difference between revisions of "Team:Aalto-Helsinki/Laboratory Theory"

Heterologous Production of Dermcidin

In many studies, chemically synthesized dermcidin peptides are used because of difficulties in the recombinant expression caused by toxicity of the peptide to the expression host due to its antimicrobial nature. However, chemical synthesis is expensive, costing several hundreds for mg quantities. Therefore, production of the antimicrobial peptide in the form of a fusion protein in a heterologous expression system is an important alternative to be exploited. In previously published methods, DCD-1 is produced recombinantly but CNBr is used to cleave off the fusion tag. [3] CNBr is not the most optimal method to use not only due to its volatile and toxic nature, especially in safety level 1 premises, but also because of possible side reactions in the presence of serine and threonine following methionine. With the perspective of safety, Ulp1 enzyme is utilized in our work to cleave off the His6x-Smt3 tag that is used in expression and purification. Ulp1 is known for its robust and specific proteolytic activity against SUMO fusion proteins, which makes this a promising system for recombinant protein production. [4] the His6x tag in the N-terminus is used for purification with immobilized metal ion affinity chromatography (IMAC) columns designed for histidine tagged proteins.

Employing the Smt3 tag is beneficial to our project in several aspects: it facilitates cleavage of the fusion peptide, it keeps the antimicrobial peptide in an inactive form by blocking its adhesion due to the relatively large size of His6x-Smt3 tag, and it eases the purification step due to the advantageous effects that the SUMO tag has on solubility, preventing the formation of inclusion bodies of the fusion peptides. One other major advantage of this fusion system is that it facilitates detection of the peptide with a conventional method, SDS-PAGE. Our expression system is inducible with addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to the expression culture, since IPTG induces T7 RNA polymerase promoter leading to expression of the gene of interest in the plasmid.

Immobilization and CBM

Bacterial adhesion to surfaces and biofilm formation is one of the major reasons for spreading bacterial infections especially in public places, such as the hospital environment. Preventing pathogen colonization on surfaces in such environments is crucial. Therefore, immobilization of antimicrobial peptides can be a good alternative to other bactericidal agents because of the wide antibiotic spectrum of DCD-1L, given that immobilization is a known way of increasing stability and resilience of peptides. Thus, throughout our project one of our goals was to immobilize DCD-1L as an antimicrobial coating agent. For this purpose, four different constructs were designed containing a cellulose binding domain (CBM3) from Clostridium thermocellum, to immobilize DCD-1L on cellulose based materials.

Our CBM constructs contain a 22-amino-acid long linker to avoid interference of CBM with hexameric complex formation of the antimicrobial peptides that leads to bacterial cell death. Although, after visits and discussions with healthcare professionals as part of our integrated human practices, we altered the direction of our project to fight acne causing bacteria, P. acnes. We included cellulose to our project since we discovered the versatile nature of cellulosic materials, expanding possible applications.

Figure 2. Visualization of the function of dermcidin bound to cellulose. This image was made for the postcard collaboration which we participated in.

Cellulose based materials can be used in various forms from plastic-like hard materials to hydrogels. Also, cellulose is a medically safe, ecofriendly, and abundant material that is already present or can easily be incorporated in many applications. [5] It was recently suggested that acne patients have lower expression of dermcidin, leading to progression of the skin condition. With the knowledge of DCD-1L’s activity against P. acnes, acne causing bacteria, we designed an acne product containing DCD-1L peptide with cellulose hydrogel aiming to combat P. acnes. [6] You can read more from about the application from our Applied design page.