Cultivation of Bacteria for Producing Matrix Proteins Suitable for Biomaterial Fabrication

Abstract

Amelogenin is an enamel matrix protein (EMP) that plays an important role during enamel formation. It guides apatite crystals to form in the right way and is later degraded during enamel maturation. Amelogenin has been shown to have regenerative properties in bones, teeth, and wounds, and is therefore interesting from a biomaterial perspective. The amelogenin has high self-assembly potential and creates a formation called nanoribbons under special conditions. In this master thesis an optimal process for amelogenin production by fed-batch fermentation and E.coli BL21(DE3) together with a pET11-plasmid system was investigated. Evaluation parameters of the cultivation were examined: cell dry weight (CDW), optical density (OD), total glucose consumption, glucose consumption rate, maximum specific growth rate (umax) cell doubling time during umax,( td ), yield of biomass per consumed glucose (Yxs,) yield of product per glucose consumed (Yps), yield of product per biomass (Ypx) and total production rate (Qp) was calculated. Protein expression was evaluated by Bradford assay and SDS-PAGE. The optimal strategy for production was to use a Davis-minimal broth supplemented with yeast extract, starting with a batch phase that lasts for ~7 hours then feeding the cultivation with 18-hour linear ramp-up from 1.1-2.0% of pump speed.

Popular Abstract

Amelogenin is a protein involved in the formation of enamel that makes up the outer layer on our teeth. The protein is part of the apatite crystal formation that makes up the enamel. The amelogenin is together with the other enamel matrix proteins (EMPs) degraded during maturation of the enamel. Amelogenin has a high self-assembly potential, meaning it will interact with itself or other amelogenins in the surrounding and create supramolecular structures. These structures are either monomers, nanoribbons (long fibers) or bigger aggregates called nanospheres. The unique potential of the protein to form these intricate structures, along with its proven regeneration abilities in bone, teeth, and wounds, makes it an interesting candidate for biomaterial formation that can be used in a medical setting.

The amelogenin can be produced in the bacteria E.coli BL21(DE3), with the help of a pET11-plasmid expression system that uses IPTG as an inducer. The process can be done in different scales, but the aim in this master thesis project was to make it sufficient with as high yield as possible. This is why the opportunity to produce the cells in a fed-batch cultivation was introduced, since it is a process that has shown to be able to reach high optical densities (OD), which is a good base for high protein expression later on. The fed-batch process also enables many parameters to be controlled: glucose feed, pH, stirring speed, aeration, temperature, dissolved oxygen %, which is not possible to control in e.g., shake flask cultivations. However, to be able to compare the results from the fed-batch cultivation a shake flask cultivation was also done. The protein in the shake-flask cultivation was characterized by testing its self-assembly potential. The results indicated that the protein was somewhat functional but compared to a control-protein it seemingly had lower self-assembly properties.

The fed-batch cultivation was done with two types of media. First a minimal medium called NYAT-medium was used, then a medium called Davis minimal broth (DM-medium) that additionally was supplemented with yeast extract making it a rich, complex media. Several cultivations were done with both medias, exploring both uninduced and uninduced batches, where induction also was done at different times to see its effect. The evaluation parameters measured during the cultivations were cell dry weight (CDW), OD, total glucose consumption, glucose consumption rate, maximum specific growth rate (umax), cell doubling time during umax (td), yield of biomass per consumed glucose (Yxs,) yield of product per glucose consumed (Yps), yield of product per biomass (Ypx) and total production rate (Qp). To determine the expression of protein in the induced batches SDS-PAGE and Bradford assay was conducted on the samples. All these parameters could then be used to decide the optimal strategy for producing amelogenin.

The best cultivation media for E.coli BL21(DE3) was using the enriched DM-media, which showed high optical density for the cell cultures. It was concluded to be important to induce before the cells entered the stationary phase to have a longer production window. The optimal harvest time should be monitored by measuring Bradford at-line to see when the protein concentration decreases, and the cells enters death phase. The best protein expression was achieved with induction at OD135. The total amount of amelogenin produced from a 2L fermentation was 3.98g after purification. Evaluation of acetic acid concentrations in large-scale protein purification also indicated the possibility to streamline the purification process by using higher HAc concentrations in the acid-heat treatment to reduce working volume. The thesis project both focused on maximizing protein expression and optimizing practicality and efficiency of work. A feasible fed-batch protocol was produced which focused on process control for secure protein expression.

• author: Hendberg, Wilma and Eliasson Apelqvist, Kajsa
• supervisor: Johan Svensson Bonde
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: applied biochemistry, amelogenin, e.coli BL21(DE3), recombinant protein production, fed-batch cultivation
• language: English
• id: 9163074
• date added to LUP: 2024-06-19 13:14:20
• date last changed: 2024-06-19 13:14:20