Development
Purification column EluX
To demonstrate the usage of our non-canonical amino acids 2-nitrophenylalanine we aimed to construct a purification column that can induce the elution of the target protein by irradiation with light. In the development process, our column went through different design stages. Here we document the evolution of our column from the first idea, via different models we discussed and improved over the time, up to the final version we tested and presented to experts in order to get an idea of how our system could be used properly in the future.
BSA-coated micro well plate
Before we had the idea of a light induced elution, we wanted to prove and analyze the efficiency of the light induced backbone cleavage with a micro well plate coated with biotinylated BSA, because this is very common in lab everyday life. At this stage, we already had the idea to irradiate the plate with our LED-panel and so the panel was designed in the microwell format, such that other teams can use it for other microwell experiments.
Soon we realized how big the potential of this ncAA is, and what diverse uses are thinkable. We were inspired by a paper from Peters et al. [3] which described the cleavage of a short linear model peptide. We picked up this idea and thought about this model peptide as a protein linker to design a novel elution technique as an alternative to protease restriction sides. To realize this idea, we started in designing our own purification column.
Cylindrical column
Soon we realized how big the potential of this ncAA is, and what diverse uses are thinkable. We were inspired by a paper from Peters et al. [3] which described the cleavage of a short linear model peptide. We picked up this idea and thought about this model peptide as a protein linker to design a novel elution technique as an alternative to protease restriction sides. To realize this idea, we started in designing our own purification column.
For an elution mechanism aiming on purification, it is also crucial to reach a high value of purity. We decided that it would be better to use a directly biotinylated glass plate instead of a BSA-coated carrier material, such that the BSA could be eluted in the purification process and would not be covalently bound to the surface.
Biotinylated glass slide
There is a way to directly biotinylate glass slides by hydroxylation and a treatment with APTES so that NHS-Biotin can bind covalently on the surface. We aimed at biotinylation of a whole glass slide in size of a microwell plate and irradiate it with UV-light through our LED-Panel also designed for a usage in microwell plates. Then we consider it would be better to treat a higher number of smaller glass slides and assemble them to a column.
As we were sure that this would be a practical way to bind, irradiate, and elute our protein of interest, we thought about ways to design a purification column with many parallel glass slides instead of a hollow cylindrical one. This lead to a purification concept in which the surface of the column is crucial for the yield instead of the volume compared to conventional purification techniques.
First flow model
We designed our first flow model of the column by stacking eleven biotinylated microscope slides parallelly above each other separated by blocks of acrylic glass. Two big blocks on top and at the bottom of the column close the system. There are also holes to implement a pump system. To irradiate the slides properly, we used our self-designed microwell plate like LED-panel where a row of LEDs fits precisely between two slides for the front. On the front and back side of the column scaffold, we have adjusted two UV permeable acrylic glass slides to let the light through and seal the column. On the back side, there is an aluminum plate that will reflect the light and thus ensure an evenly irradiation. The individual parts of the column are sealed through rubber.
To improve our first model, we talked to some lecturers of protein purification and related fields in order to get some feedback and ideas of how we could increase the efficiency of our column. We were advised to round up the inner sides of the acrylic glass blocks to grant a more evenly flow-through between the glass slides. We got also the required information to choose a matching port that is connectable to the pumps that are used in our labs to make the column compatible to the inventory.
Second flow model
Implementing the feedback of the experts mentioned above, we designed our second column version, which we wanted to build and test in a protein purification experiment. The second model contains the rounded inner sides of the scaffold blocks, and holes for threaded rods to tighten and seal the column up.
We were able to build this version of our purification column as a self-made 3D-printed model.. We also invented the hard- and software of an LED-panel to irradiate the column properly and control the elution process. With this test column, we met up with two experts in protein purification and analytics and discussed the further optimization of our concept. This led to another version of the column we have not been able to construct yet. However, we instead created a 3D-model to show it.
Future microfluidic / flow-bundle model
Since the consulted experts advised us to increase the area to volume ratio for efficient target protein purification, we developed a microfluidic-like model (figure 7a). Prof. Dr. Lütkemeyer proposed a bundle-like construction as an alternative. He mentioned that we could also improve our actual model by coating the glass slides with a porous material, e.g. carboxymethyl dextran, before biotinylating to increase the area of the glass surface (figure 7b).
We realize the suggested versions of our purification column via 3D-models to evaluate them. Beside the advantage of these concepts to solve the area-to-ratio-problem, both have certain challenges too. The channels of the micro-fluidic like column could be clogged by the cell fragments contained in the loaded cell lysate. To address this issue the lysate could be filtrated. Unfortunately, this could lead to a loss of the target protein and thus decrease the yield. The major challenge concerning the flow-bundle-like column is the irradiation of all channels. We designed bundles of 37 small channels to arrange them like honeycombs with LED-units between the bundles. To still guarantee appropriate elution efficiency, the minimal amount of light and the minimal irradiation time should be tested. We would like to identify conditions which guarantee the channels in the center of the bundle to get enough UV-light to induce the protein back bone cleavage through the 2-NPA.