Measurement
Since our innovative biorecycling of PET is mainly based on the secretion of MUT-PETase and MHETase by Chlamydomonas, one of the most important steps is the screening of secreted proteins and proving their activity. As the screening of unknown concentrations of enzyme in the medium was particularly complicated, we optimized this process to obtain clear results in the shortest possible time with minimum effort.
Initially we used TCA precipitation to enrich the protein concentration in the medium. We analyzed 15 ml of each culture by adding 10 ml of 20% TCA solution to cell free media, incubating the samples for 30 min on ice and then centrifuging them at 20.000 g in an ultracentrifuge. Afterwards the supernatant was discarded and the pellet was resuspended in 400 µl 1xPBS. Additionally, an acetone precipitation was performed to purge the proteins. The biggest problem in following this protocol is that, due to the high volume, only a small number of transformants can be screened at a time (by using the Avanti ultracentrifuge only six colonies including the negative control can be screened).
To speed up the process of screening, we lowered the volume of culture to 2 ml since there was no significant weakening of bands by western blot analysis. Thereby we could increase the number of screened colonies up to 24 at once by using a benchtop centrifuge for small tubes. Nevertheless, the process of screening still took too long a time.
The next step of improvement was to enrich the protein concentration by lyophilising 2 ml of cell free media overnight. Thereby we could screen a lot more colonies containing different constructs by enriching the protein concentration at night and doing western blot analysis during the day. Before, it took about two days to screen a small number of colonies, whereas this approach permitted the screening of a large number of colonies in a single day. Furthermore, we increased the yield when doing western blot analysis since the risk of protein loss through precipitation is no longer present. As we improved the screening process, we observed blurred bands due to concentrating salts of the medium. Last but not least, we could increase safety by reducing the use of toxic TCA.
The big issue of unclear bands by western blot analysis could be solved by doing a fast acetone precipitation after freeze drying the samples and thus removing the amount of salt (figure 1). This step is only necessary if the culture medium contains a large amount of salt.
Comparison of different screening methods (a) Sample preparation by using TCA precipitation. Bands are small and unclear due to loss of proteins by doing TCA precipitation. (b) Sample preparation by freeze drying in lyophilizer. Bands are unclear and blurred due to high salt concentration in the samples. (c) Sample preparation by freeze drying in lyophilizer and additional acetone precipitation. Bands are clear and easy to identify.
Now that a simple screening method for proteins in the supernatant was developed, the activity of the secreted enzymes needed to be detected. Therefore, we used reversed phase HPLC to detect BHET, MHET and TPA the metabolites of PETase and MHETase. The biggest advantage of using this approach is that it is fully automated, hence a large number of measurements can be done overnight and evaluation of results can be done during the day. Another approach of determining the enzyme activity is using chromogenic substances resulting in color reaction. Even though it is easily observable, the enzyme reaction does not resemble the reaction with the native substrate. Consequently, the HPLC is superior to chromogenic substrates thus we decided to use it.
To test the activity of our enzymes we prepared reactions in different buffers. We started by using the sodiumphosphate (NaPi) buffer, after analysing the negative control containing no or denatured enzymes we could show an active buffer substance against the tested substrates (So never forget to use negative controls!). In order to solve this problem, we searched for another buffer, which still maintains the enzyme activity. After researching various PET projects, we tested two other buffers, the glycine and HMP buffer. The use of HMP buffers was obvious as it had a similarity to the culture medium of Chlamydomonas. We could verify that both buffers are perfectly suitable since they show a very low activity against the substrates (figure 2).
Comparison of buffers for activity measurement of MHETase and PETase by reversed phase HPLC (a) Standard of 1 mM TPA, MHET and BHET dissolved in DMSO and measured by HPLC. (b) Sodiumphosphate (NaPi) buffer incubated with BHET for 96 h. A strong active buffer substance is detectable. (c) Glycine buffer and (d) HMP buffer incubated with BHET for 96 h. In both measurements only a weak activity of buffer is detectable.
Through the implementation of new measurement methods and the improvement of those the development of our project could be significantly enhanced.
Below the protocols for screening by freeze drying and the buffers for activity measurements
can be found:
Protocol
buffer HPLC
Screening for secreted proteins
