Carboxysome purification

Purification of recombinant proteins is one essential foundation of syntetic biology. For this reason, it was our first approach to purify the carboxysome from the chemolitoautotroph model organism Halothiobacillus neapolitanis, recombinant expressed in E. coli. After establishment of the methode, our constructed carboxysome should be purified with this methode. The work for purifying carboxysomes was nevertheless based on the plasmide pHnBCS1D, which is coding for the H. neapolitanis carboxysome. For cultivation and recombinant expression of the carboxysome, we followed the method for cultivation described in Bonacchi et al., 2012 and the method for purification described by So et al., 2004. To proof the expression of the carboxysomal proteins, we analysed recombinant protein expression by SDS-PAGE and MALDI-TOF. The results of the SDS-PAGE are shown in Figure 4-7:

Figure 5: Proteinexpression of pHnCBS1D induced with 0.5 mM IPTG

Figure 6: Proteinexpression of pHnCBS1D induced with 2 mM IPTG

Figure 7: Proteinexpression of pHnCBS1D induced with 5 mM IPTG

As this SDS-PAGE shows the cellular proteins at different times points in the cultivation, you can recognize, that 12 hours after induction there is a new band somewhat smaler than 55 kDa. Analytics via MALDI-TOF identified this band as the large subunit of RuBisCO (52.6 kDa) from Halothiobacillus neapolitanis with a sequence coverage (MS) of 8.9 %. When protein expression was induced with 0.5 mM IPTG, after 12 h increases a band with a size of propably 13 kDa. MALDI-TOF displayed that this is the small subunit of the RuBisCo (12.8 kDa) with a sequence coverage (MS) of 28.2 %. When IPTG was added to 2 or 5 mM, it seems like that the intensity of the band of the small subunit decreases in time of cultivation. It could be possible, that the small subunit is degrated, but inconsistent is the increase of the large subunit. Degradation of the small subunit is not described in the literature. The fact that RuBisCo composes nearly 70 % of the protein mass (by weight) in the carboxysome could explain why other proteins from the carboysome could not be found in this SDS-PAGE.

The purification of the carboxysome is carried out via several centrifugation steps. The last step of this methode is sucrose gradient centrifugation. This method did not work in our hands at all. Unfortunately, we were not able to correct the limiting step.
Two different methods for cell lysis were tryied, sonication and Frensh pressure cell, in their execution was ensured, that the mechanical stress for the cells could not destroy the microcompartiments. The following centrifugation steps seemed to be succesful, as the supernatants after centrifugation were analysed by SDS-PAGE, showing the expected result. After the first centrifugation for the seperation of cell debris, the supernatant carried the soluble proteins. After pelleting the carboxysomes, the supernatant of both centrifugation steps contained a low amount of protein.
The sample, which was layered on top of the sucrose gradient, had a slightly blue color, and it builds up a layer on the sucrose gradient, indicating that the gradient seems to be correctly formed. After ultracentrifugation there was no visble band of carboxysomes detectable. Unfortunately, we are not available of the equipment for scanning such gradients. SDS-PAGE of the fractionated gradient showed protein in every fraction, indicating that there was not enough concentrating force for concentration of the carboxysomes. A longer centrifugation periode resultet in a pellet, which had the same blue color than the sample, which was layered on the gradient. This suggest, that the carboxysomes were pelleted and the correct centrifugation time was not found.