Troubleshooting laccase production

A part of our project was to purify laccases previously produced and test their activity first against ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and then against microplastics

The main problem encountered was during purification because after production and cell lysis the laccases were found in the insoluble fraction instead of the soluble fraction as expected, this was observed in western-blot analysis. We then contacted Dr. Thierry TRON, who has worked on laccases, and he advised us to supplement with copper and also grow the cells in microaerophilic condition to have improved metal incorporation, and thus a correct folding, of our laccases.

Even following these indications and following a published protocol, "A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization - Saadia Basheer, Naeem Rashid, Muhammad Sohail Akram, Muhammad Akhtar Biosci Biotechnol Biochem . 2019", we did not manage to obtain our laccase in the soluble fraction. .

To overcome this we decided to try and refold the laccase from the insoluble fraction. To do this, we solubilized the insoluble fraction by adding 0.5 % SDS "How do surfactants unfold and refold proteins ? - Daniel E Otzen, Jannik Nedergaard Pedersen, Helena stergaard Rasmussen, Jan Skov Pedersen - Adv Colloid Interface Sci." Once solubilized, we recovered our laccases that we washed to test their activity. Unfortunately we found no activity after testing the activity against ABTS. So it is possible that the laccases cannot fold up.


We have several hypotheses that could explain our problems :

• The production was too fast resulting in the formation of inclusion bodies before the protein could fold properly. Effectively, The molecular mechanism of recombinant protein aggregation suggests that nascent polypeptide chains, adopting partially folded and unstable conformations (the folding intermediates), can follow two alternative and competitive routes, according to a kinetic partition : the correct folding, which leads to the native form, or the incorrect folding, which leads to aggregation. "Recombinant protein folding and production - Jean-Michel Betton and Alain Chaffotte - M/S: medical sciences." In some cases, aggregation results from the limited availability of chaperones and induces a stress response in the cell that produces non-physiological amounts of an unnecessary protein. "Recombinant protein folding and production - Jean-Michel Betton and Alain Chaffotte - M/S: medical sciences."
• A problem with folding of laccase, possibly associated with Cu cofactor incorporation, which also might result in the formation of inclusion bodies. In fact, copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation. Copper incorporation into CotA-laccase in the absence of copper chaperones should occur at a later step during protein folding as unfolded metal-bound states are prone to aggregation. "Unfolding pathway of CotA-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state - André T.Fernandes, Carlos Lopes, Lígia O.Martins, Eduardo Pinho Melo - Biochemical and Biophysical Research Communications"
• In inadequate refolding protocol that was unable to refold the laccases.