Most well-characterized feruloyl esterases are fungal enzymes which usually require eukaryotic expression systems, such as Pichia pastoris system. On the other hand, bacterial feruloyl esterases can be made accessible through recombinant expression using well established Escherichia coli systems. E. coli expression system is suitable for protein engineering and high-throughput structural analysis
Hi there,
The answer lies in your question : obviously fungal protein has to be expressed in eukaryotic system and bacterial protein should be expressed in prokaryotic system...
It may be worth trying expression in E. coli. With some luck, you may get your results much faster than in P. pastoris. Optimizing expression in P. pastoris can be very time-consuming.
I suggest you do a very thorough research of the relevant literature. You must know the size of your protein (there are upper limits for expression in E.coli), you must check for post-translational modifications and any influence in different codon usage between the organisms. If your protein is rather small and does not have many modification sites and if the codon usage of E. coli does not destroy any critical sites, then an expression in E. coli may be worth a try (secretion into the medium, if possbible).
But you are well advised to set yourself a limit. If expression in E. coli is not successful after X months, you should switch to another system. Pichia pastoris is one option, Saccharomyces cerevisiae another one. There are also expression systems available for Schizosaccharomyces pombe, Trichoderma and Aspergillus sp.
It depends to a great degree on your time-frame and available ressources. Do you want to establish an expression system in your own lab, do optimizations on your own or do you need it to be done rather quickly, in a narrow time-frame?
If you ask the right people, maybe you can have your expression done in hands already experienced in a specific expression system. Offering "cooperation", for example co-authorship for the resulting paper or some kind of payment is always a good idea. ;)
Good luck.
Thanks Dominique for your explanation, however the answer is not obvious. Fungal protein may be expressed in eukariotic and prokariotic system. Several glicosyl hydrolases have been efficiently expressed in E. coli. Nevertheless, E. coli expression system is suitable for protein engineering and high-throughput structural analysis.
Thanks all
Actually it is mainly depending on if your eukaryotic protein needs eukaryotic postranslational processing/maturation. If not, expression in bacteria may be tried. If yes it needs eukariotic system...
I don't think that you need to necessarily go with a eukaryotic expression system, we produce active mammalian, insect and fungal proteins using E. coli expression systems (both membrane bound and cytosolic). Controlling the expression conditions, using the correct promoter and choosing the correct strain of E. coli can be critical. We have found most things express best in JM109 but we use other strains for some proteins.
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