Protein expression in bacteria is a common method to obtain high quantities of recombinant proteins. It is used in industry and research to a great extent and proteins from many different organisms are produced this way. However, some recombinant proteins are hard and tedious to express in bacteria. Factors that impact this can be size, toxicity or folding properties. Prokaryotes are not optimized to fold all recombinant proteins. This can be a preventing factor when attempting to produce a high protein yield[1]. The goal of our project this year is to investigate the folding process and the necessity of chaperones by creating and expressing our own chaperone plasmids containing GroES in E.coli.
Background
Chaperones assist the protein folding in all organisms. They are very conserved proteins and high similarities of individual chaperones can be found between different organisms. They assist the folding of proteins by interacting in different subsystems that target different problems of the folding pathway [2].
This year we focused on the GroE-system, and more specifically the chaperone GroES. GroES is a co-chaperone that is known to interact with the GroEL chaperone, and turn misfolded and unfolded proteins into natively folded proteins. However, it is hypothesised that GroES can interact with a substrate protein on its own. The thought being that GroES acts as a holdase and thereby prohibits unfolded proteins from interacting with aggregates [3].
How do we plan to solve the project?
Our project revolves around creating a chaperone plasmid containing the chaperone GroES. The chaperone will then be utilized in co-expression with aggregation-prone proteins. The idea is not a groundbreaking one, e.g. Takara created chaperone plasmids that can be used in co-expression alongside client-proteins. They achieve this by utilizing a B-class origin of replication (ORI) [4]. Takara-plasmids are thereby compatible with A-class ORIs. For this reason, we placed our GroES-part in a plasmid containing C-class ORIs, more specifically pSB_4A5. This enables co-expression with all B class plasmids, like for instance Takara plasmids and co-expression with A class plasmids, like pSB_1C3.
Applications
Our project illustrates the necessity of chaperones, more specifically GroES, in protein production. Our plasmid could be utilized for research purposes of neurodegenerative diseases. It can also be used in other fields where a higher and more correctly folded amount of protein is needed such as industrial protein production. We showed that our part can enhance the expression of the aggregation-prone fluorescent fusion protein such as EGFP-Ađ· 1-42 and mNG-Ađ· 1-42. Similar results could be achieved when co-expressing other proteins. Our part can also be combined with the Takara plasmids as an add-on. The co-expression of chaperones also gives insight into the folding pathway of the client proteins.
References
[1] Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172.
[2] Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Ulrich Hartl F. Molecular Chaperone Functions in Protein Folding and Proteostasis. Annu Rev Biochem. 2013 Jun 2;82(1):323â55.
[3] Moparthi SB, Sjölander D, Villebeck L, Jonsson B-H, Hammarström P, Carlsson U. Transient conformational remodeling of folding proteins by GroESâindividually and in concert with GroEL. J Chem Biol. 2014;7(1):1â15.
[4] Chaperone Plasmid Set For Research Use v201701Da [Internet]. [cited 2018 Aug 12]. Available from: http://www.takara-bio.com
