Project Description

Why is this project needed?

One of the major problems with expressing proteins in bacteria such as E. coli for manufacturing and for industrial use is the limit of expressing large and complex proteins. Bacteria are usually only able to produce recombinant proteins smaller than 60 kDa. However, there are proteins among these, smaller, proteins that are still difficult to fold. One reason for this is that prokaryotic cells are not optimized to fold proteins that are complex or eukaryotic. It might be assumed that proteins will fold spontaneously to their native structures since structures with low free energy are favoured. However, there are situations where this is not the case. Local minima in free energy causes the proteins to fold incorrectly. As a result, energy is needed in order for the proteins to achieve their native structure. One way to make the proteins achieve their native structure is to co-express chaperones and to let them assist in the folding process (1). The goal of LiU iGEM 2018 is therefore 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 do this by interacting in different subsystems that targets different problems of the folding pathway. 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 holdas and there by prohibits unfolded proteins from interacting with aggregates (2).

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 the difficult to express proteins. Co-expression is essential in order to enable easy and sufficient protein expression of different proteins. The idea is not a groundbreaking one, e.g. Takara created chaperone plasmids that can be used similarly (3). However, our plasmid can be co-expressed both alongside a client protein, but also in combination with Takara plasmids and the client protein. This was done by placing GroES in a plasmid with a C class Origin of replication. More specifically pSB_4A5. This enables co-expression with all B class plasmids, like for instance Takara plasmids. Or 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 in research purposes of neurodegenerative diseases, and also 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 protein such as EGFP-A𝜷 1-42 and mNG-A𝜷 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. Moparthi SB, Sjölander D, Villebeck L, Jonsson B-H, Hammarström P, Carlsson U. Transient conformational remodeling of folding proteins by GroES—individually and in concert with GroEL. J Chem Biol. 2014;7(1):1–15.

3. Chaperone Plasmid Set For Research Use v201701Da [Internet]. [cited 2018 Aug 12]. Available from: http://www.takara-bio.com