Difference between revisions of "Part:BBa K3037001"

Revision as of 18:45, 12 October 2019

Maltose Binding Protein (MBP-tag)

Overview

The TU Dresden 2019 team design this biobrick in order to make a fusion protein with dCas9 in accordance to the RFC 25 standard. (more information)

MBP was inserted into the pSB1C3 vector for transformation and expressed in E. coli.

https://parts.igem.org/Assembly_standard_25

Description

MBP is a well-established, reliable protein-tag that can increase the solubility of proteins it is fused to, therefore limits the risk of accumulation of overexpressed recombinant protein in inclusion bodies and increases the total protein yield. It can also improve expression of difficult enzymes like Cas9. It can further be used to purify proteins via affinity chromatography in amylose resin.(1) Additionally we included a pre-scission site upstream and downstream of the coding sequence, so the tag can be easily removed by purification in a digest. Additionally the pre-siccon sequence acts as a linker and thereby limits the risk of steric hindrance.

Many times in synthetic biology we design our own proteins, enzymes or new fusions of already existing proteins for our projects. This newly designed protein will require an expression host and E.coli is most of the time the first choice for this purpose. It can easily be used to produce large quantities of protein by overexpressing it. A recurring problem here can be insoluble expression, a phenomenon in which the overexpressed recombinant protein forms insoluble aggregates in the cell. These are called inclusion bodies. An easy way to circumvent this problem is to tag the protein with MBP. It was originally developed in the 1980s as an expression tag and was in the further research found to significantly increase the solubility of proteins it is fused to. (1) The exact mechanism by which this is happening is still unclear. Additionally it strongly increases the cytoplasmic yield of the tagged protein. A common approach is to fuse it to the N-terminus of the protein in question. Even though it has been shown to increase recombinant soluble expression in N- and C-terminal fusions.(1)

The other advantage of using a MBP-fusion strategy is its ability to bind to amylose resin. This way it can be used for fast and effective single step affinity purification. This resin is much more affordable than other affinity purification methods. The only issue that can occur with MBP is that it can create steric hindrance with the protein it is fused to due to its size. This can be avoided by adding a linker sequence. Our BioBrick is designed in a way that avoids this problem from the beginning so if you use it you don’t have to worry about it. We added an additional sequence upstream and downstream of the MBP coding sequence, which is a recognition site of the PreScission Protease recombinase. PreScission Protease is a fusion protein of human rhinovirus (HRV) 3C protease and GST. It allows for on-column cleavage of tagged proteins in one step. (2) This way if you are worried that this relatively large tag might influence the activity of your purified protein you can cleave it off by digesting it with the prescission protease. It is suitable for on-column and off-column cleavage.

Biology

Maltose Binding Protein is approximately 42 kDa in size and naturally occurs in E.coli. It is encoded in the malE gene. MBP is responsible for the uptake, breakdown and transport of a special carbohydrate, maltodextrin. (1)

Sequence

Design Notes

- RBS in between NgoMIV and RCF10 enzymes, Stop as well (in between A and RCF10), this way can be translationally fused N-or C-terminally.

- Prescission site upstream and downstream for same reason

- RCF25 used to ensure translational fusion so downstream proteins are read in frame

- Codon optimized for E.coli with all forbidden restriction enzyme sites removed

References

[1] https://www.genscript.com/bacterial-soluble-protein-expression-MBP-tag.html

[2] https://www.gelifesciences.com/en/us/shop/chromatography/resins/affinity-tagged-protein/prescission-protease-for-gst-tag-removal-p-00248