Team:Uppsala/Contribution
Basic parts to enable easier purification of FGF2
FGF2 is an epidermal growth factor with applications in medicine, research and cellular agriculture. We have created several soluble FGF2 composite biobrick parts with flanking restriction sites suitable for insertion in certain pET vectors. FGF2 is most successfully expressed by using inducible expression, and a pET vector is designed so that the FGF2 biobrick parts are preceded by an inducible promoter. The FGF2 constructs are flanked by the restriction sites NdeI and SpeI which can be cut and inserted into the vector of choice. The solubility is increased with these constructs as the FGF2 is attached to thioredoxin, making it easier to extract during purification [1]. They also contain 6 his-tags to enable his-tag-column purification and an enterokinase site to isolate the protein of choice [1], in our case FGF2.
Our bronze requirement contribution to future iGEM teams are three basic parts, a thioredoxin basic part, a 6-his-tag basic part and an enterokinase-site basic part to enable creation of a composite part for easy purification of a protein. Their use in helping to purify proteins is documented on our FGF2 composite parts pages, links to these composite parts can be found on our parts page here. A composite part including the thioredoxin part, 6-his-tag part, enterokinase site and FGF2 part is annotated in Figure 1 below.
Figure 1. FGF2 construct.
An easily expressed soluble FGF2 part can be extremely useful to teams looking to work with epidermal growth factors, especially as cellular agriculture takes off in the coming years. Furthermore, it is also very useful for teams working on tissue engineering for medical applications. Expression of FGF2 can be quite difficult, but with detailed lab notes which we have provided, future teams will not have to waste time trying to express an FGF2 part for their experiment.
The expression has only been tested in 1 vector, i.e. the same pET vector, promoter, RBS and terminator were used for expression of all parts.
References
[1] Gasparian ME, Elistratov PA, Drize NI, Nifontova IN, Dolgikh DA, Kirpichnikov MP. Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2). Biochemistry (Mosc). 2009 Feb;74(2):221-5. doi: 10.1134/s000629790902014x. PMID: 19267679.
