Thank you.

Hi everyone.

First, I would like to thank Johannes and Alexander for gathering us all here at this

beautiful location and all of the other organizers and sponsors who have invested their time

and financial support so that we could be here today.

Before I dive into the project that I worked on a bit earlier this year, I wanted to mention

that when I'm not working in Bo's lab spinning samples in the AUC, I am doing my master's

for Dr. Trichard Patel and in this lab we work a lot with viruses and specifically viral

RNA and protein interactions.

So because of this, I'm doing a lot of protein expression and protein purification and so

when I was given the opportunity to work on this collaboration and to express or characterize

this new newer version of protein purification using GFP nanobody complex formation, I was

very excited to start on that.

So in the past few years, there's been a few publications that have come out that talk

about this new strategy or system and basically what you do is you take GFP or some other

kind of fluorescent molecule and you take their anti-nanobody and use that to purify

your protein of interest.

So what you would have is your protein of interest fused to GFP and in between that

you would have a protease cutting site and this would be an alternative to the traditional

nickel affinity purification method where you would use a his tag.

So even in this purification method, you can still add a his tag and still use nickel purification.

In this particular case, you would have your anti-GFP nanobody covalently attached to your

beads in your column and then you can imagine that then the GFP would attach there and then

there to the column and then you can add a protease which will then release either

just your protein of interest or the protein of interest plus the his tag and alternatively

you can use nickel affinity and then keep your protein of interest fused to the GFP

and then be able to use fluorescence methods to track your protein.

So then you end up hopefully with a high purity and high yield and anyone who's expressed

difficult to express proteins and purified them might know that that's a very tedious

procedure. So if this one is able to maybe even cut down on one step from that, it's

a very useful technique.

So green fluorescent protein was originally discovered in the Aquaria victoria jellyfish

and it has a beta barrel structure with a unique chromophore that doesn't need any additional

cofactors and because of this you can fuse it to proteins and then track them not only

in vitro but in vivo.

So we actually didn't work specifically with GFP. There's a few variations. There's eGFP

and SFGFP, enhanced GFP and superfolded GFP and eGFP has a 99% conservation but it has

a few mutations in it which has helped increase the brightness of it and also become more

stable and SFGFP has a few more mutations which have an even brighter quantum yield

and it's also, as the name suggests, has really good folding kinetics in systems where

normal proteins might misfold. And we actually started our characterization with eGFP and

eventually moved to SFGFP and redid all of our experiments so today I'll be talking about

the characterizations we did with SFGFP.

So the nanobodies, a brief overview on that. A typical antibody would be immunoglobin G,

that's the one we would all be aware of and then there's also H-cap which is very similar

but it is missing its light chain, the light chain and one heavy chain as well so the antigen

recognition is all based off of this one heavy chain and if you express that one heavy chain

by itself you have a nanobody and these nanobodies are highly stable. You can use them in various

PHs and temperatures which again make them ideal candidates for protein purification

and the nanobodies are expressed in camelids.