Protein Engineering Assistance...Thx

[dan0909]

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I'm trying to find a protein that has what one might consider a "tail," that is, it is in its usual globular shape, but also has a pertruding sequence of amino acids that the Laman would instinctively call a "tail."

Thanks in Advance for Your Replies,
Daniel

 

[coldchemist]

Most integral membrane proteins (of the transporter variety) have short AA stretches at the C and N termini. Since the internal helices serve the major functions of supporting the protein within the membrane and moving solutes across it, it could be casually stated that these termini don't have much of a function, at least once they have been translated. Obviously, they are extremely important during the transcription and translation processes.

 

[tofurious]

Most membrane proteins that require a transmembrane anchor protein have a tail (vs. transmembrane proteins). What do you need the protein for?

 

[dan0909]

I want to add the tail to another protein (vie recomb tech)opposite its active site, so that i can bind the protein selectively, such that the active site is still accessible to substrates...

long shot, i know...its for a presentation...all hypothetical, so i dont have to prove anything...

 

[coldchemist]

Why don't you just add a histidine tag to the end? That would solve your problem, wouldn't it?

Poly-histidine tags bind to nickel-nitriloacetic acid (A.K.A. Ni-NTA). You attach a 6x or 10x his tag to either the C or N terminus, depending on which is more accessible. Then you get some Ni-NTA beads (in an agarose gel--we use Qiagen's stuff), put them in a mini-column. Run your protein solution through the column, wash it with some sort of wash buffer (be careful--binding diminishes as you approach ph 5). After the washes your protein should still be on the column. Then you simply run your substrate through the column. Probably would want to use gravity rather than a pump since it's important that your substrate has a chance to see the protein. The eluted mixture should contain the modified substrate.

 

[Hermeone]

What class did you guys take to know about all these proteins?

I don't have a single clue about it?
😕 😕

 

[coldchemist]

It's lab experience, not classes, that REALLY teaches us about this stuff. That said, you can still learn quite a bit from a cell biology course (highly recommended) although you probably won't be able to apply it too well until you've actually worked with the concepts.

 

[Hermeone]

Thanks! What a speedy reply!

I am going to change my schedule and enrol in one of those labs.

Sorry. I am an engineering major. That's why I don't know much about all these cool courses.

 

[NE_Cornhusker1]

DNA polymerase B has a big, fat C-terminal domain.

 

[Gleevec]

GPI anchor? I think its added after the protein is folded. I think GPI is glycophosphatidylinositol, and can run transmembrane. It can be cleaved by some enzymes as well I believe.

 

[tofurious]

I think we need more clarification on "selective binding". If you just want to do your experiments in vitro, what coldchemist suggested could work (never done it myself thus "could"). You can also apply the same principle and use an antibody on an affinity column. If you want to express this enzyme on either side of a biological membrane in a living cell, the accessibility of the active site can really only be determined by trial and error (along with the length of the "tail" and the spacer sequence you'd probably need). Theoretically, the probability of failure in an in vitro system exists as well, as the active site of any enzyme can close due to small changes in intermolecular interactions.

If you are only doing this for a presentation, a simpler approach might even be to directly link your enzyme of interest to a transmembrane sequence such that you eliminate the need to deal with protein-protein interaction, which can be as unpredictable as protein folding.

 

[shwtime11]

The problem with running His tagged proteins over a Ni++ column is that often the elution is done under denaturing conditions which will change the protein structure. Upon renaturing the protein, you may alter the active site and thus lose specific activity. I've been working in a Micro lab for the past two years and I tried fruitlessly to purify an enyme with a his tag, and always lost activity. You might want to try a GST or MBP tag....they elute under much less stringent conditions.

 

[dan0909]

Actually, my grand plan (or maybe not so) is to put an enzyme such as hexokinase on a cantilever, such that it might be possible to monitor the reaction with a substrate (ie glucose) based on cantilever deflection. However, as for any experiment, it would be important that the enzyme layer on the cantilever be consistent and reproducible. I'd imagine that adding anything to either of the termini would alter the structure significantly. So, I was thinking about the possibility of trying to add the sequence of some kind of protein "tail" that seems to be present on another protein and creating a hybrid hexokinase + useful tail. A bit far-fetched (as this addition would probably kill the original structure), but fun, presentable, perhaps...

Tail is the wrong word, as I'd like for the "protruding part" to simply be on the opposite side of the enzyme's active site (not necessarily on a terminus), so that one could find a bit of consistency on layering these enzymes on a cantilever. The "tail" would also have a cys at its farthest point for making a disulfide bond with a material on the cantilever, perhaps achieving stability...

This is a Protein Engineering class, as I'm doing my PhD in Biomed Engin...Hermeone, Biochem Lab was one of the best classes I ever took...the harder it is the more you will learn...but it's an excellent intro into protein engineering techniques...

Update: I actually found a protein from a papaya plant that looks like it has potential...


Thanks Again for Your Replies,
Dan

 

[tofurious]

I think you may be complicating the picture unnecessarily as every smart young scientist tends to do. You are basically describing an ELISA plate. You just need to find an antibody that does not change the kinetics of the catalysis (if it doesn't change the kinetics, it doesn't interfere with the subtrate entry into and product departure out of the active site). Use this antibody as the primary capture antibody and use your enzyme as the reporter. ELISA can be made to be quantitatively reproducible WITHIN the same plate (ie, you have to wash your enzymes off), and can be somewhat comparable from plate to plate if you always run it with a set of concentration controls.

 

[coldchemist]

Originally posted by tofurious
I think you may be complicating the picture unnecessarily as every smart young scientist tends to do. You are basically describing an ELISA plate. You just need to find an antibody that does not change the kinetics of the catalysis (if it doesn't change the kinetics, it doesn't interfere with the subtrate entry into and product departure out of the active site). Use this antibody as the primary capture antibody and use your enzyme as the reporter. ELISA can be made to be quantitatively reproducible WITHIN the same plate (ie, you have to wash your enzymes off), and can be somewhat comparable from plate to plate if you always run it with a set of concentration controls.

I agree. The antibody approach would probably be most useful because you wouldn't actually have to alter the amino acid sequence. Without doing tons of extra experimentation, you probably will not know how an AA alteration will affect activity.


shwtime11: You're right, elution of the enzyme would be done under denaturing conditions, but that isn't relevant here. You would be running the substrate through the column while keeping the enzyme bound to the Ni-NTA column...this would occur at physiological pH.

 

[coldchemist]

Originally posted by Hermeone
Thanks! What a speedy reply!

I am going to change my schedule and enrol in one of those labs.

Sorry. I am an engineering major. That's why I don't know much about all these cool courses.

I want to make sure something is clear. As I said, a cell biology course (or a biochem lab course for that matter) are great ways to begin learning this stuff. But when I said the best way to learn is in the lab, I meant that most of us probably know this stuff from research experience in the lab. I.e., were talking about actually WORKING in a laboratory--not simply spending time in a lab due to course requirements.

 

[Hermeone]

Don't worry about it. I know what you meant.
Since I can't be working in another lab now so taking lab course is the best option left for me.
Thanks for clarifying