1.which vector is best for protein expression?

This is completely a matter of choice, and may not matter as far as your protein is expressed and gets into the soluble fraction of cell lysate. pET32a has T7 promoter which is a very strong promoter, and expresses a protein with N-terminus His-tag. pGEX-KG has a tac promoter (Ptac), relatively weaker than T7 and expresses a protein with N-terminus GST tag. Thus pGEX-KG vector will be suitable for proteins with lower solubility and having tendency to get into inclusion bodies.

2.As we know that, pGEX-KG contains GST-tag and pE-T32a contains His-tag.After purification how to remove these tags in order to get native protein for crystallization? is there any enzyme for the remove off tags?

There is a thrombin cleavage site (LVPRGS) between the tag and the protein of interest in both cases. Though you can start the proteolysis right after elution from affinity column (Ni-NTA or Talon for His tag) and glutathione sepharose column (for GST tag) post proteolysis, the buffers need to be completely dialyzed to get rid of imidazole/reduced glutathione, so that the the product can be reloaded on affinity or glutathione sepharose column to remove the tag (His or GST). You rprotein of interest should come in flow through during the second round of purification.

NOTE: Thrombin will also come in flow through and needs to be removed by an additional method.

3. At the initial stage of crystallization, which screening buffer are suitable?

MCSG suite should give you enough conditions (384) for your initial screen.

4. kindly let me know, which method is better hanging-drop vapour diffusion or sitting-drop vapour diffusion.

It's a personal choice. Preparation of sitting drop vapor diffusion set up is easier by robots.

Hi Farman,

If the proteins express well with good solubility you have solved half of your protein expression / purification problems already - congratulations.

I have used both pET and pGEX family vectors for protein expression - both can give excellent results on a case by case basis.  I have also used His-Trx and GST tags on many occasions with good results as you would expect from their classic status.

Regarding your specific question on tag cleavage : there is also an Enterokinase cleavage site in pET32 between the Trx-His tag and the protein reading frame.  Personally, I've never had a good experience with any of the classic protease cleavage sites (Thrombin, Enterokinase etc) - my preferred options would be to use TEV or HRV3C proteases as they are generally more specific and have less off-target cleavage incidents.

If you wanted to use these you'd need to use either pGEX family or some of the more recent pET family versions of the vector that contain recognition sites for these proteases, or alternatively re-clone the reading frame with a modification to the junction to contain a viral protease recognition site.  If you need any advice on this, please do not hesitate to come back and I will clarify this point.

Best of luck with your experiments.

I used to use the Hampton screening kit.   When you get your first crystal, you then would move on to the optimization kit,  i.e., it is a two-stage screening protocol.  

Welcome to dark art of protein crystallography!

I would recommend that you try crystallisation experiments with all the soluble constructs that you have. This will increase the diversity of your experiments making a crystal hit more likely. More work, but given the stochastic nature of crystallisation it increases the likelihood of success.

The standard final buffer I used in the Structural Genomics Consortium was 10 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 1 mM TCEP

On final Gel Filtration of your protein be selective of your fraction selection, pooling too widely will likely result in a poor quality protein from which to crystallise.

There is often no better way in crystallisation as there are so many unpredictable parameters affecting crystallisation that diversity increases the likelihood of success.

As mentioned before access to robotic liquid handlers for drop setup is very desirable. There are plates which allow you to setup multiple drops per crystallisation condition again increasing the diversity of the experiments through condition to protein ratio (2:1, 1:1 and 1:2).

If not there are plates for both hanging and sitting drop, which is the way i learnt and appreciated the robotics later. Sitting drop is easier and less accident prone for those new to these techniques.

Hampton Index Screen and  JCSG+ screen from Molecular Dimensions are both good initial screens.

SGC vectors this link will take you to vectors that have been engineered by the SGC and are available from Source Biosciences for non-commercial use. These generally have the TEV cleavable tag, and TEV is available with a his-tag so it makes cleaning up post cleavage very up easy.

http://www.lifesciences.sourcebioscience.com/clone-products/mammalian/orfs/structural-genomics-consortium-expression-clones/sgc-vectors/?rd=1