We are doing crystalization for a protein, which could exist as rod-like oligomer in cells. We expressed the protein in bacterial and in insect Sf9 cells. After purification procedures, protein was examined in size-exclusion chromatography (SEC) and was found that most(>50%) of protein is polymer/aggregate(?). This protein is easy to form polymer/aggregate(?) during purification manipulation. We don't know if the polymer in SEC is natural oligomer or misfolded aggregate.
My questions:
1. Is protein monomer in buffer solution required for crystal formation, even for a naturally potential oligomerized protein?
2. Can protein form oligomer complex normally and correctly in buffer solution?
3. Are there any samples that naturally protein oligomer (?) in buffer solution formed successfully a crystal oligomer structure?
Thank you for your time.
Hi, proteins can crystallize as monomers or defined multimers. So for a protein to crystallize it doesn't necessarily have to be monomeric. An important parameter for successful crystallization is polydispersity - if all the protein molecules are present in the same monomeric or oligomeric state(a monodisperse sample) the chances of obtaining crystals are much higher. This is because for the crystalline lattice to form, the interactions between the entities need to be the same for all entities. Ways to assay monodispersity areDLS (dynamic light scattering) and/or native PAGE.
If your protein easily forms aspecific aggregates, the chances of obtaining good crystals are low. You could try to screen for an additive (e.g. salt, glycerol) that lowers the aggregation.
Best, Tjaard
Many Thanks to Tjaard:
My understanding from your points:
Crystal structure could be formed as long as the protein in buffer solution exists in monomer or oligomer uniformly instead of a mixture of monomer and oligomer, which can be examined with "dynamic light scattering" or native PAGE.
Size-exclusion chromatography (SEC) is able to separate distribution of protein polymer/aggregate and monomer, but it does not discern oligomer or aggregate, and does not discern dimer, trimer, tetramer and larger oligomer.
So, for protein crystalization, purity and uniformity of monomer or oligomer is the most important thing, either all are monomer or all are natural oligomer with same size. Oligomer mixture with different size(dimer, trimer, tetramer) is not good for crystal formation.
We will identify with PAGE if the polymer part in SEC is natural oligomer or problematic aggregate.
Thank you very much again.
Hi, yes your understanding is correct. Purity and monodispersity are crucial for increasing the success of crystallization. However I wonder how you are going to determine the size of the oligomer/aggregate by native PAGE since with this technique the mobility of your protein is not only determined by size but also by charge.
Best, Tjaard
In some cases proteins that naturally form fibrils and are very difficult to crystallise can be crystallised as defined monomers by either masking the oligomerization interface with crystallisation chaperones such as antibody fragments, DARPins or other binding partners (e.g.. Tubulin complexes crystallised in the presence of DARPins that prevent microtubule formation), or by introducing mutations in the oligomerization interface.
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