Purifying a simple protein

Achim Recktenwald, PhD achim at ibex.ca
Mon Mar 3 08:10:26 EST 1997


Quasi26 at concentric.net wrote:
> 
> I am a student at WPI in Mass. and was wondering if you could help me
> purify this protein.
> 
> The protein has a MW of 6,000, pI @ 4.9  and is intracellular
> The contaminants have MW of  113,000;  13,000;   and 39,000
> Acetate is an inhibitor and the protein is being used for the food
> industry
> It is good at temp. from 22 - 62 C, with the highest activity between
> 40-50 C
> It is good at ph from 4 - 10, with high activity between 6 and 9
> 
> I am just looking for a simple scheme, using tangental flow, ion
> exchanges, HIC, Gel filtration, ultra, PPT etc...
> 
> This is a scheme that I have chosen but I was wondering if you could
> help me get more in depth with it or show something that would work
> better. I use simple flow sheets, but I will write this out because I
> can not think of a way to make a flow sheet on this.
> 
> From the cells, I choose to centrifuge and then break them (French
> Pressure cell), Succinate buffer (pKa of 5.6).  From the homogenate, I
> choose ultrafiltration, using 100K at cutoff (getting rid of 113,000).
> then discard pellet and take your low level supernatant and use cation
> exchanger with succinate again.  pool the active fractions and maybe
> use Sephadex G 25 or HIC.
> 
> This is just a simple scheme, but I was hoping that you could critique
> it and help me on it.  I am taking a purification lab next term, and
> this was given to us to show us what we will be seeing in the future.
> I also have another question,  What ph range should I operate from and
> how do you determine that?
> 
> Hope to hear from you soon.  My email is  davido at wpi.edu       Thank
> you.


I would say, try it, looks good. I don't know how stable your protein
is, but if it is not easily deactivated by the proteases in your
homogenate, try using a lower cut-off ultrafiltration, e.g., 10000 or
20000. 
Another possiblility: skip the ultrafiltration step and go directly to
the cation exchanger. If the homogenate is too turbid because of
colloidal particles, use a 'Big Beads-material' with a larger pore-size;
we do this routinely, I am not quite sure, but I think we buy the gel
from Pharmacia. 


Achim



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