Fusion protein and Enterokinase

Jennifer Lyon jennyann at ix.netcom.com
Thu May 1 10:03:48 EST 1997

>Huang wrote:

>>I am experessing some genes in E. coli by using pET32, it gave me
>>good result. But the problem is when I tried to relase the native
>>protein from fusion protein using enterokinase, I could not get the
>>amount what I want although I followed the protocol Novagen provided.


Ted replied:

><snip>check the activity of the Novagen single chain, Pichia
>expresssed enterokinase, it is far less active than the mammalian
>derived enzyme available from a number of other sources. <snip>Try
>some of the purified native enterokinase and see if it works better.

Actually, Novagen's recombinant EK is expressed in E. coli, not in
Pichia. And it has a high specific activity in cutting pET-expressed
fusion proteins (it is quality controlled for that specific function).
However, the nature of every fusion protein is different, which can
have an effect on the efficiency of cleavage under given conditions.

Huang, feel free to call Novagen technical service (800-207-0144) for
assistance. Also, check your Novagen protocol for help with digestion
conditions - it may be that you need to use a low level denaturant
(urea, up to 2M, not more) to open up your protein structure to allow
EK to find its target site. Also, a list of factors that affect
digestion is listed in the Novagen protocols, including protease-target
ratio, sensitivity to salt concentrations, denaturants and chaotropes,
detergents, incubation time and temperature, etc. 

I would strongly recommend using the cleavage control protein that is
supplied with the rEK to verify the function of the enzyme. Like any
enzyme, rEK can occasionally go bad due to mishandling during
dispensing, shipping, receiving and storage. So make sure that the rEK
will cut the control protein properly (preferably under the same
conditions you are cutting your own fusion protein).

The rEK from Novagen is quality controlled for the absence of other
contaminating protease activities, so those should not be a problem.
Please contact Novagen if you have any further questions or problems.

Novagen Technical Services

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