I've been pretty happy with an MBP fusion of my 7.5 kDa protein that has an
H3C protease site between the 2. The vector system was published in a
recent biotechniques (sorry dont have it handy). I get reasonable
expression (but not the 100 mg/L NEB claims. ha!). You express your own
H3C protease as well, or buy "Prescission" from Pharmacia. The fusion
protein can be purified via IMAC or amylose resin (It is simple to make
your own amylose resin if you want to save a few pennies).
dk at no.email.thankstospam.net (D.K.) wrote in
news:ab7fes$6nl$2 at news.doit.wisc.edu:
> Scott <scj-13 at deletemecharter.net> wrote:
>>I need to express a 5 KDa and a 10 KDa protein in E. coli. I've tried a
>>number of candidates, but they always express very poorly.
>>Interestingly, if I clone two tandem copies(of a 10 KDa) of the same
>>thing it works very well. E. coli seems to not really tollerate such
>>small protein molecules very well.
>> This is a well-known fact.
>>>Has anybody expressed anything like this?
>>It doesn't really matter what it is. It can be a full length
>>protein or just a portion.
>> People do it all the time. Most frequently as a fusion with
> something else (GST, MBP), but sometimes as a tandem just
> like you describe. In all cases a protease-sensitive linker
> is introduced in between. Thrombin is the cheapest choice
> if it does not cleave the peptide of interest.
>> DK
>
