Let me make three short notes:
1. If the protein does not bind in a 'normal' buffer try first under
denaturing conditions to see if everything is expressed correctly
(a repeat of Artem's suggestion basically!). Protocols for that are
in all materials manuals.
150 kD in E.coli is very very tough. 190 is even tougher. If you use
a C-term tag,
which has also the advantage it will select 'full length' proteins,
take care of the first few codons; His- and other Tags are
optimized for that. A silent mutation at position 4-5 (after the ATG)
has made for us an easy 10-20 fold difference in the past.
2. Most buffer change pH depending on temperature.
See for example:
http://www.neb.com/nebecomm/tech_reference/general_data/tris_buffer.asp
Always measure pH at the temperature you plan to work at, or make
sure you always measure at the same temperature
and be aware of the problem, look-up tables as above exist for many
buffers.
3. For such a long protein an optimized-codon synthetic gene worked
really well for us for a 100 kD protein,
boosting expression 10-fold. consider it if it ends up to be low-
yield trouble.
A.
On 11 Oct 2007, at 19:02, Jacob Keller wrote:
Recently, a make-or-break difference for me was making sure the pH
was what I thought it was:
In the presence of 50mM HEPES pH 7.0 (at room temp), the pH of my
protein sample was actually
nearer to 6 at 4degC, as judged by pH paper. I am not sure whether
the pH difference was mainly
because of the temperature drop or because of other components in
the mixture, but adding an
additional 50mM TRIS pH 8.5 (RT) made all the difference. The pH
paper after this addition
indicated a pH of 7-8. In the former case, the protein was
completely undetectable in elution
fractions, and in the latter, was successfully purified. Of course
this makes complete biochemical
sense, but nevertheless I am very glad I checked the pH.
Jacob Keller
On Wed, 10 Oct 2007 8:12:46 pm CDT changrui lu wrote:
Dear all,
I am trying to express a 150 kd protein in E coli. I have it in two
constructs, one with pmal-his and other with only his tag at N
terminus. The
full length protein can be detected both by sds and western using
anti-his
(190kd and 150kd respectively) but strangely neither binds to his-
column
very well. The majority of the full length comes through the column
either
at loading step or low salt wash step. The major species that gets
trapped
and eluted is the mbp-his truncation (~40kd). Some, though very
little, full
length protein did make it out the his column. The pmal-his
construct does
not bind amylose resin any better with majority flows right
through. All
purification are carried out under standard conditions as mentioned
in the
manuals. The protein is soluble and does not precipitate in the
columns. I
appreciate and ideas or explanations.
Thanks in advance.
Ray
Cornell Univerisity
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Jacob Keller
Northwestern University
6541 N. Francisco #3
Chicago IL 60645
(847)491-2438
[EMAIL PROTECTED]
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