Hi Reza,
A few month ago I had a the exact same problem and we checked everything
we could think of but without any improvement. Finally we were able to
solve the problem only by subcloning the ORF into another plasmid (pET9a
or pET29b). The big difference being the His tag position (C-ter or
N-ter) might be critical for some protein solubility in large scale
protein expression.
Hope it could help
Abbas
Le 20/03/2015 11:56, Reza Khayat a écrit :
Hi,
I’ve received a number of concurring suggestions. Some have requested
more detail about the experiments. Here are the details.
1. Cells: BL21(DE3)
2. Plasmid: pET28a (T7 promoter)
3. Media: TB
4. Protein: cytoplasmic
5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce
with 1mM IPTG, grow <16hrs at 20degC, centrifuge.
6. Lysis method: Sonication via micro-tip/macro-tip. Small culture
produces soluble protein where as large culture produces insoluble
protein. We first thought it may be due to poor lysis, thus equivalent
amount of cells from 3 and 500ml cultures were lysed with the
micro-tip. Same results were observed.
Best wishes,
Reza
Reza Khayat, PhD
Assistant Professor
City College of New York
160 Convent Ave, MR-1135
New York, NY 10031
(212) 650-6070
[email protected] <mailto:[email protected]>
On Mar 19, 2015, at 11:15 PM, John Fisher <[email protected]
<mailto:[email protected]>> wrote:
Hi Reza.
Clearly nobody needs to know anything about what protein you are
specifically working on; that being said, in order to avoid a
potentially endless email string of expert advices, please include
everything detail-wise regarding your expression system, culture
conditions, induction, and lysis method for BOTH the 3 ml and 500 ml
expression trials. You will get, I imagine, amazing advices likely
specific enough to solve the problem without your having to chase
your tail.
Best,
John Fisher
John C. Fisher, M.D./PhD
On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat <[email protected]
<mailto:[email protected]>> wrote:
Hi,
We can express quite a bit of soluble protein when growing 3ml
cultures. However, the protein becomes insoluble (inclusion
bodies) when we scale up to 500ml cultures. Has anyone
experienced such a problem, and found a solution to it? Thanks.
Best wishes,
Reza
Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
New York, NY 10031
http://www.khayatlab.org/
212-650-6070 <tel:212-650-6070>
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Ph.D. Student
(+33) 01.69.82.42.49
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