n***w
发帖数: 2405 | 1 Hi, all,
I am using Rosetta-Gami DE3 pLySs to expression my fusion protein which is
predicted as 91kDa.
I first did the expression assay and found protein expression was induced
after adding IPTG by testing the whole cell lysate. (bacteria at certain
time points, add 2X sample buffer, boil, SDS-PAGE).
Then I moved to culture more and lysed the cells using a sonicator, after
resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
the supernatant and pellet side by side and stained by GelCode and a bulk of
protein (I assume it was protein) was detected in the pellet part.
Then I tried 1L culture and repeated the 2nd experiment. This time, I
incubated the supernatant with glutathione sepharose 4b beads and cleaved
the mix with thrombine afterwards. I did not see a clear pattern of fusion+
GST, fusion, GST.
I doubt inclusion bodies formed during expression. Someone here suggested me
to add lysozyme lysis buffer before sonication.
My question is since this strain already contains pLySs which encode
lysozyme, shall I add additional lysozyme? Do you guys have other
recommendations?
Thank you so much. Pardon me that I cannot type in Chinese. |
T**********t
发帖数: 1604 | 2 pLysS只表达少量的Lysozyme用于抑制T7 RNA polymerase，而且表达出来的lysozyme在
胞内而不是细胞外，无法作用在细胞壁上。所以在lysis buffer里加lysozyme是必需的。
只不过我觉得lysozyme对于inclusion body本身的溶解应该没什么作用吧，它的作用估
计只是让细胞降解的更彻底，释放出更多的inclusion body而已。
of
【在 n***w 的大作中提到】
: Hi, all,
: I am using Rosetta-Gami DE3 pLySs to expression my fusion protein which is
: predicted as 91kDa.
: I first did the expression assay and found protein expression was induced
: after adding IPTG by testing the whole cell lysate. (bacteria at certain
: time points, add 2X sample buffer, boil, SDS-PAGE).
: Then I moved to culture more and lysed the cells using a sonicator, after
: resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
: the supernatant and pellet side by side and stained by GelCode and a bulk of
: protein (I assume it was protein) was detected in the pellet part.
|
s********n
发帖数: 2939 | 3 Then I moved to culture more and lysed the cells using a sonicator, after
resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
the supernatant and pellet side by side and stained by GelCode and a bulk of
protein (I assume it was protein) was detected in the pellet part.
你有没有在supernatant中检测到你的target protein？如何检测的？WB or activity?
从你的表述你好像没有做WB。
Then I tried 1L culture and repeated the 2nd experiment. This time, I
incubated the supernatant with glutathione sepharose 4b beads and cleaved
the mix with thrombine afterwards. I did not see a clear pattern of fusion+
GST, fusion, GST.
这一步你是已经确定在supernatant中也有你的蛋白么?
I doubt inclusion bodies formed during expression. Someone here suggested me
to add lysozyme lysis buffer before sonication.
就像ls说的，lysozyme能够帮助cell lysis，最好能加点EDTA （1 mM），而且要确保
pH在7-8之间。
My question is since this strain already contains pLySs which encode
lysozyme, shall I add additional lysozyme? Do you guys have other
recommendations?
这个strain的确表达T7 lysozyme，但是是在胞内，你可以用冻融的方法，可以参照pET
手册。 |
n***w
发帖数: 2405 | 4 Thanks. I did WB as well. Supernatants did show the specific band.
I will try freeze and thaw method rather than sonication tomorrow.
of
activity?
【在 s********n 的大作中提到】
: Then I moved to culture more and lysed the cells using a sonicator, after
: resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
: the supernatant and pellet side by side and stained by GelCode and a bulk of
: protein (I assume it was protein) was detected in the pellet part.
: 你有没有在supernatant中检测到你的target protein？如何检测的？WB or activity?
: 从你的表述你好像没有做WB。
: Then I tried 1L culture and repeated the 2nd experiment. This time, I
: incubated the supernatant with glutathione sepharose 4b beads and cleaved
: the mix with thrombine afterwards. I did not see a clear pattern of fusion+
: GST, fusion, GST.
|
e****s
发帖数: 1125 | 5 那你加Beads以后的检测结果呢？WB检测的吗？
【在 n***w 的大作中提到】
: Thanks. I did WB as well. Supernatants did show the specific band.
: I will try freeze and thaw method rather than sonication tomorrow.
:
: of
: activity?
|
N2
发帖数: 81 | 6 ask a question not relevant to this, R U from NANTONG?
of
【在 n***w 的大作中提到】
: Hi, all,
: I am using Rosetta-Gami DE3 pLySs to expression my fusion protein which is
: predicted as 91kDa.
: I first did the expression assay and found protein expression was induced
: after adding IPTG by testing the whole cell lysate. (bacteria at certain
: time points, add 2X sample buffer, boil, SDS-PAGE).
: Then I moved to culture more and lysed the cells using a sonicator, after
: resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
: the supernatant and pellet side by side and stained by GelCode and a bulk of
: protein (I assume it was protein) was detected in the pellet part.
|
n***w
发帖数: 2405 | 7 Yes. How do you know?
LOL
【在 N2 的大作中提到】
: ask a question not relevant to this, R U from NANTONG?
:
: of
|
g*********r
发帖数: 9366 | 8 这一步要WB和commasie同时跑
大多数inclusion body的蛋白在WB 上也会显现，这个没什么意义
要看在supernatant 中有多少
而且要充分离心，取真正的清液
办法， 降温，少加IPTG，等等
实在不行denature
【在 n***w 的大作中提到】
: Thanks. I did WB as well. Supernatants did show the specific band.
: I will try freeze and thaw method rather than sonication tomorrow.
:
: of
: activity?
|
i***0
发帖数: 160 | 9 Microfluidizer or French Press will break the cell more efficiently compared
to Sonication. |
n***w
发帖数: 2405 | 10 Thanks.
We don't have such device here =(
We are not protein engineering lab so my boss has no plan to purchase one in
the near future.
I repeated my experiments and modified my protocol a little bit and am
running gels now. Let's see how it
goes.
Thank you again!
compared
【在 i***0 的大作中提到】
: Microfluidizer or French Press will break the cell more efficiently compared
: to Sonication.
|
|
|
g*********r
发帖数: 9366 | 11 no, they can not "break" inclusion body
compared
【在 i***0 的大作中提到】
: Microfluidizer or French Press will break the cell more efficiently compared
: to Sonication.
|
N2
发帖数: 81 | 12 From your name."nt"gxw
【在 n***w 的大作中提到】
: Yes. How do you know?
: LOL
|
N2
发帖数: 81 | 13 Check your mailbox at mitbbs!
of
【在 n***w 的大作中提到】
: Hi, all,
: I am using Rosetta-Gami DE3 pLySs to expression my fusion protein which is
: predicted as 91kDa.
: I first did the expression assay and found protein expression was induced
: after adding IPTG by testing the whole cell lysate. (bacteria at certain
: time points, add 2X sample buffer, boil, SDS-PAGE).
: Then I moved to culture more and lysed the cells using a sonicator, after
: resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
: the supernatant and pellet side by side and stained by GelCode and a bulk of
: protein (I assume it was protein) was detected in the pellet part.
|
i***0
发帖数: 160 | 14 For inclusion bodies, you have to reduce temperature and IPTG amount. One of
my proteins has inclusion bodies and very small amount of soluble protein.
But when I tried with 10 uM IPTG induction, the soluble part has about equal
amount of my protein compared to inclusion bodies. But when you have
efficient cell breaking method you will release almost all of the soluble
protein from the cells. So try with more expression conditions before you
enlarge culture size. Personally, I like French Press, since it lyse E.coli
very well without freeze and thaw. |
g*********r
发帖数: 281 | 15 Maybe it is not necessary to ask, where did you put your GST? Normally N-
terminal GST will help your protein folding and solubility.
In term of inclusion body, like people mentioned, lower temperature and IPTG
concentration might help.
Or try some other Tag, like MBP. |
w*********e
发帖数: 98 | 16 Try low temperature at 18 C, which should start right after you inoculate
the overnight culture into the final culture. Then wait until OD is 0.6-1.0,
add low concentration of IPTG such as 0.1 mM IPTG for 16-20 hours at 300rpm
. I just tried this protocol, it increased a lot the ratio of GST fused
protein in soluble fraction compared with that at 37 C. |
n***w
发帖数: 2405 | 17 Thank you guys.
I use PGEX2T vector so GST is on the N-terminus.
I normally do 100ml culture size. Here is how I did this:
1. small culture of the bacteria from glycerol stock O/N, about 6-8ml.
2. transfer the small culture to 100ml 2xYTA medium in the next day morning
, shaking at 300rpm, 37degrees.
3. it ususally takes about 1hr 40min - 2 hrs to have an OD600 around 0.75.
4. add IPTG (stock 100mM) 100ul to get a final concentration of 0.1mM.
5. Lower the temp to 22degrees and shake at 300 rmp for another 3-4 hours.
6. harvest cells at 8000g for 10min.
7. freeze the pellet and thaw on ice.
8. resuspend the pellet with 2ml 1x PBS, add 1% Protease Inhibitor
9. Sonication on ice, 30s continuously followed by 4min incubation on ice, repeat about 8 times.
10. dilute the sonicant to about 8ml and add 10% Triton X-100 to final concentration 1%.
11. incubate at RT on shaker for about 30min.
12. centrifuge at 8000g for 30min, collect the supernatant. (soluble fraction)
13. treat the pellet with detergent buffer (1.5% N-lauroylsarcosine, 25mM triethanolamine and 1mM EDTA pH 8.0)
14. mix on ice for about 10min and centrifuge as above, collect the supernatant (solubilized pellet)
15. SDS-PAGE analysis or WB.
Anything wrong with my current protocol? Thanks.
I will try lower IPTG concentration (final conc. 10uM) as suggested and
lower to the temp to 18degrees.
Thanks. |
n***w
发帖数: 2405 | 18 Hi, how much did you inoculate into the final culture? 0.1mM IPTG is the
final concentration or the stock concentration? Thanks!
0,
300rpm
【在 w*********e 的大作中提到】
: Try low temperature at 18 C, which should start right after you inoculate
: the overnight culture into the final culture. Then wait until OD is 0.6-1.0,
: add low concentration of IPTG such as 0.1 mM IPTG for 16-20 hours at 300rpm
: . I just tried this protocol, it increased a lot the ratio of GST fused
: protein in soluble fraction compared with that at 37 C.
|
w*********e
发帖数: 98 | 19 I inoculated 5ml overnight culture (37 C)into 30ml medium because I was
using 18 C to shake ( cell grows much slower than 37C) until OD is 0.8 and
then I added IPTG at final 0.1mM. The speed of shaker when I started using
18 C is 350 rpm.
|
w*********e
发帖数: 98 | 20 I used 30ml because I was trying to see if my protein could be expressed.
After IPTG was added, the culture was shaked at 18 C for 20 hours in order
to get enough cells.
The big difference based on your protocol is that I started low temperature
the next morning when I did final inoculation, while you still used 37 C.
I compared the soluble and the pellet fractions at 18, 28 and 37 C,so I know
18 C is the best one. |
