Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Peter Hsu]

Hi Sutapa,

With regards to insect cell expression, how do you know it wasn't expressed? 
You didn't see a band by coomassie staining or by western? 

If by staining, I'd recommend to just try expressing a limited amount through 
infection of a few 150mm dishes of your favorite cell line (I use High fives), 
and then do a quick lysis and purification over a small (200uL) column. In my 
experience, most of my proteins (including some big ones around 100kda) don't 
show up readily on a whole cell lysate, but you can actually see it and have 
quite a bit of it after seeing it on a column. While it's a pain, I find it far 
easier to get larger yields of protein from monolayer plates of insect cells vs 
suspension culture. Something about plates produces far happier cells = more 
protein. 

Are you using a His or GST tag for insect cells? My experience has always been 
use GST for insect cells, the purification is far simpler and your protein is 
much, much cleaner as a result. 

Hope it helps,
P

Re: [ccp4bb] FW: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Lakshmi SwarnaMukhi Pidugu]

Hi Sutapa,

Slow expression did work in my case. I had to induce with 100uM IPTG, and
grow cells at 10C for 48 hours. It left at least 50% of expressed protein
in soluble fraction.

Best,
Swarna

On Wed, Apr 5, 2017 at 1:52 AM, AJV  wrote:

> Hi Sutapa,
>
> The input from other commenters has been great, so I'll stick to
> discussing only the portion of your question regarding baculovirus.
>
> In our experience, codon-optimization has resulted in little to no
> improvement of total protein yields or protein solubility in baculovirus.
> We routinely work on challenging targets (mammalian membrane proteins), and
> while we don't have mounds of data on the subject, we have expressed enough
> eukaryotic protein families to say this with some confidence. Our
> workaround to bad expressers has been to screen orthologs of the protein of
> interest, using life's natural codon diversity to our advantage.
>
> That being said, my suggestion would be to attempt baculovirus again, as
> there must have been a good reason for you to try it in the first place.
> Not knowing whether your gene is eukaryotic, specifically mammalian, or if
> post-translational modifications could be important; but if any of these
> are the case, baculovirus would be one of the best systems to pound on.
>
> We test several variables that always result in "seeing" some amount of
> protein expression with baculovirus (depending on detection method) --
> time, temperature, and MOI. Once high titer / low passage virus is obtained
> and titered (can't stress the importance of titering enough), I usually set
> up six 50mL suspension cultures of Sf9 cells and infect them at 2x10^6
> cells/mL, using MOIs of 0.2, 2.0, and 20.0, in duplicate. After 24hrs at
> 27°C, I place three of the cultures at each MOI in an identical shaking
> incubator at 20-23°C, and leave the other three at 27°C. I then remove ~5mL
> of culture at various time points (about every 12 or 24hrs depending on
> scheduling), up to ~120hrs for all six samples. Then, crack the cells and
> use your detection method of choice (GFP, Western, Coomassie) to assess
> expression. This workflow can be repeated using other insect cell types
> (Sf21, Tn5), as they all will vary in their protein expression capabilities.
>
> Hope this answers part of your question, and results in some quality
> sample.
>
> Best,
>
> Alex
> _
> Alex J. Vecchio, Ph.D.
> Postdoctoral Fellow
> University of California, San Francisco
> Department of Biochemistry & Biophysics
> Laboratory of Robert M. Stroud, Ph.D.
>

Re: [ccp4bb] FW: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[AJV]

Hi Sutapa,

The input from other commenters has been great, so I'll stick to discussing 
only the portion of your question regarding baculovirus.

In our experience, codon-optimization has resulted in little to no improvement 
of total protein yields or protein solubility in baculovirus. We routinely work 
on challenging targets (mammalian membrane proteins), and while we don't have 
mounds of data on the subject, we have expressed enough eukaryotic protein 
families to say this with some confidence. Our workaround to bad expressers has 
been to screen orthologs of the protein of interest, using life's natural codon 
diversity to our advantage.

That being said, my suggestion would be to attempt baculovirus again, as there 
must have been a good reason for you to try it in the first place. Not knowing 
whether your gene is eukaryotic, specifically mammalian, or if 
post-translational modifications could be important; but if any of these are 
the case, baculovirus would be one of the best systems to pound on. 

We test several variables that always result in "seeing" some amount of protein 
expression with baculovirus (depending on detection method) -- time, 
temperature, and MOI. Once high titer / low passage virus is obtained and 
titered (can't stress the importance of titering enough), I usually set up six 
50mL suspension cultures of Sf9 cells and infect them at 2x10^6 cells/mL, using 
MOIs of 0.2, 2.0, and 20.0, in duplicate. After 24hrs at 27°C, I place three of 
the cultures at each MOI in an identical shaking incubator at 20-23°C, and 
leave the other three at 27°C. I then remove ~5mL of culture at various time 
points (about every 12 or 24hrs depending on scheduling), up to ~120hrs for all 
six samples. Then, crack the cells and use your detection method of choice 
(GFP, Western, Coomassie) to assess expression. This workflow can be repeated 
using other insect cell types (Sf21, Tn5), as they all will vary in their 
protein expression capabilities. 

Hope this answers part of your question, and results in some quality sample.

Best,

Alex
_
Alex J. Vecchio, Ph.D.
Postdoctoral Fellow
University of California, San Francisco
Department of Biochemistry & Biophysics
Laboratory of Robert M. Stroud, Ph.D.

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Mark J van Raaij]

Hello Sutapa,

With codon optimisation you would get more and faster expression and likely 
even more insolubility in E. coli (less time for folding). To get the protein 
more soluble perhaps slower production might help. I guess you could try codon 
de-optimisation :-). Cheaper possibilities are growth at lower temperature, 
using a slower-growing E coli strain, using less IPTG or even no IPTG and just 
relying on the leaky expression, but perhaps you have tried all these already.
Or another expression plasmid with tighter control or a different system like 
yeast.
Codon optimisation for insect cells might be worth it if your analysis shows 
that the current sequence is really badly optimised.
Or perhaps your protein needs a specific natural chaperone...it  might be worth 
studying the natural production of your protein more before designing an 
expression strategy.
Without knowing more details about your protein it's hard to give more help - 
you could try to contact other researchers studying similar proteins.

Greetings,

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser .cnb.csic.es/~mjvanraaij 




> On 03 Apr 2017, at 07:49, Sutapa Chakrabarti  
> wrote:
> 
> Dear All,
> 
> We’re trying to express and purify a 1000 residue long protein and have run 
> into the problem that it is completely insoluble when expressed in E.coli and 
> is not expressed at all in insect cells. The usual tricks for improving 
> solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
> media and induction conditions and use of different cell strains, have not 
> led to any improvement. 
> 
> We are now looking into ordering a codon-optimised synthetic gene for this 
> protein and are trying to decide whether it would be worthwhile to 
> codon-optimise for expression in E.coli (given that the protein was expressed 
> but not soluble) or if we should attempt baculovirus expression again with a 
> gene that has been codon-optimised for insect cells. 
> 
> My question is:
> has anyone observed an improvement in the solubility of their target protein 
> using a codon optimised gene? 
> 
> I know of several instances where the use of a codon-optimised gene has led 
> to expression where the native gene sequence did not but am unable to find 
> any references for improvement in solubility. Since codon optimisation 
> significantly alters the translation rate of a gene, I believe this should 
> affect solubility as well; but I’d like to know what the community thinks/has 
> observed before I order an exorbitantly priced gene! 
> 
> Thank you in advance,
> Sutapa
> 
> --
> Sutapa Chakrabarti, Ph.D.
> Institute of Chemistry and Biochemistry
> Freie Universität Berlin
> Takustr. 6 
> 14195 Berlin
> Germany
> Phone: +49-(0)30-83875094
> 
> 
> 
> 
> 
> 

Re: [ccp4bb] FW: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[zaigham mahmood khan]

Dear Sutapa

There has been wonderful suggestions, particularly from Bert, size matters!

Considering that all is in order, i would share my experience with
retroviral integrase, a sparingly soluble protein when expressed in E.
coli. The protein exists in mammalian cells as a multi-protein complex
assembly. When expressed alone, many of the surface-exposed residues were
available for non-specific interaction. Surprisingly, mutation of one
hydrophobic residue to lysin turned the table around, and we got ample
protein in soluble form. We also tried super-charging algorithm from
Rosetta, as well mutation of those surface-exposed residues that are
involved in the protein-protein interaction.

There must be a way to express these challenging proteins in E. coli. We
always start with the easy and handy tricks, but some proteins are just
notorious, and demand more attention!

Best of luck!


-Z


Zaigham Mahmood Khan, PhD

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York

On Mon, Apr 3, 2017 at 6:30 AM, Noam Adir  wrote:

> Dear Supta,
>
>
>
> If I may make another type of suggestion, we have had success in
> crystallizing sparingly soluble proteins in the presence of up to 2M urea
> (Dines et al. Journal of Structural Biology, 2007). It is enough to avoid
> non-specific associations, but not enough to denature the protein.  It is
> certainly worth a try.
>
>
>
> Noam
>
>
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Sutapa
> Chakrabarti
> *Sent:* Monday, April 3, 2017 9:50 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Using a codon-optimised gene to improve protein
> solubility
>
>
>
> Dear All,
>
>
>
> We’re trying to express and purify a 1000 residue long protein and have
> run into the problem that it is completely insoluble when expressed in *E.coli
> *and is not expressed at all in insect cells. The usual tricks for
> improving solubility in *E.coli, *such as addition of GST/MBP tags,
> optimising expression media and induction conditions and use of different
> cell strains, have not led to any improvement.
>
>
>
> We are now looking into ordering a codon-optimised synthetic gene for this
> protein and are trying to decide whether it would be worthwhile to
> codon-optimise for expression in *E.coli *(given that the protein was
> expressed but not soluble) or if we should attempt baculovirus expression
> again with a gene that has been codon-optimised for insect cells.
>
>
>
> *My question is:*
>
> *has anyone observed an improvement in the solubility of their target
> protein using a codon optimised gene?*
>
>
>
> I know of several instances where the use of a codon-optimised gene has
> led to expression where the native gene sequence did not but am unable to
> find any references for improvement in solubility. Since codon optimisation
> significantly alters the translation rate of a gene, I believe this should
> affect solubility as well; but I’d like to know what the community
> thinks/has observed before I order an exorbitantly priced gene!
>
>
>
> Thank you in advance,
>
> Sutapa
>
>
>
> --
>
> Sutapa Chakrabarti, Ph.D.
>
> Institute of Chemistry and Biochemistry
>
> Freie Universität Berlin
>
> Takustr. 6
>
> 14195 Berlin
>
> Germany
>
> Phone: +49-(0)30-83875094 <+49%2030%2083875094>
>
>
>
>
>
>
>
>
>
>
>

[ccp4bb] FW: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Noam Adir]

Dear Supta,

If I may make another type of suggestion, we have had success in crystallizing 
sparingly soluble proteins in the presence of up to 2M urea (Dines et al. 
Journal of Structural Biology, 2007). It is enough to avoid non-specific 
associations, but not enough to denature the protein.  It is certainly worth a 
try.

Noam

[cid:image001.jpg@01D2AC7E.67A52A60]

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sutapa 
Chakrabarti
Sent: Monday, April 3, 2017 9:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Using a codon-optimised gene to improve protein solubility

Dear All,

We’re trying to express and purify a 1000 residue long protein and have run 
into the problem that it is completely insoluble when expressed in E.coli and 
is not expressed at all in insect cells. The usual tricks for improving 
solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
media and induction conditions and use of different cell strains, have not led 
to any improvement.

We are now looking into ordering a codon-optimised synthetic gene for this 
protein and are trying to decide whether it would be worthwhile to 
codon-optimise for expression in E.coli (given that the protein was expressed 
but not soluble) or if we should attempt baculovirus expression again with a 
gene that has been codon-optimised for insect cells.

My question is:
has anyone observed an improvement in the solubility of their target protein 
using a codon optimised gene?

I know of several instances where the use of a codon-optimised gene has led to 
expression where the native gene sequence did not but am unable to find any 
references for improvement in solubility. Since codon optimisation 
significantly alters the translation rate of a gene, I believe this should 
affect solubility as well; but I’d like to know what the community thinks/has 
observed before I order an exorbitantly priced gene!

Thank you in advance,
Sutapa

--
Sutapa Chakrabarti, Ph.D.
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustr. 6
14195 Berlin
Germany
Phone: +49-(0)30-83875094

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Bert Van-Den-Berg]

I think it is very unlikely codon optimisation will improve solubility, so I'd 
save my money and use it to try other things. Assuming you have tried (much) 
lower temperatures for expression you could consider dialing down expression 
via a different promoter or low-copy number plasmid. I assume your protein is 
eukaryotic given you have tried insect cells. What about yeasts? I also assume 
your protein is not a membrane protein?


It's also good to keep in mind that funstional expression of such large protein 
is challenging to say the least.


Bert



From: CCP4 bulletin board  on behalf of Sutapa 
Chakrabarti 
Sent: 03 April 2017 07:49
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Using a codon-optimised gene to improve protein solubility

Dear All,

We’re trying to express and purify a 1000 residue long protein and have run 
into the problem that it is completely insoluble when expressed in E.coli and 
is not expressed at all in insect cells. The usual tricks for improving 
solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
media and induction conditions and use of different cell strains, have not led 
to any improvement.

We are now looking into ordering a codon-optimised synthetic gene for this 
protein and are trying to decide whether it would be worthwhile to 
codon-optimise for expression in E.coli (given that the protein was expressed 
but not soluble) or if we should attempt baculovirus expression again with a 
gene that has been codon-optimised for insect cells.

My question is:
has anyone observed an improvement in the solubility of their target protein 
using a codon optimised gene?

I know of several instances where the use of a codon-optimised gene has led to 
expression where the native gene sequence did not but am unable to find any 
references for improvement in solubility. Since codon optimisation 
significantly alters the translation rate of a gene, I believe this should 
affect solubility as well; but I’d like to know what the community thinks/has 
observed before I order an exorbitantly priced gene!

Thank you in advance,
Sutapa

--
Sutapa Chakrabarti, Ph.D.
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustr. 6
14195 Berlin
Germany
Phone: +49-(0)30-83875094

[ccp4bb] AW: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Herman . Schreuder]

Dear Sutapa,

I fully agree with Grant, the first question is whether the naturally-produced 
protein is soluble and whether your protein is not a membrane protein, or a 
domain, cut out of a much larger protein? The other question is whether your 
protein is toxic for E.coli and only the bacteria producing it in inclusion 
bodies survive.

Another thing is to consider is to produce the protein in the same class of 
organism as the natural producer, e.g. prokaryotic proteins in a bacterial 
system, mammalian in proteins in mammalian cells etc.

My 2 cents,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Vipul 
Panchal
Gesendet: Montag, 3. April 2017 09:06
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

There is also lack of information here.  Do you really expect this protein in 
soluble fraction? Is it a membrane associated or transmembrane protein?

Well, I don't have any experience in protein expression using codon 
optimization.
However, Considering the fact that in E.coli it is getting expressed in 
insoluble fraction, I would recommend to co-express this protein with groEL/ES 
system. I have plasmid expressing groEL/ES operon.

On Mon, Apr 3, 2017 at 12:22 PM, Hansman, Grant 
mailto:g.hans...@dkfz-heidelberg.de>> wrote:
Hi,

Why don't you try adding GST/MBP tags first? This is a easy quick test.

We have a nice fusion (MBP) vector for e.coli expression if you want.

Grant

From: Sutapa Chakrabarti 
mailto:chakr...@zedat.fu-berlin.de><mailto:chakr...@zedat.fu-berlin.de<mailto:chakr...@zedat.fu-berlin.de>>>
Reply-To: Sutapa Chakrabarti 
mailto:chakr...@zedat.fu-berlin.de><mailto:chakr...@zedat.fu-berlin.de<mailto:chakr...@zedat.fu-berlin.de>>>
Date: Monday 3 April 2017 08:49
To: 
"CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK><mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>"
 
mailto:CCP4BB@JISCMAIL.AC.UK><mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>>
Subject: [ccp4bb] Using a codon-optimised gene to improve protein solubility

Dear All,

We're trying to express and purify a 1000 residue long protein and have run 
into the problem that it is completely insoluble when expressed in E.coli and 
is not expressed at all in insect cells. The usual tricks for improving 
solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
media and induction conditions and use of different cell strains, have not led 
to any improvement.

We are now looking into ordering a codon-optimised synthetic gene for this 
protein and are trying to decide whether it would be worthwhile to 
codon-optimise for expression in E.coli (given that the protein was expressed 
but not soluble) or if we should attempt baculovirus expression again with a 
gene that has been codon-optimised for insect cells.

My question is:
has anyone observed an improvement in the solubility of their target protein 
using a codon optimised gene?

I know of several instances where the use of a codon-optimised gene has led to 
expression where the native gene sequence did not but am unable to find any 
references for improvement in solubility. Since codon optimisation 
significantly alters the translation rate of a gene, I believe this should 
affect solubility as well; but I'd like to know what the community thinks/has 
observed before I order an exorbitantly priced gene!

Thank you in advance,
Sutapa

--
Sutapa Chakrabarti, Ph.D.
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustr. 6
14195 Berlin
Germany
Phone: +49-(0)30-83875094



--
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Vipul Panchal]

There is also lack of information here.  Do you really expect this protein
in soluble fraction? Is it a membrane associated or transmembrane protein?

Well, I don't have any experience in protein expression using codon
optimization.
However, Considering the fact that in E.coli it is getting expressed in
insoluble fraction, I would recommend to co-express this protein with
groEL/ES system. I have plasmid expressing groEL/ES operon.

On Mon, Apr 3, 2017 at 12:22 PM, Hansman, Grant <
g.hans...@dkfz-heidelberg.de> wrote:

> Hi,
>
> Why don't you try adding GST/MBP tags first? This is a easy quick test.
>
> We have a nice fusion (MBP) vector for e.coli expression if you want.
>
> Grant
>
> From: Sutapa Chakrabarti  chakr...@zedat.fu-berlin.de>>
> Reply-To: Sutapa Chakrabarti  chakr...@zedat.fu-berlin.de>>
> Date: Monday 3 April 2017 08:49
> To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" <
> CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>
> Subject: [ccp4bb] Using a codon-optimised gene to improve protein
> solubility
>
> Dear All,
>
> We're trying to express and purify a 1000 residue long protein and have
> run into the problem that it is completely insoluble when expressed in
> E.coli and is not expressed at all in insect cells. The usual tricks for
> improving solubility in E.coli, such as addition of GST/MBP tags,
> optimising expression media and induction conditions and use of different
> cell strains, have not led to any improvement.
>
> We are now looking into ordering a codon-optimised synthetic gene for this
> protein and are trying to decide whether it would be worthwhile to
> codon-optimise for expression in E.coli (given that the protein was
> expressed but not soluble) or if we should attempt baculovirus expression
> again with a gene that has been codon-optimised for insect cells.
>
> My question is:
> has anyone observed an improvement in the solubility of their target
> protein using a codon optimised gene?
>
> I know of several instances where the use of a codon-optimised gene has
> led to expression where the native gene sequence did not but am unable to
> find any references for improvement in solubility. Since codon optimisation
> significantly alters the translation rate of a gene, I believe this should
> affect solubility as well; but I'd like to know what the community
> thinks/has observed before I order an exorbitantly priced gene!
>
> Thank you in advance,
> Sutapa
>
> --
> Sutapa Chakrabarti, Ph.D.
> Institute of Chemistry and Biochemistry
> Freie Universität Berlin
> Takustr. 6
> 14195 Berlin
> Germany
> Phone: +49-(0)30-83875094
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Hansman, Grant]

Hi,

Why don't you try adding GST/MBP tags first? This is a easy quick test.

We have a nice fusion (MBP) vector for e.coli expression if you want.

Grant

From: Sutapa Chakrabarti 
mailto:chakr...@zedat.fu-berlin.de>>
Reply-To: Sutapa Chakrabarti 
mailto:chakr...@zedat.fu-berlin.de>>
Date: Monday 3 April 2017 08:49
To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Using a codon-optimised gene to improve protein solubility

Dear All,

We're trying to express and purify a 1000 residue long protein and have run 
into the problem that it is completely insoluble when expressed in E.coli and 
is not expressed at all in insect cells. The usual tricks for improving 
solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
media and induction conditions and use of different cell strains, have not led 
to any improvement.

We are now looking into ordering a codon-optimised synthetic gene for this 
protein and are trying to decide whether it would be worthwhile to 
codon-optimise for expression in E.coli (given that the protein was expressed 
but not soluble) or if we should attempt baculovirus expression again with a 
gene that has been codon-optimised for insect cells.

My question is:
has anyone observed an improvement in the solubility of their target protein 
using a codon optimised gene?

I know of several instances where the use of a codon-optimised gene has led to 
expression where the native gene sequence did not but am unable to find any 
references for improvement in solubility. Since codon optimisation 
significantly alters the translation rate of a gene, I believe this should 
affect solubility as well; but I'd like to know what the community thinks/has 
observed before I order an exorbitantly priced gene!

Thank you in advance,
Sutapa

--
Sutapa Chakrabarti, Ph.D.
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustr. 6
14195 Berlin
Germany
Phone: +49-(0)30-83875094

[ccp4bb] Using a codon-optimised gene to improve protein solubility

[Sutapa Chakrabarti]

Dear All,

We’re trying to express and purify a 1000 residue long protein and have run 
into the problem that it is completely insoluble when expressed in E.coli and 
is not expressed at all in insect cells. The usual tricks for improving 
solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
media and induction conditions and use of different cell strains, have not led 
to any improvement. 

We are now looking into ordering a codon-optimised synthetic gene for this 
protein and are trying to decide whether it would be worthwhile to 
codon-optimise for expression in E.coli (given that the protein was expressed 
but not soluble) or if we should attempt baculovirus expression again with a 
gene that has been codon-optimised for insect cells. 

My question is:
has anyone observed an improvement in the solubility of their target protein 
using a codon optimised gene? 

I know of several instances where the use of a codon-optimised gene has led to 
expression where the native gene sequence did not but am unable to find any 
references for improvement in solubility. Since codon optimisation 
significantly alters the translation rate of a gene, I believe this should 
affect solubility as well; but I’d like to know what the community thinks/has 
observed before I order an exorbitantly priced gene! 

Thank you in advance,
Sutapa

--
Sutapa Chakrabarti, Ph.D.
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustr. 6 
14195 Berlin
Germany
Phone: +49-(0)30-83875094