Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Parthasarathy Sampathkumar]

Hi Radu,

Many Thanks for your clarifications, and reference for background
materials. I will implement all suggestions in my next expression batch.

Best Wishes,
Partha

On Mon, Jul 16, 2018 at 1:53 PM,  wrote:

> Hi Partha,
>
> In this case (un-treated HEK293), you cannot use EndoH with already
> purified
> proteins (as per your reply to Artem's email). The N-glycans will be
> fucosylated, hence EndoH-insensitive (see PMID: 23623336 for background
> infos). You can still use PNGase. But, as Artem suggested, this often
> causes
> protein precipitation when it can access the sites on folded proteins.
> Apart
> from shaving the glycan shield, PNGase causes unwanted mutations at the
> N-linked sites: Asn -> Asp, which is where most problems stem from.
>
> Nevertheless, since your fully glycosylated protein doesn't crystallize,
> there's not much else you can do with the current prep Except for
> example
> masking your glycans with a nice lectin such as griffithsin, which would be
> very elegant for crystallography. Or indeed trying cryo-EM, where wild-type
> N-linked glycans are extremely helpful! Why would anyone crystallize
> proteins
> these days anyway :-)
>
> Best wishes,
>
> Radu
> --
> Radu Aricescu
> MRC Laboratory of Molecular Biology
> Francis Crick Avenue
> Cambridge Biomedical Campus
> Cambridge CB2 0QH, U.K.
> tel: +44-(0)1223-267049
> fax: +44-(0)1223-268305
> www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu
>
> > Hi Savvas,
> > Many Thanks for your inputs and references. This cell line used is not
> HEK293S *MGAT1-/-. *This particular batch was purified from HEK293
> cell-line
> stably expressing (created using lenti-methods by a
> > former colleague) the protein of interest. In future, I will plan to do
> expression in the presence of Kifunensine, followed by EndoH treatment
> before complexation and crystallization.
> > Best Wishes,
> > Partha
> > On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides <
> savvas.savvi...@ugent.be>
> wrote:
> >> Dear Partha
> >> you do not specify which HEK293 cell line you have used, but if it so
> happens that it is the very handy HEK293S *MGAT1-/- *cell line
> >> (previously known as HEK293S *GnTI-/- ) *which produces
> >> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH
> (e.g.
> see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
> alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
> immuni.2017.12.008).
> >> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
> quite well for protein expression in HEK293T and renders N-linked glycans
> digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
> et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et al.
> doi:10.1038/nsmb.2367).
> >> If resources and protein material allow, you might also want to consider
> the permutation exercise of subjecting the complex to deglycosylation, or
> the individual components followed by complex formation/purification, or
> just one of the two components followed by complex formation/purification,
> or even one of the two components followed by further deglycosylation of
> the complex. We are becoming more and more apprehensive of the possible
> role of glycans in complex formation.
> >> And then there is of course the option to apply mutagenesis, e.g. via
> N—>Q, to eliminate certain N-linked glycans either as a standalone
> approach
> >> or in combination with enzymatic glycan digestions as described above.
> Best
> wishes
> >> Savvas
> >> *---*
> >> *Savvas Savvides*
> >> VIB Center for Inflammation Research
> >> Dept. Biochemistry & Microbiology, Ghent University
> >> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
> >> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
> savvas.savvides_skype
> >> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx
> On
> 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar 
> wrote:
> >> Dear All,
> >> I am in a situation, almost for the first time within my limited
> experience, that deglycosylation might be necessary to obtain crystal. So,
> I thought of tapping to vast experience of CCP4BBers, while I am searching
> literature.
> >> I have protein that has been expressed in HEK293 cells, secreted into
> media, purified over IMAC and SEC columns. Crystallization-screens with its
> binding partners (they form good complexes based on analytical SEC) have
> not produced any useful hits (whereas complexes with related proteins
> worked well). So, I plan to re-try complex formation and \crystallization
> screen after deglysosylation.
> >> My question is: In practice, Does a kit (for example here: https://www.
> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
> containing
> Endo F1, F2, F3 be sufficient or should this be tried in combination with
> PNGase (which requires
> >> desaturating conditions)?!!
> >> Many Thanks in advance for your suggestions, and reference.
> >> 

Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[radu]

Oops,

I made a mistake in my previous email, apologies! Griffithsin would also
require high-Man glycans, so kifunensine treatment or GnTI-/- cells So
yes, it's either PNGase or cryo-EM...

Best wishes,

Radu



> Hi Savvas,
>
> Many Thanks for your inputs and references. This cell line used is not
> HEK293S *MGAT1-/-. *This particular batch was purified from HEK293
> cell-line stably expressing (created using lenti-methods by a
> former colleague) the protein of interest. In future, I will plan to do
> expression in the presence of Kifunensine, followed by EndoH treatment
> before complexation and crystallization.
>
> Best Wishes,
> Partha
>
>
> On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides 
> wrote:
>
>> Dear Partha
>> you do not specify which HEK293 cell line you have used, but if it so
>> happens that it is the very handy HEK293S *MGAT1-/- *cell line
>> (previously known as HEK293S *GnTI-/- ) *which produces
>> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH (e.g.
>> see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
>> alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
>> immuni.2017.12.008).
>> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
>> quite well for protein expression in HEK293T and renders N-linked glycans
>> digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
>> et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et
>> al. doi:10.1038/nsmb.2367).
>>
>> If resources and protein material allow, you might also want to consider
>> the permutation exercise of subjecting the complex to deglycosylation, or
>> the individual components followed by complex formation/purification, or
>> just one of the two components followed by complex formation/purification,
>> or even one of the two components followed by further deglycosylation of
>> the complex. We are becoming more and more apprehensive of the possible
>> role of glycans in complex formation.
>> And then there is of course the option to apply mutagenesis, e.g. via
>> N—>Q, to eliminate certain N-linked glycans either as a standalone
>> approach
>> or in combination with enzymatic glycan digestions as described above.
>>
>> Best wishes
>> Savvas
>>
>>
>> *---*
>> *Savvas Savvides*
>> VIB Center for Inflammation Research
>> Dept. Biochemistry & Microbiology, Ghent University
>> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
>> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
>> savvas.savvides_skype
>> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx
>>
>> On 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar 
>> wrote:
>>
>> Dear All,
>>
>> I am in a situation, almost for the first time within my limited
>> experience, that deglycosylation might be necessary to obtain crystal. So,
>> I thought of tapping to vast experience of CCP4BBers, while I am searching
>> literature.
>>
>> I have protein that has been expressed in HEK293 cells, secreted into
>> media, purified over IMAC and SEC columns. Crystallization-screens with its
>> binding partners (they form good complexes based on analytical SEC) have
>> not produced any useful hits (whereas complexes with related proteins
>> worked well). So, I plan to re-try complex formation and \crystallization
>> screen after deglysosylation.
>>
>> My question is: In practice, Does a kit (for example here: https://www.
>> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
>> containing Endo F1, F2, F3 be sufficient or should this be tried in
>> combination with PNGase (which requires
>> desaturating conditions)?!!
>>
>> Many Thanks in advance for your suggestions, and reference.
>>
>> Best Wishes,
>> Partha
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>>
>>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
fax: +44-(0)1223-268305
www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[radu]

Hi Partha,

In this case (un-treated HEK293), you cannot use EndoH with already purified
proteins (as per your reply to Artem's email). The N-glycans will be
fucosylated, hence EndoH-insensitive (see PMID: 23623336 for background
infos). You can still use PNGase. But, as Artem suggested, this often causes
protein precipitation when it can access the sites on folded proteins. Apart
from shaving the glycan shield, PNGase causes unwanted mutations at the
N-linked sites: Asn -> Asp, which is where most problems stem from.

Nevertheless, since your fully glycosylated protein doesn't crystallize,
there's not much else you can do with the current prep Except for example
masking your glycans with a nice lectin such as griffithsin, which would be
very elegant for crystallography. Or indeed trying cryo-EM, where wild-type
N-linked glycans are extremely helpful! Why would anyone crystallize proteins
these days anyway :-)

Best wishes,

Radu
-- 
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
fax: +44-(0)1223-268305
www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu

> Hi Savvas,
> Many Thanks for your inputs and references. This cell line used is not
HEK293S *MGAT1-/-. *This particular batch was purified from HEK293 cell-line
stably expressing (created using lenti-methods by a
> former colleague) the protein of interest. In future, I will plan to do
expression in the presence of Kifunensine, followed by EndoH treatment
before complexation and crystallization.
> Best Wishes,
> Partha
> On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides 
wrote:
>> Dear Partha
>> you do not specify which HEK293 cell line you have used, but if it so
happens that it is the very handy HEK293S *MGAT1-/- *cell line
>> (previously known as HEK293S *GnTI-/- ) *which produces
>> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH (e.g.
see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
immuni.2017.12.008).
>> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
quite well for protein expression in HEK293T and renders N-linked glycans
digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et al.
doi:10.1038/nsmb.2367).
>> If resources and protein material allow, you might also want to consider
the permutation exercise of subjecting the complex to deglycosylation, or
the individual components followed by complex formation/purification, or
just one of the two components followed by complex formation/purification,
or even one of the two components followed by further deglycosylation of
the complex. We are becoming more and more apprehensive of the possible
role of glycans in complex formation.
>> And then there is of course the option to apply mutagenesis, e.g. via
N—>Q, to eliminate certain N-linked glycans either as a standalone
approach
>> or in combination with enzymatic glycan digestions as described above. Best
wishes
>> Savvas
>> *---*
>> *Savvas Savvides*
>> VIB Center for Inflammation Research
>> Dept. Biochemistry & Microbiology, Ghent University
>> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
>> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
savvas.savvides_skype
>> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx On
16 Jul 2018, at 20:53, Parthasarathy Sampathkumar 
wrote:
>> Dear All,
>> I am in a situation, almost for the first time within my limited
experience, that deglycosylation might be necessary to obtain crystal. So,
I thought of tapping to vast experience of CCP4BBers, while I am searching
literature.
>> I have protein that has been expressed in HEK293 cells, secreted into
media, purified over IMAC and SEC columns. Crystallization-screens with its
binding partners (they form good complexes based on analytical SEC) have
not produced any useful hits (whereas complexes with related proteins
worked well). So, I plan to re-try complex formation and \crystallization
screen after deglysosylation.
>> My question is: In practice, Does a kit (for example here: https://www.
sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US) containing
Endo F1, F2, F3 be sufficient or should this be tried in combination with
PNGase (which requires
>> desaturating conditions)?!!
>> Many Thanks in advance for your suggestions, and reference.
>> Best Wishes,
>> Partha
>> --
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>  To
unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Parthasarathy Sampathkumar]

Hi Savvas,

Many Thanks for your inputs and references. This cell line used is not
HEK293S *MGAT1-/-. *This particular batch was purified from HEK293
cell-line stably expressing (created using lenti-methods by a
former colleague) the protein of interest. In future, I will plan to do
expression in the presence of Kifunensine, followed by EndoH treatment
before complexation and crystallization.

Best Wishes,
Partha


On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides 
wrote:

> Dear Partha
> you do not specify which HEK293 cell line you have used, but if it so
> happens that it is the very handy HEK293S *MGAT1-/- *cell line
> (previously known as HEK293S *GnTI-/- ) *which produces
> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH (e.g.
> see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
> alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
> immuni.2017.12.008).
> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
> quite well for protein expression in HEK293T and renders N-linked glycans
> digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
> et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et
> al. doi:10.1038/nsmb.2367).
>
> If resources and protein material allow, you might also want to consider
> the permutation exercise of subjecting the complex to deglycosylation, or
> the individual components followed by complex formation/purification, or
> just one of the two components followed by complex formation/purification,
> or even one of the two components followed by further deglycosylation of
> the complex. We are becoming more and more apprehensive of the possible
> role of glycans in complex formation.
> And then there is of course the option to apply mutagenesis, e.g. via
> N—>Q, to eliminate certain N-linked glycans either as a standalone approach
> or in combination with enzymatic glycan digestions as described above.
>
> Best wishes
> Savvas
>
>
> *---*
> *Savvas Savvides*
> VIB Center for Inflammation Research
> Dept. Biochemistry & Microbiology, Ghent University
> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
> savvas.savvides_skype
> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx
>
> On 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar 
> wrote:
>
> Dear All,
>
> I am in a situation, almost for the first time within my limited
> experience, that deglycosylation might be necessary to obtain crystal. So,
> I thought of tapping to vast experience of CCP4BBers, while I am searching
> literature.
>
> I have protein that has been expressed in HEK293 cells, secreted into
> media, purified over IMAC and SEC columns. Crystallization-screens with its
> binding partners (they form good complexes based on analytical SEC) have
> not produced any useful hits (whereas complexes with related proteins
> worked well). So, I plan to re-try complex formation and \crystallization
> screen after deglysosylation.
>
> My question is: In practice, Does a kit (for example here: https://www.
> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
> containing Endo F1, F2, F3 be sufficient or should this be tried in
> combination with PNGase (which requires
> desaturating conditions)?!!
>
> Many Thanks in advance for your suggestions, and reference.
>
> Best Wishes,
> Partha
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
>



To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Parthasarathy Sampathkumar]

Many Thanks Artem. I will use Kifunensine in culture for next batch
expression, and will use EndoH with already purified proteins.

Best Wishes,
Partha

On Mon, Jul 16, 2018 at 12:26 PM, Artem Evdokimov  wrote:

> Dear Partha
>
> Treat your culture with Kifunensine prior to transfection (or throughout
> growth if you're using stables) and then treat purified protein with EndoH.
> Pretty cheap and effective.
>
> PNGase F does not *require* denaturing conditions. It just likes the
> protein to be 'loose' - in my experience about half the time the accessible
> sites will fall off even under native conditons. However, PNGase F is
> somewhat expensive and after treatment certain proteins have fallen out of
> solution (presumably because not a single carbohydrate remained, as opposed
> to 'shielding' provided by the single remaining sugar left behind by
> EndoH). Caveat emptor.
>
> Artem
>
> - Cosmic Cats approve of this message
>
> On Mon, Jul 16, 2018 at 2:53 PM, Parthasarathy Sampathkumar <
> spart...@gmail.com> wrote:
>
>> Dear All,
>>
>> I am in a situation, almost for the first time within my limited
>> experience, that deglycosylation might be necessary to obtain crystal. So,
>> I thought of tapping to vast experience of CCP4BBers, while I am searching
>> literature.
>>
>> I have protein that has been expressed in HEK293 cells, secreted into
>> media, purified over IMAC and SEC columns. Crystallization-screens with its
>> binding partners (they form good complexes based on analytical SEC) have
>> not produced any useful hits (whereas complexes with related proteins
>> worked well). So, I plan to re-try complex formation and \crystallization
>> screen after deglysosylation.
>>
>> My question is: In practice, Does a kit (for example here:
>> https://www.sigmaaldrich.com/catalog/product/SIGMA/
>> NDEGLY?lang=en=US) containing Endo F1, F2, F3 be sufficient or
>> should this be tried in combination with PNGase (which requires
>> desaturating conditions)?!!
>>
>> Many Thanks in advance for your suggestions, and reference.
>>
>> Best Wishes,
>> Partha
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
>



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Savvas Savvides]

Dear Partha
you do not specify which HEK293 cell line you have used, but if it so happens 
that it is the very handy HEK293S MGAT1-/- cell line (previously known as 
HEK293S GnTI-/- ) which produces N-linked Man5GlcNAc2 glycans you might want to 
consider using EndoH (e.g. see Verstraete et al. DOI: 10.1038/ncomms14937) or 
even Jack-bean alpha-mannosidase (e.g. see Bloch et al. 
https://doi.org/10.1016/j.immuni.2017.12.008 
).
Kifunensine, an inhibitor of N-glycosylation processing, tends to work quite 
well for protein expression in HEK293T and renders N-linked glycans digestible 
by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix et al. 
http://dx.doi.org/10.1016/j.str.2015.06.019 
 ; Elegheert et al. 
doi:10.1038/nsmb.2367).

If resources and protein material allow, you might also want to consider the 
permutation exercise of subjecting the complex to deglycosylation, or the 
individual components followed by complex formation/purification, or just one 
of the two components followed by complex formation/purification, or even one 
of the two components followed by further deglycosylation of the complex. We 
are becoming more and more apprehensive of the possible role of glycans in 
complex formation.
And then there is of course the option to apply mutagenesis, e.g. via N—>Q, to 
eliminate certain N-linked glycans either as a standalone approach or in 
combination with enzymatic glycan digestions as described above.

Best wishes
Savvas


---
Savvas Savvides
VIB Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 


> On 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar  
> wrote:
> 
> Dear All, 
> 
> I am in a situation, almost for the first time within my limited experience, 
> that deglycosylation might be necessary to obtain crystal. So, I thought of 
> tapping to vast experience of CCP4BBers, while I am searching literature. 
> 
> I have protein that has been expressed in HEK293 cells, secreted into media, 
> purified over IMAC and SEC columns. Crystallization-screens with its binding 
> partners (they form good complexes based on analytical SEC) have not produced 
> any useful hits (whereas complexes with related proteins worked well). So, I 
> plan to re-try complex formation and \crystallization screen after 
> deglysosylation. 
> 
> My question is: In practice, Does a kit (for example here: 
> https://www.sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US 
> )
>  containing Endo F1, F2, F3 be sufficient or should this be tried in 
> combination with PNGase (which requires
> desaturating conditions)?!! 
> 
> Many Thanks in advance for your suggestions, and reference. 
> 
> Best Wishes,
> Partha 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Artem Evdokimov]

Dear Partha

Treat your culture with Kifunensine prior to transfection (or throughout
growth if you're using stables) and then treat purified protein with EndoH.
Pretty cheap and effective.

PNGase F does not *require* denaturing conditions. It just likes the
protein to be 'loose' - in my experience about half the time the accessible
sites will fall off even under native conditons. However, PNGase F is
somewhat expensive and after treatment certain proteins have fallen out of
solution (presumably because not a single carbohydrate remained, as opposed
to 'shielding' provided by the single remaining sugar left behind by
EndoH). Caveat emptor.

Artem

- Cosmic Cats approve of this message

On Mon, Jul 16, 2018 at 2:53 PM, Parthasarathy Sampathkumar <
spart...@gmail.com> wrote:

> Dear All,
>
> I am in a situation, almost for the first time within my limited
> experience, that deglycosylation might be necessary to obtain crystal. So,
> I thought of tapping to vast experience of CCP4BBers, while I am searching
> literature.
>
> I have protein that has been expressed in HEK293 cells, secreted into
> media, purified over IMAC and SEC columns. Crystallization-screens with its
> binding partners (they form good complexes based on analytical SEC) have
> not produced any useful hits (whereas complexes with related proteins
> worked well). So, I plan to re-try complex formation and \crystallization
> screen after deglysosylation.
>
> My question is: In practice, Does a kit (for example here: https://www.
> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
> containing Endo F1, F2, F3 be sufficient or should this be tried in
> combination with PNGase (which requires
> desaturating conditions)?!!
>
> Many Thanks in advance for your suggestions, and reference.
>
> Best Wishes,
> Partha
>
> --
>
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>



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[ccp4bb] Suggestions for deglycosylaiton enzymes

[Parthasarathy Sampathkumar]

Dear All,

I am in a situation, almost for the first time within my limited
experience, that deglycosylation might be necessary to obtain crystal. So,
I thought of tapping to vast experience of CCP4BBers, while I am searching
literature.

I have protein that has been expressed in HEK293 cells, secreted into
media, purified over IMAC and SEC columns. Crystallization-screens with its
binding partners (they form good complexes based on analytical SEC) have
not produced any useful hits (whereas complexes with related proteins
worked well). So, I plan to re-try complex formation and \crystallization
screen after deglysosylation.

My question is: In practice, Does a kit (for example here:
https://www.sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
containing Endo F1, F2, F3 be sufficient or should this be tried in
combination with PNGase (which requires
desaturating conditions)?!!

Many Thanks in advance for your suggestions, and reference.

Best Wishes,
Partha



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