Re: [ccp4bb] Design of SUMO fusion proteins

2021-04-27 Thread Iwai, Hideo 

 Hi,


I would like ask your advice on designing SUMO fusion proteins. 
Specifically, is it recommended to include a linker between the 
C-terminus of SUMO (-QIGG) and the N-terminus of the target protein? 
Figure 3 of Guerrero et al. (https://pubmed.ncbi.nlm.nih.gov/26297996/ 
) suggests that including a 
linker of SGG or SGGG greatly improves cleavage efficiency by Ulp1. 
However, the often-stated advantage of SUMO is that, in the absence of 
a linker, the target protein contains no extra residues after cleavage.



As the author,

1) GGG is not required when the globular domain is not so close to the 
Ulp1 cutting site.   For example, if you have a flexible N-terminus, the 
insertion like GG, GGG is irrelevant.


We used an Intein domain in this manuscript, the N-terminus is very 
close to the core of the Hint fold. Without a short linker, Ulp1 does 
not cut at all. The required length for cleavages


actually varies depending on different inteins.

if the cutting site is immediately adjacent to a well-folded region, it 
will not be cut. I recommend 2-3 flexible sequence such as GG,. This 
will change the efficiency of the cleavage.


you could also increase the temperature from 25 to 37 degree if one has 
a problem with cleavage.


However, the fused proteins aggregates or form oligomers, preventing 
Ulp1 to access, then again, Ulp1 will not cut and might need even a 
longer linker for the cleavage.



I’ll also take any advice on your favorite N-terminal His tag for SUMO 
fusions. I am considering MGSSHHG, based on two Acta F papers 
(https://pubmed.ncbi.nlm.nih.gov/22949189/ 
, 
https://pubmed.ncbi.nlm.nih.gov/18391421/ 
), or perhaps 
MGSSHHSSG, which is from pET14b.




GSS  tends to get the gluconoylation modification, which could be 
annoying when you need  exact Mass analysis as usually depending on  
expression condition.


https://pubmed.ncbi.nlm.nih.gov/9918669/


My recommendation is to pay attentions to DNA sequences which could 
affect transcription initiation or translation initiation. Commercial 
vectors widely used are usually fine.



best regards,

Hideo




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Re: [ccp4bb] Design of SUMO fusion proteins

2021-04-27 Thread Patrick Loll 
Hi Jack,

We routinely include a single glycine downstream of the SUMO (so, …QIGGG). This 
essentially always gives good cleavage with UD, assuming the construct isn’t 
aggregated or otherwise unhappy.

Re the His tag: Presumably you mean at the N-terminus of SUMO? In this case 
we’ve had good luck with MGHHG. If I was going to redesign this from 
scratch, I might make it a His-10 tag, but that’s really just a theoretical 
notion; practically speaking, we’ve never had the impetus to change things. 

Details in PMCID PMC1892228.

Cheers,

Pat

---
Patrick J. Loll, Ph. D.  (he, him, his)
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu


> On 27 Apr 2021, at 4:00 PM, Tanner, John J.  wrote:
> 
> Sorry for the non-CCP4 question…
>  
> I would like ask your advice on designing SUMO fusion proteins. Specifically, 
> is it recommended to include a linker between the C-terminus of SUMO (-QIGG) 
> and the N-terminus of the target protein? Figure 3 of Guerrero et al. 
> (https://pubmed.ncbi.nlm.nih.gov/26297996/) suggests that including a linker 
> of SGG or SGGG greatly improves cleavage efficiency by Ulp1. However, the 
> often-stated advantage of SUMO is that, in the absence of a linker, the 
> target protein contains no extra residues after cleavage.
>  
> I’ll also take any advice on your favorite N-terminal His tag for SUMO 
> fusions. I am considering MGSSHHG, based on two Acta F papers 
> (https://pubmed.ncbi.nlm.nih.gov/22949189/, 
> https://pubmed.ncbi.nlm.nih.gov/18391421/), or perhaps MGSSHHSSG, which 
> is from pET14b.
>  
> Thanks,
>  
> Jack 
>  
>  
> -- 
> John J. Tanner 
> Professor of Biochemistry and Chemistry
> Associate Chair of Biochemistry
> Department of Biochemistry
> University of Missouri
> 117 Schweitzer Hall
> 503 S College Avenue
> Columbia, MO 65211
> Phone: 573-884-1280
> Email: tanne...@missouri.edu
> https://cafnrfaculty.missouri.edu/tannerlab/
> Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
> Office: Schlundt Annex 203A
>  
>  
>  
>  
>  
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 

Patrick Loll
pjl...@gmail.com



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[ccp4bb] Design of SUMO fusion proteins

2021-04-27 Thread Tanner, John J. 
Sorry for the non-CCP4 question…

I would like ask your advice on designing SUMO fusion proteins. Specifically, 
is it recommended to include a linker between the C-terminus of SUMO (-QIGG) 
and the N-terminus of the target protein? Figure 3 of Guerrero et al. 
(https://pubmed.ncbi.nlm.nih.gov/26297996/) suggests that including a linker of 
SGG or SGGG greatly improves cleavage efficiency by Ulp1. However, the 
often-stated advantage of SUMO is that, in the absence of a linker, the target 
protein contains no extra residues after cleavage.

I’ll also take any advice on your favorite N-terminal His tag for SUMO fusions. 
I am considering MGSSHHG, based on two Acta F papers 
(https://pubmed.ncbi.nlm.nih.gov/22949189/, 
https://pubmed.ncbi.nlm.nih.gov/18391421/), or perhaps MGSSHHSSG, which is 
from pET14b.

Thanks,

Jack


--
John J. Tanner
Professor of Biochemistry and Chemistry
Associate Chair of Biochemistry
Department of Biochemistry
University of Missouri
117 Schweitzer Hall
503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280
Email: tanne...@missouri.edu
https://cafnrfaculty.missouri.edu/tannerlab/
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A








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