RE: Pepsyn: ligation

Bianchi Elisabetta (Bianchi@irbm.it)
Wed, 17 Nov 1999 08:28:37 +0100

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We have recently had experience with a chemical ligation for the synthesis
of a transmembrane protein .
We solved the problem of solubility by engineering the protein with lysine
tails at temini.
Moreover the chemical ligation reaction was run on deprotected segments,
adding detergents to the reaction medium
like those normally used for biochemical characterization.
You may find details in J. Am. Chem. Soc. 1999, 121, 7698-7699.

Moreover in order to do peptide ligation, you need to synthesize peptide
C-terminal thioesters. So far they have been prepared only
by the use of Boc/benzyl solid phase method on ad hoc prepared resin
supports.
However we have now developed a new strategy for the solid phase synthesis
of thioesters by Fmoc/t-Bu chemistry and
also compatible by Boc/benzyl. The method is based on the use of a
safety-catch linker and it is in press on J. Am. Chem. Soc.

Elisabetta Bianchi

****************************************************************************

Elisabetta Bianchi
Department of Biotechnology
IRBM P. Angeletti
via Pontina km 30,600
00040 Pomezia (Roma) Italy
tel: +39 06 91093434
fax: +39 06 91093225

> ----------
> From: Len Packman
> Sent: Tuesday, November 16, 1999 2:28 PM
> To: Recipients of ABRF List
> Subject: Pepsyn: ligation
>
> Anyone out there doing peptide ligation work to make big peptides/small
> proteins?
>
> If so, what are the currently preferred methodologies?
>
> I see Kent has a company (http://www.gryphonsci.com/) doing this now.
> Anyone use his approach of ligating unprotected peptides? Does it work
> with
> hydrophobic proteins (say, 2x40mer--> 80mer)
>
> Anyone use the approach of ligating Hmb-protected peptides?
>
> Len
>
> *********************************************************************
> Dr Len C. Packman
> Assistant Director of Research
> Protein and Nucleic Acid Chemistry Facility
> Department of Biochemistry
> University of Cambridge
> 80 Tennis Court Road
> Old Addenbrookes Site
> Cambridge, CB2 1GA, UK
> Tel: +44 (1223) 333639
> FAX: +44 (1223) 766002
> e-mail: lcp2@mole.bio.cam.ac.uk
> Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
>
>
>
>

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RE: Pepsyn: ligation

We have recently had  experience = with a chemical ligation for the synthesis
of a transmembrane protein .
We solved the problem of solubility = by engineering the protein with lysine
tails at temini.
Moreover the chemical ligation = reaction was run on deprotected segments,
adding detergents to the reaction = medium
like those normally used for = biochemical characterization.
You may find details in J. Am. Chem. = Soc. 1999, 121, 7698-7699.

Moreover in order to do peptide = ligation, you need to synthesize  peptide
C-terminal thioesters.  So far = they have been prepared only
by the use of Boc/benzyl solid phase = method on ad hoc prepared resin
supports.
However  we have now developed = a new strategy for the solid phase synthesis
of thioesters by Fmoc/t-Bu  = chemistry and
also compatible by Boc/benzyl. The = method is based on the use of a
safety-catch linker and it is in = press on J. Am. Chem. Soc.

Elisabetta Bianchi

********************************************************= ********************

Elisabetta Bianchi
Department of Biotechnology
IRBM P. Angeletti
via Pontina km 30,600
00040 Pomezia (Roma) Italy
tel:  +39 06 91093434
fax: +39 06 91093225


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