Total chemical synthesis of calstabin 2 protein
M. Jullian,a O. Melnyk,b G. Ferry, c J.A. Boutin,c L. Ronga*,a† K.
Puget,a†
a
GENEPEP SA, Saint Jean de Vedas, France; bCNRS UMR 8161, Université Lille Nord de
France, Institut Pasteur de Lille, Lille, France ; cInstitut de Recherches Servier, Croissysur-Seine, France ; † equal contribution
*E-mail:[email protected]
Introduction
Peptidyl prolyl cis/trans isomerases (PPIs) form 3 small families of proteins to which
belong FKBP and cyclophilin. These proteins are folding helper proteins. Together with
chaperones, they form receptor complexes. They catalyze the isomerization of prolyl
peptide bonds in various folding states of target proteins. Indeed, their role has been
implicated in refolding of denatured proteins or folding of de novo synthesized proteins [1].
Among them, the FKBP subclass comprises the small PPI calstabin 1 and 2. It is of interest
to try to understand the way those proteins act, in order to help the overexpression of
various types of membrane proteins, aiming at the renaturation, purification and
crystallization attempts of receptors. We chose to work on calstabin 2 because this short
(107 aa) protein has been described as a sub-family comprising 4 isoforms (from ~30 to
~100 aminoacids), some of them not being fully described to date. The relative shortness of
those proteins together with the fact that the two higher molecular weight ones are
catalytically active as prolyl isomerases, facilitate the characterization of the synthetic
proteins.
Results and Discussion
In order to obtain the full length calstabin 2, a native chemical ligation (NCL) approach was
chosen [2]. An optimized stepwise elongation allowed the obtention of the 86-mer Cterminal segment up to the Cys 22. Moreover, several methods were compared for the
synthesis of peptide 1-21 opportunely functionalized at its C-terminus for the NCL.
Firstly, we tested the in situ thioesterification of fully protected fragment 1-21 carboxylate
synthesized on 2-chlorotrityl chloride resin [3]. The desired peptide 1-21 thioester was
obtained together with a side-product probably due to the additional thioesterification of a
side chain carboxylate deprotected during 1% TFA cleavage.
Then, we synthesized the 1-21 fragment on 3-(Fmoc-amino)-4-aminobenzoyl (Dbz) resin in
order to obtain this peptide with a C-terminal N-acyl-benzimidazolinone functionality
(Nbz) which undergoes rapid thiolysis, enabling thioester peptide to be generated before
purification or in situ during a native chemical ligation [4]. The 1-21 Nbz fragment was
obtained by suppressing the acetylation step after each peptide coupling which initially
caused the acetylation of the second Dbz amine. Unfortunately, the purification of our Nbz
peptide before NCL failed due to the hydrolysis of Nbz favored by RP-HPLC conditions.
Finally, the peptide 1-21 featuring a C-terminal bis(2-sulfanylethyl)amido (SEA) group was
synthesized on an innovative solid support [5] and obtained in good overall yield and
purity.
The 1-21 SEA fragment was employed for the chemoselective and regioselective reaction
with the 22-107 cysteinyl peptide in the presence of 4-mercaptophenylacetic acid and
tris(2-carboxyethyl)phosphine in a phosphate buffer (pH 7) at 40°C. This ligation at Thr
site afforded the full length calstabin 2 in good yield (Figure 1).
Figure 1. RP-HPLC and ESI spectrum of full length calstabin 2
References
[1] Schiene-Fischer, C. and Yu, C. FEBS Lett. 2001, 495, 1.
[2] Dawson, P.E.; Muir, T.W.; Clark-Lewis, I.; Kent, S.B. Science 1994, 266, 776.
[3] Flemer, S. Jr. J Pept Sci. 2009, 15, 693.
[4] Blanco-Canosa, B. and Dawson, P. E. Angew Chem Int Ed Engl. 2008, 47, 6851.
[5] Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O., Org. Lett. 2010, 12, 5238.