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Protein Eng Des Sel. 2014 Oct;27(10):431-8. doi: 10.1093/protein/gzu042.

Affinity transfer to the archaeal extremophilic Sac7d protein by insertion of a CDR.

Author information

1
CRCNA - UMR 892 INSERM, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France 6299 CNRS, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France University of Nantes, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France Institut Pasteur, CNRS UMR 3528, Unité de Microbiologie Structurale, 25 rue du Dr. Roux, 72724 Paris Cedex 15, France.
2
CRCNA - UMR 892 INSERM, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France 6299 CNRS, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France University of Nantes, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France.
3
CRCNA - UMR 892 INSERM, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France 6299 CNRS, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France University of Nantes, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France Plate-forme IMPACT Biogenouest, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France.
4
CRCNA - UMR 892 INSERM, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France 6299 CNRS, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France University of Nantes, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France frederic.pecorari@univ-nantes.fr.

Abstract

Artificially transforming a scaffold protein into binders often consists of introducing diversity into its natural binding region by directed mutagenesis. We have previously developed the archaeal extremophilic Sac7d protein as a scaffold to derive affinity reagents (Affitins) by randomization of only a flat surface, or a flat surface and two short loops with natural lengths. Short loops are believed to contribute to stability of extremophilic proteins, and loop extension has been reported detrimental for the thermal and chemical stabilities of mesophilic proteins. In this work, we wanted to evaluate the possibility of designing target-binding proteins based on Sac7d by using a complementary determining region (CDR). To this aim, we inserted into three different loops a 10 residues CDR from the cAb-Lys3 anti-lysozyme camel antibody. The chimeras obtained were as stable as wild-type (WT) Sac7d at extreme pH and their structural integrity was supported. Chimeras were thermally stable, but with T(m)s from 60.9 to 66.3°C (cf. 91°C for Sac7d) which shows that loop extension is detrimental for thermal stability of Sac7d. The loop 3 enabled anti-lysozyme activity. These results pave the way for the use of CDR(s) from antibodies and/or extended randomized loop(s) to increase the potential of binding of Affitins.

KEYWORDS:

Affitin; CDR insertion; Sac7d; affinity transfer; extremophilic

PMID:
25301962
DOI:
10.1093/protein/gzu042
[Indexed for MEDLINE]

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