Download 6th semester-2006 Project Proposal

6th semester-2006 Project Proposal
Laurent Duroux
“Construction of plasmid vectors to tag proteins for universal
light-induced protein immobilization on surfaces”
Background: A method of light-induced immobilization of proteins(1,2) on chemically
treated surfaces has been successfully developed over the past years in the group, by
Teresa Petersen and colleagues. It essentially involves a chemical energy/electron
transfer from a light-excited tryptophan (or tyrosine) residue to a neighbouring
disulfide bridge. This results in the generation of highly reactive charged cysteine
residue which immediately cross-link to free sulfhydryl groups. The method is
currently used to generate nano-arrays of technologically relevant proteins such as
immunoglobulins, for medical diagnosis.
The main drawback of the technology is that it is only limited to these proteins
presenting a triad of amino acids, Cys-Cys / Trp (or Tyr), close neighbours in 3D
space and surface-exposed. Thus, it excludes a large number of potentially interesting
proteins for future applications in nano-biotechnology. The goal of the project is to
propose a solution to this limitation and to extend the concept developed in the group.
Project description: The general aim of the project is to generate plasmid vectors for
the production of tagged proteins, which would be immobilized via a light-induced
method. The idea is to build a system for recombinant production of any protein with
a “tag” at its N- or C-terminal end, which would allow for light-induced
immobilization, irrespective of the structural features of the target protein. The
concept of tag implicates that the peptide fused to the target protein should be as
neutral as possible regarding its folding and its biological activity. To achieve this, the
tag should be of limited size, typically no more than 30 amino-acid residues. The
basic assumption is that the smaller the tag, the lesser the probability that it interacts
with the target protein. Of course, this peptide tag will have to carry the essential CysCys / Trp triad necessary for light-induced immobilization. This is where the first (and
most likely main) challenge resides in this project.
Here, the target protein used as model would be a fatty-acid binding protein (FABP)
from human(3), for which we have a good deal of practical expertise.
Proposed strategy /methods:
(1) Designing the N-term (and/or C-term) peptide with the Cys-Cys / Trp triad. The
most likely secure route to achieve success is (again) to observe nature’s ways. A
number of dedicated specific databases for disulfide bridges already exist(4, 5, 6) and
should be used as a starting point. In particular, a class of peptides called “Knottin”(7)
appear to be suited for the purpose (http://knottin.cbs.cnrs.fr/Main.html), see figures
below.
Figure: Disulfide connectivity in the Knottin family
Figure: 3D model of a typical Knottin
(2) Design of PCR primers for molecular cloning of the peptide(s) into a plasmid
vector (pET series), Fusion to hb-FABP. Recombinant expression in E. coli.
(3) Protein purification, stability analysis (CD, fluorescence), activity analysis
(fluorescence)
(4) Immobilization to functionally activated glass plates (-SH chemistry), light
treatment (laser)
(5) Structural analysis of the chimera protein (NMR)
References:
(1) Neves-Petersen MT, Gryczynski Z, Lakowicz J, Fojan P, Pedersen S, Petersen E & Bjorn
Petersen S. 2002. High probability of disrupting a disulphide bridge mediated by an
endogenous excited tryptophan residue. Protein Sci. 11:588-600.
(2) Snabe T, Roder GA, Neves-Petersen MT, Buus S & Petersen SB. 2005. Oriented coupling of
major histocompatibility complex (MHC) to sensor surfaces using light assisted
immobilisation technology. Biosens. Bioelectron. In press.
(3) Balendiran, G. K.; Schnutgen, F.; Scapin, G.; Borchers, T.; Xhong, N.; Lim, K.; Godbout, R.;
Spener, F. & Sacchettini, J. C. 2000. Crystal structure and thermodynamic analysis of human
brain fatty acid-binding protein. J. Biol. Chem. 275:27045-27054.
(4) Herman W. T. van Vlijmen, Abhas Gupta, Lakshmi S. Narasimhan & Juswinder Singh. 2003.
A Novel Database of Disulfide Patterns and its Application to the Discovery of Distantly
Related Homologs. J. Mol. Biol. 335:1083–1092.
(5) http://research.i2r.a-star.edu.sg/CysView/
(6) http://www.ncbs.res.in/~faculty/mini/dsdbase/dsdbase.html
(7) Gelly JC, Gracy J, Kaas Q, Le-Nguyen D, Heitz A & Chiche L. 2004. The KNOTTIN website
and database: a new information system dedicated to the knottin scaffold. Nucleic Acids Res.
32:D156-159.
Important note: the project is ambitious and could be important for the scientific development of
the group. It is not the purpose in this project to achieve all mentioned points (6 th semester is limited in
time), but rather to lay the fundament of an overall project that will be hopefully followed-up. The
different points mentioned in the strategy are there to outline the overall project (beyond this sole 6th
semester). For example, it is obvious that more proteins will have to be included to “prove” the
“universality” of the concept.