Use of a heterodimeric coiled-coil system for biosensor application and affinity purification.
The two-stranded alpha-helical coiled-coil is now recognized as one of nature's favorite ways of creating a dimerization motif. Based on the knowledge of protein folding studies and de novo design model systems, a novel heterodimeric coiled-coil protein was synthesized. The heterodimeric E/K coiled-coil was constructed with two distinct peptides (E and K) that will spontaneously associate into a full helical coiled-coil structure in solution. Equilibrium CD, NMR and real time biosensor kinetics experiments showed that the E/K coiled-coil is both structurally (deltaG(unfold)=11.3 kcal/mol) and kinetically (Kd approximately 1 nM) stable in solution at neutral pH. The engineered coiled-coil had been applied as a dimerization and capture domain for biosensor based applications and used in an expression/detection/affinity chromatography system. Specific test examples demonstrated the usefulness of the E/K heterodimeric system in these applications. The universality of coiled-coil as a dimerization motif in nature and our ability to design and synthesize these proteins suggest a wide variety of applications.
Chao
H
H
Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Canada.
Bautista
D L
DL
Litowski
J
J
Irvin
R T
RT
Hodges
R S
RS
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D002846:Chromatography, Affinity / Q000295:instrumentation*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification
D011506:Proteins