Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy.
This manuscript introduces a versatile system for construction of multimeric proteins to be used as substrates for atomic force microscopy. The construction makes use of a cassette system that allows modules to be cut and ligated in any combination in eight different positions. The modules can be sequenced in situ after construction. A three-module fragment can be produced that is of a size amenable to structural and biophysical analysis to check the effect of placing a protein into a multimeric construct. We show that if the parent titin modules are retained in a construct, they can act both as linkers and as an internal standard for the force measurements. Proteins that cannot be expressed solubly in an eight-module homopolymer have been expressed and subject to force measurements using this system.
Department of Chemistry, MRC Centre for Protein Engineering, Lensfield Road, Cambridge CB2 1EW, UK.
Annette
A
Steward
José Luis
JL
Toca-Herrera
Jane
J
Clarke
D003001:Cloning, Molecular / Q000379:methods*; D064211:Connectin; D005822:Genetic Vectors; D018625:Microscopy, Atomic Force; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011108:Polymers; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D021122:Protein Subunits
D064211:Connectin; D009124:Muscle Proteins; D011108:Polymers; D021122:Protein Subunits; D011494:Protein Kinases