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Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy
Author(s) -
Steward Annette,
TocaHerrera José Luis,
Clarke Jane
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0212702
Subject(s) - atomic force microscopy , construct (python library) , cloning (programming) , titin , fragment (logic) , nanotechnology , computational biology , chemistry , biophysics , physics , computer science , materials science , biology , microbiology and biotechnology , algorithm , programming language , sarcomere , myocyte
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.