Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy - PubMed (original) (raw)

Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy

Annette Steward et al. Protein Sci. 2002 Sep.

Abstract

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.

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Figures

Fig. 1.

Fig. 1.

Initial construction of an eight-module expression vector. (a) PCR product of wild-type I27 module 1 is T-cloned then subcloned into pRSET A expression vector. The T-clones can be used for site-directed mutagenesis to construct mutant proteins. (b) Module 2 is subcloned from its T-vector into Sac I – _Eco_R I site in the pRSET A expression vector containing module 1. Module 3 now can be subcloned into _Bss_H II – _Eco_RI site. (c) Final eight-module construct in pRSET A, showing position of the two cysteine codons in module 8. (d) The expressed protein showing the position of the histidine tag, (H)6, the linking amino acids encoded by the restriction enzyme site (one letter code), and the C-terminal cysteine residues.

Fig. 2.

Fig. 2.

Sample atomic force microscopy traces of homopolymers constructed by this method. All traces collected at 600 nm/sec. (a) Wild-type TI I27. (b) Mutant TI I27 (each module with the mutation V86A). Note that the forces are significantly lower than for wild type. (c) A TI I27 construct with TNfn3 cloned at positions 2, 4, and 6. TNfn3 can be distinguished from TI I27 on the grounds of both peak height and distance between the peaks in the worm-like chain fit (as described in Rief et al. 1997) with the Δ_L_, increase in contour length from one peak to another, being 324 Å on unfolding of a TNfn3 module and 293Å on unfolding of a TI I27 module. The TI I27 and TNfn3 unfolding forces are the same as in the respective homopolymers.

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