Engineering a de novo-designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins

Abstract

Using the techniques of genetic engineering and the principles of protein de novo design, we have developed a unique affinity matrix protein tag system as a rapid, convenient and sensitive method to detect, purify and characterize newly expressed recombinant peptides or proteins from cell extracts. The method utilizes two de novo-designed linear peptide sequences that can selectively dimerize to form the stable protein motif, the two-stranded alpha-helical coiled-coil. In this method, a recombinant bacterial expression vector pRLDE has been engineered so that one of the dimerization strands (E-coil) is expressed as a C-terminal fusion tag on newly expressed peptides or proteins, while the other (K-coil) is either biotin-labeled for detection in a Western blot-type format or immobilized on an insoluble silica support for selective dimerization affinity chromatography. Recombinantly expressed peptides from Escherichia coli containing the dimerization tag have been produced, detected and purified using this method. The recombinant peptides were easily and clearly identified using the biotin-labeled coil, while the single-step affinity purification results indicated the purity of the affinity purified expressed peptides to be > 95%, as assessed by reversed-phase chromatography. The stability of the dimerization domain also allows for the purified peptide to be left attached to the matrix, thus creating a new peptide-bound column that can be used to study peptide-protein or peptide-ligand interactions. Therefore this system offers a new alternative to existing peptide or protein fusion tags and demonstrates the utility of a de novo-designed system.

Structure of a Pilin Monomer fromPseudomonas aeruginosa

“…Protein Samples-A DNA sequence encoding P. aeruginosa strain K122-4 pilin (29 -150) was polymerase chain reaction-amplified from the full-length K122-4 pilin cDNA and cloned into the pRLD expression vector such that it had an in-frame OmpA leader sequence fused to the truncated pilin (30) (29 -150) was extracted from the periplasm by osmotic shock and purified by cation exchange with a CM-cellulose column (utilizing a linear gradient of 0 -0.8 M NaCl) in 10 mM sodium acetate, pH 4.5. K122-4 pilin (29 -150) was subsequently desalted on a Sephadex G75 column and lyophilized.…”

Designing Heterodimeric Two-stranded α-Helical Coiled-coils

“…We have designed the E/K coil, a heterodimeric coiled-coil for use in biotechnological applications including as an expression and purification tag and as a universal dimerization domain for biosensors (14,25,38). Heterodimerization is based on the placement of charged residues at the e and g positions.…”

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

“…We have designed a heterodimeric coiled-coil system formed by two peptides (designated the E coil and K coil) (19,20) and have shown that this heterodimerization motif is promising not only as a protein expression and purification tag (21), but also as a universal dimerization domain for biosensor applications (22). The stability and affinity of this E/K system have been successfully modified by varying the R-helical propensity (serine vs alanine), the hydrophobicity of residues in the coiled-coil core (valine vs isoleucine), and the chain length (three to five heptads, or 21 to 35 residues) (23,24).…”