title: Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide.
abstract: We report a new method to reversibly bind proteins to a surface in a functionally active orientation directly from cell lysate by exploiting a thermodynamically reversible hydrophilic-hydrophobic lower critical solution temperature (LCST) transition exhibited by a recombinant, stimuli-responsive elastin-like polypeptide (ELP). An ELP is covalently micropatterned on a glass surface against an inert BSA background. The ELP-patterned surface is incubated with the soluble fraction of E. coli lysate containing an expressed ELP fusion protein, which is appended with the same ELP as on the surface. The LCST transition of the grafted ELP and the ELP fusion protein is simultaneously triggered by an external stimulus. The LCST transition results in capture of the ELP fusion protein from solution onto the immobilized ELP by hydrophobic interactions between the grafted ELP and the ELP fusion protein. The captured ELP fusion protein is oriented such that the fusion partner is accessible to binding of its target from solution. We also demonstrate that TRAP is reversible; the bound protein-ligand complex is released from the surface by reversing the LCST transition. The triggered control of interfacial properties provided by an immobilized stimuli-responsive polypeptide at the solid-water interface is an enabling technology that allows reversible and functional presentation of ELP fusion proteins on a surface directly from cell lysate without the necessity of intermediate purification steps and subsequent recovery of the protein-ligand complex for downstream analysis by other analytical techniques. TRAP has application in lab-on-a-chip bioanalytical devices as well as in the fabrication of peptide and protein arrays.
authors.affiliation: Department of Biomedical Engineering, Duke University, Box 90281, Durham, North Carolina 27708, USA.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Nath
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Chilkoti
mesh_terms: D002474:Cell-Free System / Q000737:chemistry; D004549:Elastin / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D005898:Glass; D010455:Peptides / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004549:Elastin