Method and device for integrated protein expression, purification and detection

A method for carrying out the presentation of a plurality of target proteins, comprising

• (a) adding to each of a plurality of wells in a substrate, each well having a first coil-forming peptide therein, a selected one of a plurality of different-sequence nucleic acid molecules, each having a common-sequence capture portion encoding a second coil-forming peptide and a different-sequence target portion encoding a target protein;
• (b) filling said wells with a solution comprising protein synthesis components capable of expressing the different-sequence nucleic acid molecules under selected protein-synthesis conditions;
• (c) promoting expression of said different-sequence nucleic acid molecules under such conditions, wherein the target protein expressed in each well binds to the well through coil-coil heterodimer formation and is thus presented for analysis in the well in captured form;
• and
• (d) washing the wells to remove unbound components.

A kit for presenting one or more target proteins for solid-phase analysis for use with a cell free in vitro translation system, comprising

• (a) a substrate containing a plurality of wells, wherein each well is functionalized with a first coil-forming peptide having a selected charge and being capable of interacting with a second, oppositely charged coil-forming peptide to form a stable α-helical coiled-coil heterodimer;
• (b) a cloning vector comprising in the 5′ to 3′ direction and operably linked
• (i) a transcription and translation initiation region,
• (ii) a nucleic acid sequence which encodes said second coil-forming peptide,
• (iii) a transcription and translation termination region;
• (c) said vector also having a cleavable site at which a nucleic acid encoding a heterologous protein can be inserted between (i) and (ii) or between (ii) and (iii).

A multiplexed in vitro cell free protein synthesis system, comprising

• (a) a substrate comprising a plurality of wells, each well having bound thereto a first coil-forming peptide having a selected charge and being capable of interacting with a second, oppositely charged coil-forming peptide to form a stable α-helical coiled-coil heterodimer;
• (b) contained in each of said wells,
• (i) a coding sequence comprising,
• (A) a first nucleic acid sequence which encodes a first coil-forming peptide having a selected charge and being capable of interacting with a second, oppositely charged coil-forming peptide to form a stable α-helical coiled-coil heterodimer;
• and
• (B) a second nucleic acid sequence encoding the target protein;
• (ii) protein-synthesis components capable of expressing the target protein under selected protein-synthesis conditions in the well, said well having a surface which has been functionalized with the second coil-forming peptide;
• whereby the mixture reacts under conditions such that the target protein is synthesized and binds to the well through coil-coil heterodimer formation, and is thus presented for analysis in each of the wells in captured form.