Combinatorial protein library screening by periplasmic expression

Abstract

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in the periplasm of gram negative bacteria and mixed with a labeled ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the concentration of the labeled ligand bound to the binding protein is increased and allows the cells to be isolated from the rest of the library. Where fluorescent labeling of the target ligand is used, cells may be isolated by fluorescence activated cell sorting (FACS). The approach is more rapid than prior art methods and avoids problems associated with the outer surface-expression of ligand fusion proteins employed with phage display.

Description

[0001] This application claims the priority of U.S. Provisional Patent App. No. 60 / 396,058, filed Jul. 15, 2002, and is also a continuation-in-part of U.S. patent application Ser. No. 09 / 699,023, filed Oct. 27, 2000. The entire disclosures of the foregoing applications are incorporated herein by reference.[0002] The government may own rights in the present invention pursuant to the U.S. Army ARO MURI program and the Texas Consortium for Development of Biological Sensors and in connection with contract number DADD17-01-D-0001 with the U.S. Army Research Laboratory.BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention relates generally to the field of protein engineering. More particularly, it concerns improved methods for the screening of combinatorial libraries of polypeptides to allow isolation of ligand binding polypeptides. [0005] 2. Description of Related Art [0006] The isolation of polypeptides that either bind to ligands with high affinity a...

AI Technical Summary

Benefits of technology

The invention provides a method for obtaining a bacterium that expresses a nucleic acid sequence encoding a binding polypeptide with specific affinity for a target ligand. This method involves contacting the bacterium with a labeled ligand capable of diffusing into the bacterium and selecting the bacterium based on the presence of the labeled ligand. The invention also provides an isolated antibody or fragment thereof that binds immunologically to a target ligand. The technical effects of the invention include improved efficiency in obtaining bacteria with specific binding polypeptides and increased permeability of the outer membrane to the labeled ligand.

Problems solved by technology

However, even with robotic automation and digital image systems for detecting binding in high density arrays, it is not feasible to screen large libraries consisting of tens of millions or billions of clones.

However, several spectacular successes notwithstanding, the screening of phage-displayed libraries can be complicated by a number of factors.

As a result, the clones isolated after several rounds of panning are frequently difficult to produce on a preparative scale in E. coli.

Second, although phage displayed proteins may bind a ligand, in some cases their un-fused soluble counterparts may not (Griep et al., 1999).

Third, the isolation of ligand-binding proteins and more specifically antibodies having high binding affinities can be complicated by avidity effects by virtue of the need for gene III protein to be present at around 5 copies per virion to complete phage assembly.

Finally, even though pIII and to a lesser extent the other proteins of the phage coat are generally tolerant to the fusion of heterologous polypeptides, the need to be incorporated into the phage biogenesis process imposes biological constraints that can limit library diversity.

This imposes several potential limitations.

For example, the requirement for display of the protein on the surface of a cell imposes biological constraints that limit the diversity of the proteins and protein mutants that can be screened.

Also, complex proteins consisting of several polypeptide chains cannot be readily displayed on the surface of bacteria, filamentous phages or yeast.

Images