Combinatorial protein library screening by periplasmic expression
First Claim
1. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:
- (a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm;
said bacterium comprising a nucleic acid sequence encoding a candidate binding polypeptide comprising an inner membrane anchor polypeptide;
wherein the bacterium further comprises a nucleic acid sequence encoding a target ligand and wherein the target ligand is exported to the periplasm;
(b) allowing the target ligand to bind to the candidate binding polypeptide in said periplasm;
(c) removing unbound target ligand from said periplasm; and
(d) selecting the bacterium based on the presence of the target ligand bound to the candidate binding polypeptide.
1 Assignment
0 Petitions
Accused Products
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, may be expressed in the periplasm of gram negative bacteria with at least one target ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the ligand becomes bound and retained by the cell even after removal of the outer membrane, allowing the cell to be isolated from cells not expressing a binding polypeptide with affinity for the target ligand. The target ligand may be detected in numerous ways, including use of direct fluorescence or secondary antibodies that are fluorescently labeled, allowing use of efficient screening techniques such as fluorescence activated cell sorting (FACS). The approach is more rapid and robust than prior art methods and avoids problems associated with the outer surface-expression of ligand fusion proteins employed with phage display.
-
Citations
54 Claims
-
1. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:
-
(a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm;
said bacterium comprising a nucleic acid sequence encoding a candidate binding polypeptide comprising an inner membrane anchor polypeptide;
wherein the bacterium further comprises a nucleic acid sequence encoding a target ligand and wherein the target ligand is exported to the periplasm;
(b) allowing the target ligand to bind to the candidate binding polypeptide in said periplasm;
(c) removing unbound target ligand from said periplasm; and
(d) selecting the bacterium based on the presence of the target ligand bound to the candidate binding polypeptide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 54)
-
-
45. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:
-
(a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm;
said bacterium comprising a nucleic acid sequence encoding a candidate binding polypeptide, wherein the candidate binding polypeptide is anchored to the outer side of the inner membrane with an inner membrane anchor polypeptide;
wherein the bacterium further comprises a nucleic acid sequence encoding a target ligand, wherein the target ligand is exported to the periplasm;
(b) allowing the target ligand to bind to the candidate binding polypeptide;
(c) removing the outer membrane of said bacterium; and
(c) selecting the bacterium based on the presence of the target ligand bound to the candidate binding polypeptide on the outer side of the inner membrane.
-
-
46. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:
-
(a) providing a population of Gram negative bacteria the members of which comprise an inner membrane, an outer membrane and a periplasm;
said population collectively comprising nucleic acid sequences encoding plurality of candidate binding polypeptides, wherein the candidate binding polypeptides are anchored to the outer side of the inner membrane of said bacteria;
wherein the bacteria further comprise nucleic acid sequences encoding a target ligand, wherein the target ligand is exported to the periplasm;
(b) allowing the target ligand to bind to the candidate binding protein in said periplasm;
(c) removing the outer membrane of said bacterium; and
(d) selecting the bacterium from said population based on the presence of the target ligand bound to the candidate binding polypeptide on the outer side of the inner membrane. - View Dependent Claims (47, 48)
-
-
49. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:
-
(a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm;
said bacterium comprising a nucleic acid sequence encoding a candidate binding polypeptide, wherein the candidate binding polypeptide is anchored to the outer side of the inner membrane;
wherein the bacterium further comprises a nucleic acid sequence encoding a fusion polypeptide comprising a target ligand, a periplasmic export signal, a fluorescent label and a cytoplasmic degradation signal;
(b) allowing the target ligand to bind to the candidate binding polypeptide;
(c) removing the outer membrane of said bacterium; and
(d) selecting the bacterium based on the presence of the target ligand bound to the candidate binding polypeptide on the outer side of the inner membrane using fluorescence activated cell sorting (FACS). - View Dependent Claims (50, 51, 52, 53)
-
Specification