Directed evolution of enzymes and antibodies

Abstract

The invention relates to methods of selecting proteins, out of large libraries, having desirable characteristics. Exemplified are methods of expressing enzymes and antibodies on the surface of host cells and selecting for desired activities. These methods have the advantage of speed and ease of operation when compared with current methods. They also provide, without additional cloning, a source of significant quantities of the protein of interest.

Description

[0001] The present application is a continuation of copending U.S. patent application Ser. No. 09 / 813,444, filed Mar. 20, 2001, which is a continuation of copending U.S. patent application Ser. No. 09 / 782,672, filed Feb. 12, 2001, which is a continuation-in-part of U.S. Pat. No. 5,866,344 filed May 23, 1995 and issued Feb. 2, 1999, which is a continuation-in-part of U.S. Ser. No. 08 / 258,543, filed Jun. 10, 1994 is abandoned, which is a divisional of U.S. Pat. No. 5,348,867 issued Sep. 20, 1994. The entire text of each of the above-referenced applications and patents are specifically incorporated by reference herein without disclaimer. The government owns rights in the present invention pursuant to grant number BCS-9412502 from the National Science Foundation.BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of biochemistry, immunology and molecular biology. More particularly, it concerns the use of rapid selecti...

Examples

example 1

Surface Expression of Anti-digoxin Single Chain Fv Antibodies

[0217]E. coli strain JM109 [endA1 recA1 gyrA thi-1 hsdR17 (rk−, Mk+) relA1 supE44 Δ(lac-proAB) / F′ traD36proAB laclq lacZΔM15] was used for all studies. pTX101 codes for an Lpp-OmpA-β-lactamase fusion (Francisco et al., 1992). pTX152 codes for an Lpp-OmpA-scFv(digoxin) fusion, where the scFv(digoxin) is an anti-digoxin single chain Fv consisting of the heavy- and light-chain variable regions (VH and VL). The VH and VL, joined by a 15 amino acid [(Gly)4Ser]3 linker (Huston et al., 1988), were amplified from messenger RNA isolated from two separate anti-digoxin hybridomas. An 11 amino acid peptide from the Herpes Simplex Virus glycoprotein (Novagen) was introduced at the C-terminus of the scFv for analytical purposes. The presence of the HSV peptide allowed detection of the scFv(digoxin) protein by reaction with a monoclonal antibody specific for the 11 amino acid epitope. The sequence of the single chain Fv antibody fragmen...

example 2

Enrichment of Cells Displaying scFv Antibodies by FACS

[0232] Antibody expressing cells were sorted essentially quantitatively from an excess of control E. Coli in a single step. Specifically, in mixtures containing JM109 / pTX101 control cells at an excess of either 100:1 or 1,000:1, the fraction of the total population that was sorted in the high fluorescence intensity window was 1.1% and 0.1% respectively (after subtracting the background), as expected from the ratio of input cells.

[0233] The use of FACS for isolating rare clones from a very large excess of background was also demonstrated. JM109 / pTX101 (cells displaying an unrelated protein on the cell surface) and JM101 / pTX152 (cells displaying the scFv(digoxin) antibodies) were mixed at a ratio of 100,000:1 and labeled with digoxin-FITC. Following washing to remove any non-specific binding of the digoxin-fluorescein conjugate on the control E. Coli, 500,000 cells were run through the FACSort flow cytometer. A wide sorting gate,...

example 3

Surface Expression of Anti-Digoxin Antibody Incorporating Protease Cleavage Site

[0238] A construct similar to that used for surface expression of scFv (digoxin) can be modified to incorporate a protease cleavage site. For example, the recognition sequence of enterokinase [(Asp)4-Ile-Arg] can be introduced in the Lpp-OmpA(46-159)-scFv between the OmpA(46-159) and the scFv domains. The protease cleavage site at the N-terminal of the scFv antibody domain of the fusion protein is then used to release the scFv antibody in soluble form following treatment of the cells with the appropriate proteolytic enzyme. Because the outer membrane of E. coli serves as a protective barrier to the action of externally added proteases, very few contaminating proteins will be present in the culture supernatant. A single colony expressing a desired single chain Fv antibody can be grown in liquid media and harvested by centrifugation after overnight growth at 24° C. The cells are resuspended in buffer to m...

[0001] The present application is a continuation of copending U.S. patent application Ser. No. 09 / 813,444, filed Mar. 20, 2001, which is a continuation of copending U.S. patent application Ser. No. 09 / 782,672, filed Feb. 12, 2001, which is a continuation-in-part of U.S. Pat. No. 5,866,344 filed May 23, 1995 and issued Feb. 2, 1999, which is a continuation-in-part of U.S. Ser. No. 08 / 258,543, filed Jun. 10, 1994 is abandoned, which is a divisional of U.S. Pat. No. 5,348,867 issued Sep. 20, 1994. The entire text of each of the above-referenced applications and patents are specifically incorporated by reference herein without disclaimer. The government owns rights in the present invention pursuant to grant number BCS-9412502 from the National Science Foundation.BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of biochemistry, immunology and molecular biology. More particularly, it concerns the use of rapid selecti...

Examples

example 1

Surface Expression of Anti-digoxin Single Chain Fv Antibodies

[0217]E. coli strain JM109 [endA1 recA1 gyrA thi-1 hsdR17 (rk−, Mk+) relA1 supE44 Δ(lac-proAB) / F′ traD36proAB laclq lacZΔM15] was used for all studies. pTX101 codes for an Lpp-OmpA-β-lactamase fusion (Francisco et al., 1992). pTX152 codes for an Lpp-OmpA-scFv(digoxin) fusion, where the scFv(digoxin) is an anti-digoxin single chain Fv consisting of the heavy- and light-chain variable regions (VH and VL). The VH and VL, joined by a 15 amino acid [(Gly)4Ser]3 linker (Huston et al., 1988), were amplified from messenger RNA isolated from two separate anti-digoxin hybridomas. An 11 amino acid peptide from the Herpes Simplex Virus glycoprotein (Novagen) was introduced at the C-terminus of the scFv for analytical purposes. The presence of the HSV peptide allowed detection of the scFv(digoxin) protein by reaction with a monoclonal antibody specific for the 11 amino acid epitope. The sequence of the single chain Fv antibody fragmen...

example 2

Enrichment of Cells Displaying scFv Antibodies by FACS

[0232] Antibody expressing cells were sorted essentially quantitatively from an excess of control E. Coli in a single step. Specifically, in mixtures containing JM109 / pTX101 control cells at an excess of either 100:1 or 1,000:1, the fraction of the total population that was sorted in the high fluorescence intensity window was 1.1% and 0.1% respectively (after subtracting the background), as expected from the ratio of input cells.

[0233] The use of FACS for isolating rare clones from a very large excess of background was also demonstrated. JM109 / pTX101 (cells displaying an unrelated protein on the cell surface) and JM101 / pTX152 (cells displaying the scFv(digoxin) antibodies) were mixed at a ratio of 100,000:1 and labeled with digoxin-FITC. Following washing to remove any non-specific binding of the digoxin-fluorescein conjugate on the control E. Coli, 500,000 cells were run through the FACSort flow cytometer. A wide sorting gate,...

example 3

Surface Expression of Anti-Digoxin Antibody Incorporating Protease Cleavage Site

[0238] A construct similar to that used for surface expression of scFv (digoxin) can be modified to incorporate a protease cleavage site. For example, the recognition sequence of enterokinase [(Asp)4-Ile-Arg] can be introduced in the Lpp-OmpA(46-159)-scFv between the OmpA(46-159) and the scFv domains. The protease cleavage site at the N-terminal of the scFv antibody domain of the fusion protein is then used to release the scFv antibody in soluble form following treatment of the cells with the appropriate proteolytic enzyme. Because the outer membrane of E. coli serves as a protective barrier to the action of externally added proteases, very few contaminating proteins will be present in the culture supernatant. A single colony expressing a desired single chain Fv antibody can be grown in liquid media and harvested by centrifugation after overnight growth at 24° C. The cells are resuspended in buffer to m...

Images