Method of generating multiple protein variants and populations of protein variants prepared thereby

Claim 3

The method of claim 1, wherein the donor vector is a phagemid.

Claim 4

The method of claim 2, wherein the host cell is a bacterium.

Claim 5

The method of claim 3, wherein the bacterium is Escherichia coli.

Claim 6

The method of claim 2, further comprising separating phage particles which contain a helper phage genome from phage particles which contain a phagemid having a mutant encoding nucleotide sequence.

Claim 7

The method of claim 5, wherein the phage particles which contain a helper phage genome are separated from the phage particles which contain a phagemid by size difference.

Claim 8

The method of claim 2, further comprising maintaining a multiplicity of infection of at least one and a transductant-forming-unit to plaque-forming-unit ratio (tfu/pfu) of at least one.

Claim 9

The method of claim 2, wherein each recipient vector expresses a distinct genetic marker.

Claim 10

The method of claim 2, further comprising repeating steps (c) to (i) using additional cloned donor pools of mutant encoding nucleotide sequences.

Claim 11

The method of claim 2, wherein each phagemid expresses a distinct genetic marker.

Claim 12

The method of claim 2, wherein the helper phage is a filamentous phage.

Claim 13

The method of claim 11, wherein the filamentous phage is M13.

Claim 14

The method of claim 1, further comprising screening intermediate populations.

Claim 15

A population of multiple protein variants prepared by a process comprising the steps of: a) providing first and second sets of allelic mutants, wherein said first set of allelic mutants comprises at least one recipient pool of mutant encoding nucleotide sequences and said second set of allelic mutants comprises at least one donor pool of mutant encoding nucleotide sequences; b) cloning each recipient pool of mutant encoding nucleotide sequences into a recipient vector; c) transforming or transducing the recipient vector into a host cell to generate a pool of recipient cells; d) cloning each donor pool of mutant encoding nucleotide sequences into a donor vector selected from the group consisting of phagemids and cosmids; e) transforming or transducing the donor vector into a host cell to generate a pool of donor cells; f) infecting the pool of donor cells with a helper phage to generate a mixture of donor phage particles, wherein said mixture comprises donor phage particles which contain said helper phage genome and phage particles which contain donor vectors having a mutant encoding nucleotide sequence; g) clonally amplifying the pools of donor phage and recipient cells; h) transducing the recipient cells with the mixture of donor phage particles to generate a set of recombinants; and i) screening the recombinants.

Claim 16

A population of multiple protein variants prepared by a process comprising the steps of: a) providing first and second sets of allelic mutants, wherein said first set of allelic mutants comprises at least one recipient pool of mutant encoding nucleotide sequences and said second set of allelic mutants comprises at least one donor pool of mutant encoding nucleotide sequences; b) cloning each recipient pool of mutant encoding nucleotide sequences into a recipient vector; c) transforming or transducing the recipient vector into a host cell to generate a pool of recipient cells; d) cloning each donor pool of mutant encoding nucleotide sequences into a donor vector selected from the group consisting of phagemids and cosmids; e) transforming or transducing the donor vector into a host cell to generate a pool of donor cells; f) infecting the pool of donor cells with a helper phage to generate a mixture of donor phage particles, wherein said mixture comprises donor phage particles which contain said helper phage genome and phage particles which contain donor vectors having a mutant encoding nucleotide sequence; g) clonally amplifying the pools of donor phage and recipient cells; h) transducing the recipient cells with the mixture of donor phage particles to generate a set of recombinants; and i) screening the recombinants.

Claim 17

The population of claim 14, prepared by the process wherein the donor vector is a phagemid.

Claim 18

The population of claim 15, prepared by the process wherein the host cell is a bacterium.

Claim 19

The population of claim 16, prepared by the process wherein the bacterium is Escherichia coli.

Claim 20

The population of claim 15, prepared by the process further comprising separating phage particles which contain a helper phage genome from phage particles which contain a phagemid having a mutant encoding nucleotide sequence.

Claim 21

The population of claim 18, prepared by the process wherein the phage particles which contain a helper phage genome are separated from the phage particles which contain a phagemid by size difference.

Claim 22

The population of claim 15, prepared by the process further comprising maintaining a multiplicity of infection of at least one and a transductant-forming-unit to plaque-forming-unit ratio (tfu/pfu) of at least one.

Claim 23

The population of claim 15, prepared by the process wherein each recipient vector expresses a distinct genetic marker.

Claim 24

The population of claim 15, prepared by the process further comprising repeating steps (c) to (i) using additional cloned donor pools of mutant encoding nucleotide sequences.

Claim 25

The population of claim 15, prepared by the process wherein each phagemid expresses a distinct genetic marker.

Claim 26

The population of claim 15, prepared by the process wherein the helper phage is a filamentous phage.

Claim 27

The population of claim 24, prepared by the process wherein the filamentous phage is M13.

Claim 28

The population of claim 15, wherein the process further comprises screening intermediate populations.