Method of generating multiple protein variants and populations of protein variants prepared thereby
AssigneeLeland Stanford Junior University
InventorsChaitan Khosla, Robert Caren
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Abstract
A method based on generalized recombination is provided for generating multiple protein variants. The method involves introducing into host cells at least two pools of mutant encoding nucleotide sequences, recipient and donor, transducing the donor pool into the recipient cells and subsequently screening and selecting the recombinants thereby produced for expressed proteins exhibiting desired characteristics. A population of such protein variants is also provided.
Claims26
A method of generating multiple protein variants, wherein said method comprises:
- 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;
- and
- h) transducing the recipient cells with the mixture of donor phage particles to generate a set of recombinants;
- i) screening the recombinants.
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.
Dependencies are inferred from the claim text.