Display library process
View Patent ↗Disclosed are methods for identifying desired members from a display libraries, including bacteriophage display libraries. Display library members can be amplified in the presence of a target compound so that cycles of selection can be rapidly completed.
1. A method comprising:
(i) maintaining a first plurality of bacterial host cells in the presence of a target compound, each host cell containing a nucleic acid encoding a candidate protein that is attachable to a bacteriophage particle and that varies among the first plurality of host cells, the maintaining being under conditions wherein the host cells of the first plurality produce a first plurality of bacteriophage particles that display the candidate proteins; and
(ii) immobilizing a first subset of the bacteriophage particles, wherein the particles of the first subset display candidate proteins that bind to the target compound.
2. The method of claim 1 , wherein the method further comprises:
(iii) separating the first subset of the bacteriophage particles that bind to the target compound from the first plurality of bacteriophage particles produced by the first plurality of bacterial host cells.
3. The method of claim 2 , further comprising: (iv) amplifying the separated first subset of bacteriophage particles in the presence of the target compound to yield a population of amplified bacteriophage particles, wherein during or after the amplifying, at least a subset of the amplified bacteriophage particles physically interact with the target compound.
4. The method of claim 1 , wherein the first plurality of bacteriophage particles is characterized by a first titre; and wherein the first subset is less than 0.01% of the first titre.
5. The method of claim 3 , wherein the amplifying step is performed by a process comprising:
immobilizing the separated first subset of bacteriophage particles on a support that includes the target compound, wherein members of the first subset display candidate proteins on their surface;
contacting a host cell to the support under conditions that allow an immobilized member of the first subset to infect the host cell; and
culturing the host cell in the presence of the support under conditions that enable production of replicates of the immobilized member of the first subset, thereby amplifying the first subset of bacteriophage particles.
6. The method of claim 5 , wherein the amplifying step is performed in a defined nutritive medium that supports growth of the host cell.
7. The method of claim 2 , further comprising:
contacting the amplified bacteriophage particles to the target compound;
immobilizing a second subset of the bacteriophage particles, wherein the bacteriophage particles of the second subset display candidate proteins that bind to the target compound;
separating the second subset of the bacteriophage particles; and
amplifying the separated second subset in bacterial host cells to produce a second plurality of bacterial host cells.
8. The method of claim 1 , wherein the maintaining step includes a target-presenting cell that includes the target compound accessible to medium surrounding the cell.
9. The method of claim 7 , wherein the second plurality of bacterial host cells produces a second plurality of bacteriophage particles characterized by a second titre, which is less than one tenth of the first titre.