Method of enabling pooled-library based nucleic acid constructs screening
The invention relates to a method for generating in a single step a library of recombinant baculoviruses for screening for a protein of interest, starting from a pooled-library of poly-nucleotides each encoding a different protein. Insect cells are infected with a pooled-library of baculoviruses at a very low multiplicity of infection (MOI) leading to the situation where the majority of cells are infected by a single baculovirus. The pool of cells is then treated with a viral inhibitor which prevents secreted baculoviruses from penetrating other cells without affecting, either the capacity of the cell to express the target protein, nor intracellular viral replication and accumulation.
1 . A method for screening proteins and polypeptides to identify a protein or polypeptide of interest, which comprises the sequential steps of:
i) generating a pooled-library of genes or genomic fragments cloned in baculovirus as a vector,
(ii) transducing host cells with the pooled-library of baculovirus vectors at a multiplicity of infection (MOI) of less than 0.1,
(iii) washing the host cells 2-4 hours post infection in a washing step,
(iv) adding a viral inhibitor to the host cells,
(v) culturing the cells for at least 2 days, so that the protein of interest is expressed in the host cells,
wherein the viral inhibitor is selected from the group consisting of chloroquine, ammonium chloride, oseltamivir, or a neuraminidase inhibitor.
2 . The method of claim 1 , further comprising step (vi) identifying and isolating the protein of interest.
3 . The method of claim 1 , wherein the host cell is an insect cell.
4 . The method of claim 1 , wherein the host cells are pooled together and transduced in a batch.
5 . The method of claim 1 , wherein the viral inhibitor's inhibition of viral infection is reversible.
6 . The method of claim 1 , wherein the viral inhibitor does not affect production of the proteins and polypeptides.
7 . The method of claim 1 , wherein the viral inhibitor is chloroquine.
8 . The method of claim 7 , wherein the chloroquine is added at a concentration of between 0.1 mM and 2 mM.
9 . The method of claim 1 , wherein the protein of interest is identified using flow cytometry.
10 . The method of claim 1 , wherein the host cells are cultured to expand cells to levels of protein or polypeptide required for screening and/or analysis.
11 . The method of claim 1 , wherein said method comprises transducing host cells with the pooled-library of baculovirus vectors at a multiplicity of infection (MOI) of 0.1-0.01.
12 . The method of claim 1 , wherein said method comprises culturing the cells for 2 to 5 days.
13 . The method of claim 1 , wherein the viral inhibitor is selected from the group consisting of chloroquine, ammonium chloride, and oseltamivir.
14 . The method of claim 1 , wherein the viral inhibitor is selected from the group consisting of chloroquine and oseltamivir.
15 . The method of claim 1 , wherein the viral inhibitor is selected from the group consisting of chloroquine and ammonium chloride.
16 . The method of claim 1 , wherein the viral inhibitor is a neuraminidase inhibitor.