Expression of toxic genes in vivo in a non-natural host
View Patent ↗Compositions and methods are provided for expression of a toxic protein in a host cell preferably a bacterial host cell where at least one T7 RNA polymerase gene Is contained on the host cell chromosome and one or more genes encoding a T7 RNA polymerase inhibitor is located on an F′ plasmid or on the chromosome.
1. A genetically engineered host cell capable of expressing a toxic protein, the host cell, comprising:
(a) a chromosome in which is contained one or more T7 RNA polymerase genes; and
(b) one or more genes encoding a T7 RNA polymerase inhibitor located in an F′ plasmid or on the chromosome.
2. The host cell according to claim 1 , wherein the one or more T7 RNA polymerase genes are present in a ratio of 1:1 with the one or more T7 RNA polymerase inhibitor genes.
3. The host cell according to claim 1 , further comprising: a gene expressing LacI or Laclq.
4. The host cell according to claim 1 , wherein the T7 RNA polymerase inhibitor gene is the T7 lysozyme gene.
5. The host cell according to claim 4 , wherein the T7 lysozyme gene expresses a mutant T7 lysozyme.
6. The host cell according to claim 4 , wherein the mutant T 7 lysozyme has a mutation selected from Y46F, K128Q, K128Y, K128M, K128W and K128I.
7. The host cell according to claim 1 , wherein the cell is competent for receiving foreign DNA.
8. The host cell according to claim 2 , wherein the cell is an E. coil cell.
9. An F′ plasmid, comprising: a wild type T7 lysozyme gene or a mutant thereof capable of expressing a T7 lysozyme protein wherein the protein expressed by the mutant gene is characterized by a mutation at position Y46 or K128, the wild type or mutant gene being operably linked to a constitutive promoter.
10. The F′ plasmid according to claim 9 , wherein the T7 lysozyme mutant gene expresses a protein having a mutation selected from Y46F, K128Q, K128Y, K128M, K128W and K128I.
11. A method, comprising the steps of:
(a) providing a genetically engineered host cell according to claim 1 ;
(b) transforming the host cell with a plasmid containing a target gene;
(c) incubating the host cell to a cell density sufficient to permit expression of the target gene; and
(d) inhibiting T7 RNA polymerase activity until initiation of induction so as to permit expression of the target gene in the genetically engineered host cell.
12. A method according to claim 11 , wherein the target gene expresses a toxic protein.
13. A method according to claim 11 , wherein the toxic gene product is a membrane protein.
14. A method according to claim 11 , wherein the T7 RNA polymerase inhibitor is selected from a wild type T7 lysozyme gene, LysY or mutants thereof.
15. A method according to claim 14 , wherein the mutant T7 lysozyme gene expresses a protein having a mutation selected from Y46F, K128Q, K128Y, K128M, K128W and K128I.
16. An F′plasmid according to claim 9 , inserted into a genetically engineered host cell capable of expressing a toxic protein, and containing a chromosome in which is contained one or more of the T7 RNA polymerase genes.