Secretion of antibodies without signal peptides from bacteria
View Patent ↗The present invention is directed generally to compositions and methods for obtaining secretion of antibodies or antigen-binding antibody fragments from prokaryotes without the need for a signal peptide through making use of mutant host strains with altered secretory properties. In particular, the invention provides host cells and methods for obtaining secretion of antibodies or antigen-binding antibody fragments from bacteria without the need for a signal peptide and provides diverse libraries of antibody sequence resulting from such methods. The invention additionally provides diverse libraries.
1. A method for producing an antibody, comprising the steps of:
(a) culturing an E. coli host cell containing a prlA4 mutation (SEQ ID NO: 1) and at least one expression vector comprising a polynucleotide molecule encoding a heavy chain polypeptide of the antibody and a polynucleotide molecule encoding a light chain polypeptide of the antibody, under conditions that express the polynucleotide encoding the heavy chain polypeptide and the polynucleotide molecule encoding the light chain polypeptide of the antibody, wherein at least one of the polynucleotides encodes a polypeptide lacking a signal peptide,
(b) secreting the heavy chain polypeptide and the light chain polypeptide across a cytoplasmic membrane, and
(c) forming the antibody from the heavy chain polypeptide and the light chain polypeptide.
2. The method of claim 1 , wherein the antibody is selected from the group consisting of a human antibody, a mouse antibody, a rat antibody, a rabbit antibody, a camel antibody, a sheep antibody, a chimeric antibody, a humanized antibody, a fusion antibody and an epitope-focused antibody.
3. The method of claim 1 , wherein the polynucleotides encoding the heavy chain polypeptide and the light chain polypeptide are present on different expression vectors.
4. The method of claim 1 , wherein the polynucleotides encoding the heavy chain polypeptide and the light chain polypeptide are both present on the same vector.
5. The method of claim 1 , wherein the polynucleotide encodes the heavy chain polypeptide lacking a signal peptide.
6. The method of claim 1 , wherein the polynucleotide encodes the light chain polypeptide lacking a signal peptide.
7. The method of claim 1 , wherein the polynucleotides encode the heavy and light chain polypeptides lacking signal peptides.
8. The method of claim 1 , further comprising a plurality of other polynucleotide molecules encoding a plurality of different heavy chain polypeptides and a plurality of other polynucleotide molecules encoding a plurality of different light chain polypeptides, under conditions that express the polynucleotides encoding the heavy chain polypeptides and the polynucleotide molecules encoding the light chain polypeptides, wherein each E. coli host cell contains polynucleotides encoding one pair of one heavy chain polypeptide and one light chain polypeptide, wherein at least one polypeptide of each pair of polypeptides lacks a signal peptide, and whereby the representation of V H and V L subclasses in the polypeptide pairs is improved.
9. The method of claim 1 , wherein the SecY gene having a prlA4 mutation phenotype is carried on a plasmid or other vector.
10. The method of claim 1 , wherein the SecY gene having a prlA4 mutation phenotype is carried on the E. coli host cell's chromosome.
11. A method for producing an antibody, comprising the steps of:
(a) culturing an E. coli host cell containing a prlA4 mutation (SEQ ID NO: 1) and an expression vector comprising a polynucleotide molecule encoding the antibody under conditions that express the polynucleotide encoding the antibody wherein the polynucleotide encodes a polypeptide lacking a signal peptide, and
(b) secreting the antibody across a cytoplasmic membrane.
12. The method of claim 11 , wherein the mutant gene is carried on a vector.
13. The method of claim 12 , wherein the antibody is selected from the group consisting of a single-chain antibody, a scFv, a dAB, a VHH, a camelid antibody, and a nanobody.
14. A method for producing an antibody, comprising the steps of:
(a) culturing an E. coli host cell containing a prIA4 mutation (SEQ ID NO: 1) and at least one expression vector that comprises one or more polynucleotide molecules encoding the antibody under conditions that express the polynucleotide or polynucleotides encoding the antibody,
(b) transporting the polypeptide or polypeptides across a cytoplasmic membrane in a signal-independent manner, and
(c) forming the antibody, wherein said antibody is capable of binding antigen.
15. A method for making an antibody, comprising the steps of:
(a) expressing an antibody or antibody fusion from at least one expression vector encoding the antibody or antibody fusion in an E. coli host cell containing a prlA4 mutation (SEQ ID NO: 1),
(b) forming the antibody or antibody fusion, and
(c) identifying the E. coli host cells that make the antibody or antibody fusion.
16. The method of 15 , further comprising the step of isolating the antibody or antibody fusion.