Methods of host cell modification

Patent metadata

Field	Value
Publication number	US-11220676-B2
Application number	US-201415028483-A
Country	US
Kind code	B2
Filing date	Oct 13, 2014
Priority date	Oct 11, 2013
Publication date	Jan 11, 2022
Grant date	Jan 11, 2022

Abstract

Described herein are methods of inserting nucleic acid sequences into host cells. Also described herein are genetically stable host cells comprising inserted nucleic acid sequences and methods of using such host cells in the generation of proteins.

First claim

What is claimed is:

1. An isolated Escherichia coli ( E. coli ) host cell comprising an E. coli host cell genome comprising (i) a heterologous DNA sequence comprising a gene that encodes an oligosaccharyl transferase wherein said oligosaccharyl transferase is heterologous to the E. coli host cell and (ii) a heterologous DNA sequence comprising one or more genes encoding one or more glycosyltransferases, wherein said one or more glycosyltransferases are heterologous to the E. coli host cell wherein the heterologous DNA sequence comprising the heterologous gene that encodes an oligosaccharyl transferase and the heterologous DNA sequence comprising the one or more heterologous genes encoding one or more glycosyltransferases have been inserted into the genome of the E. coli host cell, wherein the heterologous DNA sequence comprising the one or more heterologous genes encoding one or more glycosyltransferase genes is at least 12 kb and encodes one or more glycosyltransferases capable of making an S. aureus capsular polysaccharide or an E. coli O-antigen, wherein the heterologous gene encoding the oligosaccharyl transferase is flanked by a homology region; and wherein the E. coli host cell further comprises a gene encoding a carrier protein containing at least SEQ ID NO: 72.

2. The isolated E. coli host cell of claim 1 , wherein the gene that encodes the carrier protein is heterologous to the E. coli host cell and is capable of being N-glycosylated and has been inserted into the genome of the host cell.

3. The isolated host cell of claim 1 , wherein said heterologous gene that encodes an oligosaccharyl transferase comprises the C. jejuni pgIB gene.

4. The isolated host cell of claim 1 , wherein the heterologous gene that encodes an oligosaccharyl transferase has a copy number in the host cell selected from the group consisting of 1, 2, 3, 4, or 5.

5. A method of producing an N-glycosylated protein, wherein said method comprises culturing the host cell of claim 1 under conditions suitable for the production of proteins.

6. An isolated Escherichia coli ( E. coli ) host cell comprising an E. coli host cell genome comprising (i) a heterlogous DNA sequence comprising a gene that encodes an oligosaccharyl transferase wherein said oligosaccharyl transferase is heterologous to the E. coli host cell; (ii) a heterologous DNA sequence comprising a heterologous gene that encodes an rfb cluster, wherein said heterologous gene that encodes an rib cluster has been inserted into the genome of the host cell; and (H) a heterologous DNA sequence comprising one or more genes encoding one or more glycosyltransferases, wherein said one or more glycosyltransferases are heterologous to the E. coli host cell, wherein the heterologous DNA sequence comprising the heterologous gene that encodes an oligosaccharyl transferase and the heterologous DNA sequence comprising the one or more heterologous genes encoding one or more glycosyltransferases have been inserted into the genome of the E. coli host cell, wherein the heterologous DNA sequence comprising the one or more heterologous genes encoding one or more glycosyltransferase genes is at least 12 kb and encodes one or more glycosyltransferases capable of making an S. aureus capsular polysaccharide or an E. coli O-antigen, wherein the heterologous gene encoding the oligosaccharyl transferase is flanked by a homology region; and wherein the E. coli host cell further comprises a gene that encodes a carrier protein capable of being N-glycosylated, wherein the gene encoding a carrier protein encodes a carrier protein containing at least SEQ ID NO: 72.

7. The isolated host cell of claim 6 , wherein said gene that encodes a carrier protein capable of being N-glycosylated is a heterologous gene.

8. The isolated host cell of claim 7 , wherein said gene that encodes a carrier protein capable of being N-glycosylated is expressed on a plasmid present in the host cell.

9. The isolated host cell of claim 7 , wherein said gene that encodes a carrier protein capable of being N-glycosylated has been inserted into the genome of the host cell.

10. The isolated host cell of claim 6 , wherein said heterologous gene that encodes an oligosaccharyl transferase comprises the C. jejuni pgIB gene.

11. The isolated host cell of claim 6 , wherein the heterologous gene that encodes an oligosaccharyl transferase has a copy number in the host cell selected from the groups consisting of: 1, 2, 3, 4, or 5.

12. A method of producing an N-glycosylated protein, wherein said method comprises culturing the host cell of claim 6 under conditions suitable for the production of proteins.

Assignees

• Glaxosmithkline Biologicals Sa
• Glaxosmithkline Biological Sa

Inventors

• Wacker Michael
• Kowarik Michael
• Fernandez Fabiana

Classifications

• C12N2795/00041

  Use of virus, viral particle or viral elements as a vector · CPC title

• A61K39/0283

  Shigella · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12N15/90 Primary

  Stable introduction of foreign DNA into chromosome · CPC title

• A61K39/0258

  Escherichia · CPC title