Method for rapid in vitro synthesis of glycoproteins via recombinant production of N-glycosylated proteins in prokaryotic cell lysates

Patent metadata

Field	Value
Publication number	US-10829795-B2
Application number	US-201715650127-A
Country	US
Kind code	B2
Filing date	Jul 14, 2017
Priority date	Jul 14, 2016
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

First claim

We claim:

1. A method for synthesizing an N-glycosylated carrier protein for a bacterial O-antigen via coordinated transcription, translation, and N-glycosylation in vitro in a single reaction vessel comprising one or more Escherichia coli cell lysates,
wherein the one or more Escherichia coli cell lysates comprise:
(a) an exogenous bacterial oligosaccharyltransferase (OST); and
(b) an exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen;

the method comprising:
(i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction in the single reaction vessel, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline; and
(ii) N-glycosylating the carrier protein at the inserted consensus sequence with the bacterial O-antigen using the exogenous bacterial OST in a cell-free glycosylation reaction in the single reaction vessel.

2. The method of claim 1 , wherein the bacterial O-antigen is from E. coli.

3. The method of claim 1 , wherein the bacterial O-antigen is from Franciscella tularensis.

4. The method of claim 1 , further comprising formulating the N-glycosylated carrier protein as a vaccine composition comprising the N-glycosylated carrier protein.

5. The method of claim 4 , wherein the vaccine composition further comprises an adjuvant.

6. The method of claim 1 , wherein the carrier protein is an engineered recombinant protein of E. coli maltose binding protein (MBP).

7. The method of claim 1 , wherein the carrier protein is a detoxified recombinant protein of the toxin from Clostridium tetani.

8. The method of claim 1 , wherein the carrier protein is a detoxified recombinant protein of the toxin from Corynebacterium diptheriae.

9. The method of claim 1 , wherein the one or more Escherichia coli cell lysates are prepared by combining:
(a) an Escherichia coli cell lysate comprising the exogenous bacterial oligosaccharyltransferase (OST); and
(b) a separate Escherichia coli cell lysate comprising the exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen.

10. A method for synthesizing an N-glycosylated carrier protein for a bacterial O-antigen via coordinated transcription, translation, and N-glycosylation in vitro in a single reaction vessel comprising a reaction mixture prepared by adding an aqueous solution to one or more freeze-dried Escherichia coli cell lysates,
wherein the one or more freeze-dried Escherichia coli cell lysates comprise:
(a) an exogenous bacterial oligosaccharyltransferase (OST); and
(b) an exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen;

the method comprising:
(i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction in the single reaction vessel, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline; and
(ii) N-glycosylating the carrier protein at the inserted consensus sequence with the bacterial O-antigen using the exogenous bacterial OST in a cell-free glycosylation reaction in the single reaction vessel.

11. The method of claim 10 , wherein the freeze-dried Escherichia coli cell lysates comprise:
(a) a freeze-dried Escherichia coli cell lysate comprising the exogenous bacterial oligosaccharyltransferase (OST); and
(b) a separate freeze-dried Escherichia coli lysate comprising the exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen.

12. A method for synthesizing an N-glycosylated carrier protein for a bacterial O-antigen via coordinated transcription, translation, and N-glycosylation in vitro in a single reaction vessel comprising one or more Escherichia coli cell lysates,
wherein the one or more Escherichia coli cell lysates comprise:
(a) an exogenous bacterial oligosaccharyltransferase (OST) that is a naturally occurring bacterial homolog of C. jejuni PglB or an engineered recombinant protein of C. jejuni PglB; and
(b) an exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen;

the method comprising:
(i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction in the single reaction vessel, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline; and
(ii) N-glycosylating the carrier protein at the inserted consensus sequence with the bacterial O-antigen using the exogenous bacterial OST in a cell-free glycosylation reaction in the single reaction vessel.

13. The method of claim 12 , wherein the bacterial O-antigen is from E. coli.

14. The method of claim 12 , wherein the bacterial O-antigen is from Franciscella tularensis.

15. The method of claim 12 , further comprising formulating the N-glycosylated carrier protein as a vaccine composition comprising the N-glycosylated carrier protein.

16. The method of claim 12 wherein the carrier protein is an engineered recombinant protein of E. coli maltose binding protein (MBP).

17. The method of claim 12 wherein the carrier protein is a detoxified recombinant protein of the toxin from Clostridium tetani.

18. The method of claim 12 wherein the carrier protein is a detoxified recombinant protein of the toxin from Corynebacterium diptheriae.

19. The method of claim 12 , wherein the single reaction vessel comprises a reaction mixture prepared by adding an aqueous solution to one or more freeze-dried Escherichia coli cell lysates.

Assignees

• Univ Northwestern
• Univ Cornell

Inventors

• Jewett Michael Christopher
• Stark Jessica Carol
• Delisa Matthew P
• Jaroentomeechai Thapakorn

Classifications

• C12R2001/145

  Clostridium · CPC title

• C12R2001/19

  Escherichia coli · CPC title

• C12R2001/00

  Microorganisms {; Processes using microorganisms} · CPC title

• C12N1/00

  Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor · CPC title

• C12N1/205

  Bacterial isolates · CPC title