Dual Cistronic Bacterial Expression System

• US 20190309313A1
• Filed: 04/24/2019
• Published: 10/10/2019
• Est. Priority Date: 07/09/2014
• Status: Active Grant

• Current Assignee: Lupin Limited
• Original Assignee: Lupin Limited
• Inventors: Salunkhe, Shardul, Varshney, Brajesh, Soorapaneni, Sudheerbabu
• Granted Patent: US 11,365,418 B2
• Time in Patent Office: Days
• CPC Class Codes: A01H 6/342 Citrullus lanatus [watermelon]
• CPC Class Codes: C07K 14/27 Erwinia (G)
• CPC Class Codes: C07K 14/605 Glucagons
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/40 containing a tag for immunodetection, or an epitope for immunisation
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 304/24029 Aureolysin (3.4.24.29)

Abstract

The present invention relates to the dual, independent cistron expression system in a single vector for the production of protein of interest proteins and peptides expressed as insoluble inclusion bodies formed in the bacteria E. coli. The present invention also provides the process for the expression of protein of interest using said bicistronic vector.

Claims

1. A dual independent cistronic expression system for expression in a prokaryotic host cell, comprising:

a) a first cistron, comprising a T7 promoter having the sequence of SEQ ID NO;

10 operably linked to a polynucleotide sequence encoding either a heavy chain or a light chain of an antibody or a fragment thereof;

b) a second cistron, comprising an arabinose promoter having the sequence of SEQ ID NO;

14 operably linked to a polynucleotide sequence encoding either a heavy chain or a light chain of an antibody or a fragment thereof;

wherein, the first and second cistrons are positioned within the same vector and express both the heavy chain and the light chain proteins at equimolar levels in the form of inclusion bodies.

2. The dual independent cistronic expression system as claimed in claim 1, wherein the antibody or the fragment thereof is a Fab fragment.

3. The dual independent cistronic expression system as claimed in claim 1, wherein the heavy chain is encoded by the polynucleotide sequence set forth in SEQ ID NO:

1.

4. The dual independent cistronic expression system as claimed in claim 1, wherein the expressed heavy chain comprises of the amino acid sequence as set forth in SEQ ID NO:

3.

5. The dual independent cistronic expression system as claimed in claim 1, wherein the light chain is encoded by the polynucleotide sequence set forth in SEQ ID NO:

2.

6. The dual independent cistronic expression system as claimed in claim 1, wherein the expressed light chain comprises of the amino acid sequence as set forth in SEQ ID NO:

4.

7. The dual independent cistronic expression system as claimed in claim 1, wherein the Fab fragment is Ranibizumab.

8. The dual independent cistronic expression system for expression in a prokaryotic host cell as claimed in claim 1, wherein the prokaryotic host cell is E. coli.

9. The dual independent cistronic expression system as claimed in claim 1, wherein the expression system further comprises one or more elements selected from the group consisting of a polynucleotide sequence encoding, a regulator, a signal peptide, and a selection marker.

10. The dual independent cistronic expression system as claimed in claim 9, wherein the regulator is AraC gene.

11. The dual independent cistronic expression system as claimed in claim 9, wherein the signal peptide is pelB.

12. The dual independent cistronic expression system as claimed in claim 9, wherein the selection marker is ampicillin.

13. A process for producing an antibody or a fragment thereof using a dual independent cistronic expression system as claimed in claim 1.