US 11,702,456 B2
Systems and methods for the production of diphtheria toxin polypeptides
Luke Masson, Dollard-des-Ormeaux (CA); Melanie Arbour, Ste-Marthe-sur-le-Lac (CA); and Marie-Anne Gauriat, Montreal (CA)
Assigned to National Research Council of Canada, Ottawa (CA)
Appl. No. 16/639,167
Filed by NATIONAL RESEARCH COUNCIL OF CANADA, Ottawa (CA)
PCT Filed Aug. 16, 2018, PCT No. PCT/IB2018/056201
§ 371(c)(1), (2) Date Feb. 14, 2020,
PCT Pub. No. WO2019/035058, PCT Pub. Date Feb. 21, 2019.
Claims priority of provisional application 62/718,854, filed on Aug. 14, 2018.
Claims priority of application No. 17186713 (EP), filed on Aug. 17, 2017.
Prior Publication US 2020/0231635 A1, Jul. 23, 2020
Int. Cl. C07K 14/34 (2006.01); C07K 1/22 (2006.01); C12N 15/70 (2006.01); C12N 15/90 (2006.01)
CPC C07K 14/34 (2013.01) [C07K 1/22 (2013.01); C12N 15/70 (2013.01); C12N 15/902 (2013.01); C07K 2319/034 (2013.01); C07K 2319/55 (2013.01); C12N 2500/24 (2013.01); C12N 2500/34 (2013.01); C12N 2500/35 (2013.01); C12N 2500/74 (2013.01)] 17 Claims
 
1. An expression system for producing a Diphtheria toxin polypeptide or a mutated form thereof, the expression system comprising:
an Escherichia coli (E. coli) cell comprising a heterologous nucleic acid construct comprising:
(i) a rhamnose inducible promoter sequence; and
(ii) an expression sequence, said expression sequence comprising a first portion and a second portion, said first portion comprising a nucleotide sequence that encodes a periplasmic secretion signal linked to the 5′ end of the second portion, and said second portion comprising a nucleotide sequence that encodes said Diphtheria toxin polypeptide or mutated form thereof,
wherein said expression sequence is operatively linked to said rhamnose inducible promoter sequence;
wherein said E. coli cell is unable to use rhamnose as a carbon source; and
wherein:
said E. coli cell expresses a leucine/isoleucine/valine transporter subunit (LivK) fused to an affinity tag;
said E. coli cell comprises a defective livK gene;
said E. coli cell expresses a maltose transporter subunit (MalE) fused to an affinity tag;
said E. coli cell comprises a defective malE gene;
said E. coli cell expresses a LivK fused to an affinity tag and a MalE fused to an affinity tag;
said E. coli cell comprises a defective livK gene and a defective malE gene said E. coli cell expresses a LivK fused to an affinity tag and comprises a defective malE gene; or
said E. coli cell comprises a defective livK gene and expresses a MalE fused to an affinity tag.