Recombinant protein production

Patent metadata

Field	Value
Publication number	US-11268101-B2
Application number	US-201716065421-A
Country	US
Kind code	B2
Filing date	Jan 9, 2017
Priority date	Jan 12, 2016
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

The disclosure relates to recombinant protein expression systems comprising genetically modified cells wherein the cells are transformed or transfected with tRNA genes to reduce base mismatch due to genetic degeneracy in the genetic code.

First claim

The invention claimed is:

1. An isolated eukaryotic cell modified by transfection or transformation with:
(i) a first expression vector adapted for eukaryotic expression comprising a transcription cassette, the transcription cassette comprising at least one nucleotide sequence encoding a transfer RNA (tRNA) molecule, the tRNA molecule including an anticodon nucleotide sequence selected from the group consisting of AGC, GGC, CGC, UGC, ACC, GCC, CCC, UCC, AGG, GGG, CGG, UGG, AGU, GGU, CGU, UGU, AAC, GAC, CAC, UAC, AAA, GAA, AUU, GUU, CUU, UUU, AUC, GUC, CUC, UUC, AGA, GGA, CGA, UGA, ACU, GCU, ACG, GCG, CCG, UCG, CCU, UCU, AAG, GAG, CAG, UAG, CAA, UAA, AAU, GAU, UAU, CAU, AUA, GUA, ACA, GCA, AUG, GUG, CUG, UUG, or CCA,
wherein said tRNA molecule comprising said anticodon nucleotide sequence is absent from said eukaryotic cell prior to transfection or transformation with said first expression vector; and
(ii) a second expression vector encoding a recombinant protein,
wherein said tRNA molecule comprising said anticodon sequence corrects base mismatches at the third nucleotide position in a messenger RNA (mRNA) codon encoding said recombinant protein to improve translation efficiency and/or decrease amino acid mis-incorporation as a consequence of degeneracy in the genetic code; and
wherein correction of said base mismatches reduces reliance of the eukaryotic cell on wobble during translation of said mRNA.

2. The isolated eukaryotic cell according to claim 1 , wherein said first expression vector and said second expression vector are adapted for expression in a mammalian cell, fungal cell, insect cell, or plant cell.

3. The isolated eukaryotic cell according to claim 1 , wherein said isolated eukaryotic cell is an insect, plant, fungal or mammalian cell.

4. The isolated eukaryotic cell according to claim 3 wherein said isolated mammalian cell is a non-human mammalian cell.

5. The isolated eukaryotic cell according to claim 3 , wherein said isolated mammalian cell is a Chinese Hamster Ovary, (CHO), HEK293, NS0 or CAP cell.

6. The isolated eukaryotic cell according to claim 3 wherein said fungal cell is Saccharomyces cerevisae, Schizosaccharomyces pombe, Yarrowia lipolytica, Pichia pastoris, Aspergillus spp., Kluyveromyces lactis or Trichoderma reesei.

7. The isolated eukaryotic cell according to claim 1 , wherein said transcription cassette comprises nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 different tRNAs.

8. The isolated eukaryotic cell according to claim 1 , wherein said at least one nucleotide sequence encoding a tRNA molecule encodes Ala-tRNA, Arg-tRNA, Asp-tRNA, Asn-tRNA, Cys-tRNA, Glu-tRNA, Gln-tRNA, Gly-tRNA, His-tRNA, Ile-tRNA, Leu-tRNA, Lys-tRNA, Met-tRNA, Phe-tRNA, Pro-tRNA, Ser-tRNA, Thr-tRNA, Trp-tRNA, Tyr-tRNA or Val-tRNA.

9. The isolated eukaryotic cell according to claim 1 , wherein said anticodon nucleotide sequence is cytosine and/or adenine rich.

10. The isolated eukaryotic cell according to claim 1 , wherein said anticodon nucleotide sequence is uracil free.

11. The isolated eukaryotic cell according to claim 1 , wherein said second expression vector encodes a therapeutic antibody.

12. The isolated eukaryotic cell according to claim 1 , wherein said second expression vector encodes a pharmaceutical or therapeutic protein or peptide, or an industrial enzyme.

13. The isolated eukaryotic cell according to claim 1 , wherein said isolated eukaryotic cell is further modified by transformation or transfection with a nucleic acid molecule comprising a 7SL nucleotide sequence.

14. The isolated eukaryotic cell according to claim 13 , wherein said 7SL nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence comprising at least 90% nucleotide sequence identity to the nucleotide sequence of SEQ ID NO: 10.

15. An isolated mammalian cell modified by transfection or transformation with
(i) a first expression vector adapted for mammalian expression comprising a transcription cassette, the transcription cassette comprising at least one nucleotide sequence encoding a transfer RNA (tRNA) molecule(s), the tRNA molecule(s) including an anticodon nucleotide sequence selected from the group consisting of AGC, GGC, CGC, UGC, ACC, GCC, CCC, UCC, AGG, GGG, CGG, UGG, AGU, GGU, CGU, UGU, AAC, GAC, CAC, UAC, AAA, GAA, AUU, GUU, CUU, UUU, AUC, GUC, CUC, UUC, AGA, GGA, CGA, UGA, ACU, GCU, ACG, GCG, CCG, UCG, CCU, UCU, AAG, GAG, CAG, UAG, CAA, UAA, AAU, GAU, UAU, CAU, AUA, GUA, ACA, GCA, AUG, GUG, CUG, UUG, or CCA,
wherein said tRNA molecule(s) comprising said anticodon nucleotide sequence is absent from said mammalian cell prior to transfection or transformation with said first expression vector; and
(ii) a second expression vector encoding a recombinant antibody,
wherein said tRNA molecule(s) comprising said anticodon sequence corrects base mismatches at the third nucleotide position in a messenger RNA (mRNA) codon encoding said recombinant antibody to improve translation efficiency and/or decrease amino acid mis-incorporation as a consequence of degeneracy in the genetic code, and
wherein correction of said base mismatches reduces reliance of the mammalian cell on wobble during translation of said mRNA.

Assignees

• Univ York

Inventors

• White Robert

Classifications

• C12N15/67

  General methods for enhancing the expression · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/81 Primary

  for yeasts · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title