Means and methods for preparing engineered proteins by genetic code expansion in insect cells

Patent metadata

Field	Value
Publication number	US-2018346901-A1
Application number	US-201615780187-A
Country	US
Kind code	A1
Filing date	Nov 29, 2016
Priority date	Nov 30, 2015
Publication date	Dec 6, 2018
Grant date	—

Abstract

The invention relates to a method of preparing engineered target polypeptides (TP) comprising in its amino acid sequence one or more, identical or different, non-canonical amino acid (ncAA) residues, by expressing said TP in an insect cell line (ICL) and by expressing novel orthogonal bacterial aminoacyl tRNA synthetase/tRNA pairs in said ICL; a baculoviral shuttle vector (bacmid) suitable or introducing the genetic information of said orthogonal tRNA synthetase/tRNA into said ILC; particular expression cassettes for expressing said particular tRNAs in said ILC; TPs obtained by said method; as we as a kit for preparing said TPs.

First claim

1 . A method of preparing a target polypeptide (TP) comprising in its amino acid sequence one or more, identical or different, non-canonical amino acid (ncAA) residues, which method comprises the steps of
a) expressing said TP in an insect cell line (ICL), in the presence of said one or more ncAAs, wherein the TP encoding nucleotide sequence (CS TP ) comprises one or more selector codons encoding said one or more ncAA residues; and concomitantly or sequentially in any order b) expressing in said ICL one or more orthogonal bacterial aminoacyl tRNA synthetase/tRNA ncAA (O-RS/O-tRNA ncAA ) pairs required for introducing said one or more ncAA residues into the amino acid sequence of said TP, wherein the coding sequences for said one or more bacterial tRNA ncAA (CS tRNAncAA ) are under the control of an insect-cell derived regulatory sequence (RS IC ); and c) optionally recovering the expressed TP.

2 . The method of claim 1 , wherein said ICL is transfected or transduced with one or more baculoviral vectors carrying the genetic information required for expressing said TP and said one or more O-RS/O-tRNA ncAA pairs.

3 . The method of claim 1 , wherein said RS IC is selected from
a) regulatory sequences of insect snRNA U6 genes, and b) regulatory sequences of insect tRNA genes, in particular H1 regulatory sequences.

4 . The method of one of the preceding claims, wherein said RS IC comprises a U6 promoter, wherein said U6 promoter is selected from nucleotide sequences corresponding to nucleotide residues
a) 1 to 400 of SEQ ID NO. 1 b) 1 to 392 of SEQ ID NO: 2 c) 1 to 385 of SEQ ID NO: 3 to 8 d) functional fragments thereof comprising a partial sequence of anyone of the nucleotide sequences as defined in a) to c)

5 . The method of anyone of the preceding claims, wherein said ICL is selected from Spodoptera cell lines, in particular Spodoptera frugiperda cell lines, preferably Sf21 (DSMZ Nr. ACC119); or Drosophila cell lines, in particular Drosophila melanogaster cell lines preferably Schneider-2 R+ ( Drosophila Genomics Research Center (DGRC) stock number 150).

6 . A baculoviral vector comprising the coding sequences of one or more orthogonal bacterial aminoacyl tRNA synthetase/tRNA ncAA (O-RS/O-tRNA ncAA ) pairs, wherein said bacterial tRNA ncAA coding sequence (CS tRNAncAA ) is placed under the control of an insect-cell derived regulatory sequence (RS IC ).

7 . The vector of claim 6 , wherein said RS IC comprises a U6 promoter, wherein said U6 promoter is selected from nucleotide sequences corresponding to nucleotide residues
a) 1 to 400 of SEQ ID NO. 1 b) 1 to 392 of SEQ ID NO: 2 c) 1 to 385 of SEQ ID NO: 3 to 8 d) functional fragments thereof comprising a partial sequence of anyone of the nucleotide sequences as defined in a) to c)

8 . An insect cell line (ICL) capable of expressing one or more orthogonal bacterial aminoacyl tRNA synthetase/tRNA ncAA O-RS/O-tRNA ncAA pairs required for introducing at one or more ncAA residues into the amino acid sequence of a TP to be co-expressed by said ICL, wherein each bacterial tRNA ncAA coding sequence is expressed under the control of an insect-cell derived regulatory sequence (RS IC ).

9 . The ICL of one claim 8 , wherein said ICL is transfected or transduced with one or more baculoviral vectors carrying the genetic information required for expressing said TP and said one or more O-RS/O-tRNA ncAA pairs, in particular one or more vectors as defined in one of the claims 6 and 7 .

10 . The ICL of one of the claim 8 or 9 , wherein said RS IC comprises a U6 promoter, wherein said U6 promoter is selected from nucleotide sequences corresponding to nucleotide residues
a) 1 to 400 of SEQ ID NO. 1
b) 1 to 392 of SEQ ID NO: 2
c) 1 to 385 of SEQ ID NO: 3 to 8
d) functional fragments thereof comprising a partial sequence of anyone of the nucleotide sequences as defined in a) to c)

11 . The ICL of anyone of the claims 8 to 10 , wherein said ICL is selected from Spodoptera cell lines, in particular Spodoptera frugiperda cell lines, preferably Sf21 (DSMZ Nr. ACC119);
or Drosophila cell lines in particular Drosophila melanogaster cell lines preferably Schneider-2 R+ (DGRC 150).

12 . An expression cassette encoding tRNA pyl selected from U6-1 to U6-8 according to SEQ ID Nos: 1 to 8 or sequences having a degree of sequence identity of at least 40% retaining their ability to functionally express bacterial tRNA pyl in insect cells.

13 . A target protein (TP) comprising in its amino acid sequence one or more identical or different non-canonical amino acid (ncAA) residues, obtained by expressing the TP encoding nucleotide sequence (CS TP ) in an insect cell line (ICL), in the presence of said one or more ncAAs, wherein said CS TP comprises one or more selector codons encoding said one or more ncAA residues;
obtained by a method of anyone of the claims 1 to 5 , optionally further chemically modified by performing a click reaction with said at least one non-canonical amino acid (ncAA) residue contained in its amino acid sequence.

14 . A kit for preparing a recombinant target polypeptide TP having one or more ncAA residues, comprising at least one ncAA or salt thereof and further comprising at least one baculoviral vector of one of the claim 6 or 7 .

15 . A promoter sequence is selected from nucleotide sequences corresponding to nucleotide residues
a) 1 to 400 of SEQ ID NO. 1 b) 1 to 392 of SEQ ID NO: 2 c) 1 to 385 of SEQ ID NO: 3 to 8 d) functional fragments thereof comprising a partial sequence of anyone of the nucleotide sequences as defined in a) to c)

Assignees

• European Molecular Biology Laboratory

Inventors

• Berger Imre
• Lemke Edward
• Koehler Christine
• Estrada Girona Gemma

Classifications

• C12Y601/01026

  Pyrrolysine-tRNAPyl ligase (6.1.1.26) · CPC title

• C12N2710/14143

  viral genome or elements thereof as genetic vector · CPC title

• C12N9/93 Primary

  Ligases (6) · CPC title

• C12N2510/02

  Cells for production · CPC title

• C07K2317/55

  Fab or Fab' · CPC title