US 12,404,308 B2
In vivo immobilization of proteins
Michael Chan, Kowloon (CN); Bradley Heater, Shatin (CN); and Marianne Lee, Kowloon (CN)
Assigned to The Chinese University of Hong Kong, Shatin (CN)
Appl. No. 17/605,993
Filed by The Chinese University of Hong Kong, Shatin (CN)
PCT Filed Apr. 24, 2020, PCT No. PCT/CN2020/086652
§ 371(c)(1), (2) Date Oct. 22, 2021,
PCT Pub. No. WO2020/216322, PCT Pub. Date Oct. 29, 2020.
Claims priority of provisional application 62/839,400, filed on Apr. 26, 2019.
Prior Publication US 2023/0203105 A1, Jun. 29, 2023
Int. Cl. C07K 14/325 (2006.01); C12N 9/20 (2006.01); C12N 15/70 (2006.01); C12N 15/75 (2006.01)
CPC C07K 14/325 (2013.01) [C12N 15/70 (2013.01); C12N 15/75 (2013.01); C07K 2319/00 (2013.01)] 23 Claims
 
1. A method for recombinantly expressing a protein, comprising:
(1) obtaining bacterial cells comprising an expression cassette encoding the protein and an expression cassette encoding a modified Cry protein, wherein the bacterial cells are Bacillus subtilis (Bs), Bacillus thuringiensis (Bt), or Escherichia coli (E. coli) cells,
(2) culturing the bacterial cells under conditions permissible for the expression of the protein and the modified Cry protein, thereby expressing separately the protein and the modified Cry protein, and
(3) permitting the modified Cry protein to form crystal containing the protein in the bacterial cells,
wherein the modified Cry protein comprises the amino acid sequence set forth in SEQ ID NO:4, which is modified to comprise:
(i) substitutions with aspartate (D) or glutamate (E) at residues K384, N391, N395, S425, Q430, T436, Q437, K442, R443, T461, and K467;
(ii) addition of the amino acid sequence set forth in SEQ ID NO:8 in 1-3 repeats at the C-terminus;
(iii) one or more deletions in segment 110-128, 181-197, 211-226, 250-264, 423-436, 457-471, 593-598, or 627-632;
(iv) one or more substitutions at residue 5145 or H161, or
(v) the amino acid sequence set forth in SEQ ID NO:5.