ESCHERICHIA COLI COMPOSITIONS AND METHODS THEREOF
In one aspect, the invention relates to a polypeptide derived from E. coli and a fragment thereof, including compositions and methods thereof. Also disclosed herein are compositions that include a polypeptide derived from E. coli and a fragment thereof; and modified O-polysaccharide molecules derived from E. coli lipopolysaccharides and conjugates thereof. In a further aspect, disclosed herein are mammalian host cells that include sequence(s) encoding a polypeptide derived from E. coli or fragments thereof.
1 . A recombinant mammalian cell, comprising a polynucleotide encoding a polypeptide derived from E. coli or a fragment thereof.
2 . The recombinant cell according to claim 1 , wherein the polypeptide is derived from E. coli fimbrial H (FimH).
3 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a phenylalanine residue at the N-terminus of the polypeptide.
4 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a phenylalanine residue within the first 20 residue positions of the N-terminus.
5 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a phenylalanine residue at position 1 of the polypeptide.
6 . The recombinant cell according to claim 5 , wherein the polypeptide does not comprise a glycine residue immediately before the phenylalanine residue at position 1 of the polypeptide.
7 . The recombinant cell according to claim 2 , wherein the polypeptide does not comprise an N-glycosylation site at position 7 of the polypeptide.
8 . The recombinant cell according to claim 6 , wherein the polypeptide does not comprise an Asn residue at position 7 of the polypeptide.
9 . The recombinant cell according to claim 8 , wherein the polypeptide comprises a residue selected from the group consisting of Ser, Asp, Thr, and Gln at position 7.
10 . The recombinant cell according to claim 5 , wherein the polypeptide does not comprise an N-glycosylation site at position 70 of the polypeptide.
11 . The recombinant cell according to claim 10 , wherein the polypeptide does not comprise an Asn residue at position 70 of the polypeptide.
12 . The recombinant cell according to claim 10 , wherein the polypeptide does not comprise a Ser residue at position 70 of the polypeptide.
13 . The recombinant cell according to claim 1 , wherein the polypeptide comprises a residue substitution selected from the group consisting of Ser, Asp, Thr, and Gln at an N-glycosylation site of the polypeptide.
14 . The recombinant cell according to claim 13 , wherein the N-glycosylation site comprises position N235 of the polypeptide.
15 . The recombinant cell according to claim 13 , wherein the N-glycosylation site comprises position N228 of the polypeptide.
16 . The recombinant cell according to claim 13 , wherein the N-glycosylation site comprises position N235 and position N228 of the polypeptide.
17 . The recombinant cell according to claim 2 , wherein the polypeptide comprises SEQ ID NO: 3.
18 . The recombinant cell according to claim 2 , wherein the polypeptide comprises SEQ ID NO: 2.
19 . The recombinant cell according to claim 1 , wherein the polypeptide comprises an aliphatic hydrophobic amino acid residue at position 1 of the polypeptide.
20 . The recombinant cell according to claim 19 , wherein the aliphatic hydrophobic amino acid residue is selected from the group consisting of Ile, Leu, and Val.
21 . The recombinant cell according to claim 1 , wherein the polypeptide comprises a fragment of FimH.
22 . The recombinant cell according to claim 21 , wherein the polypeptide comprises a lectin domain of FimH.
23 . The recombinant cell according to claim 22 , wherein the lectin domain comprises a mass of about 17022 Daltons.
24 . The recombinant cell according to claim 1 , wherein the polypeptide is complexed with a FimC polypeptide or a fragment thereof.
25 . The recombinant cell according to claim 24 , wherein the FimC polypeptide or a fragment thereof comprises a glycine residue at position 37 of the FimC polypeptide or a fragment thereof.
26 . The recombinant cell according to claim 2 , wherein the polypeptide is in the low affinity conformation.
27 . The recombinant cell according to claim 2 , wherein the polypeptide is stabilized by FimG.
28 . The recombinant cell according to claim 2 , wherein the polypeptide is stabilized by a donor-strand peptide of FimG (DsG).
29 . The recombinant cell according to claim 28 , wherein the polynucleotide sequence further encodes a linker sequence.
30 . The recombinant cell according to claim 29 , wherein the linker comprises at least 4 amino acid residues and at most 15 amino acid residues.
31 . The recombinant cell according to claim 29 , wherein the linker comprises at least 5 amino acid residues and at most 10 amino acid residues.
32 . The recombinant cell according to claim 29 , wherein the linker comprises 7 amino acid residues.
33 . The recombinant cell according to claim 1 , wherein the polypeptide does not comprise a signal peptide selected from the group consisting of a native FimH leader peptide, influenza hemagglutinin signal peptide, and a human respiratory syncytial virus A (strain A2) fusion glycoprotein F0 signal peptide.
34 . The recombinant cell according to claim 1 , wherein the polypeptide comprises a murine IgK signal peptide sequence.
35 . The recombinant cell according to claim 1 , wherein the polypeptide comprises any one signal peptide sequence selected from human IgG receptor FcRn large subunit p51 signal peptide and a human IL10 protein signal peptide.
36 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a mutation of arginine to proline at amino acid position 60 (R60P), according to the numbering of SEQ ID NO: 3.
37 . The recombinant cell according to claim 1 , wherein the expression level of the polypeptide is greater than the expression level of the corresponding wild-type polypeptide expressed in the periplasm of a wild-type E. coli cell.
38 . The recombinant cell according to claim 1 , wherein the expression level of the polypeptide is greater than 10 mg/L.
39 . The recombinant cell according to claim 1 , wherein the polynucleotide sequence is integrated into the genomic DNA of said mammalian cell.
40 . The recombinant cell according to claim 1 , wherein the polynucleotide sequence is codon optimized for expression in the cell.
41 . The recombinant cell according to claim 1 , wherein the cell is a human embryonic kidney cell.
42 . The recombinant cell according to claim 40 , wherein the human embryonic kidney cell comprises a HEK293 cell.
43 . The recombinant cell according to claim 42 , wherein the HEK293 cell is selected from any one of HEK293T cells, HEK293TS cells, and HEK293E cells.
44 . The recombinant cell according to claim 1 , wherein the cell is a CHO cell.
45 . The recombinant cell according to claim 44 , wherein said CHO cell is a CHO-K1 cell, CHO-DUXB11, CHO-DG44 cell, or CHO—S cell.
46 . The recombinant cell according to claim 1 , wherein the polypeptide is soluble.
47 . The recombinant cell according to claim 1 , wherein the polypeptide is secreted from the cell.
48 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a N28Q substitution, according to the numbering of SEQ ID NO: 1.
49 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a N28D substitution, according to the numbering of SEQ ID NO: 1.
50 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a N28S substitution, according to the numbering of SEQ ID NO: 1.
51 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a substitution selected from any one of N28Q, V48C, and L55C, according to the numbering of SEQ ID NO: 1.
52 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a substitution N92S according to the numbering of SEQ ID NO: 1.
53 . The recombinant cell according to claim 1 , wherein the polypeptide derived from FimH or fragment thereof comprises a substation selected from any one of V48C and L55C, according to the numbering of SEQ ID NO: 1.
54 . A culture comprising the recombinant cell of claim 1 , wherein said culture is at least 5 liter in size.
55 . The culture according to claim 49 , wherein the yield of the polypeptide or fragment thereof is at least 0.05 g/L.
56 . The culture according to claim 55 , wherein the yield of the polypeptide or fragment thereof is at least 0.10 g/L.
57 . A method for producing a polypeptide derived from E. coli or a fragment thereof, comprising culturing a recombinant mammalian cell according to claim 1 under a suitable condition, thereby expressing the polypeptide or fragment thereof; and harvesting the polypeptide or fragment thereof.
58 . The method according to claim 57 , further comprising purifying the polypeptide or fragment thereof.
59 . The method according to claim 57 , wherein the cell comprises a nucleic acid encoding any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 27.
60 . The method according to claim 57 , wherein the yield of the polypeptide or fragment thereof is at least 0.05 g/L.
61 . The method according to claim 57 , wherein the yield of the polypeptide or fragment thereof is at least 0.10 g/L.
62 . A composition comprising a polypeptide having at least 70% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 29.
63 . The composition according to claim 62 , further comprising a saccharide comprising a structure selected from any one Formula in Table 1.
64 . The composition according to claim 63 , wherein the saccharide is covalently bound a carrier protein.
65 . The composition according to claim 64 , wherein the carrier protein is selected from any one of poly(L-lysine), CRM 197 , diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa ; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus , clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, and C. jejuni natural glycoproteins.
66 . The composition according to claim 64 , wherein the carrier protein is CRM 197 .
67 . The composition according to claim 64 , wherein the carrier protein is tetanus toxoid (TT).
68 . The composition according to claim 64 , wherein the carrier protein is poly(L-lysine).
69 . The composition according to claim 64 , wherein the saccharide is covalently bound a carrier protein by reductive amination.
70 . The composition according to claim 64 , wherein the saccharide is covalently bound a carrier protein by CDAP chemistry.
71 . The composition according to claim 64 , wherein the saccharide is covalently bound a carrier protein by single-end linked conjugation.
72 . The composition according to claim 64 , wherein the saccharide is covalently bound a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.
73 . A polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 27.