Methods of Controlling Cleavage of Formylglycine-Containing Polypeptides
Methods for reducing cleavage of a protein comprising a formylglycine (fGly) amino acid is provided. Such methods can involve protecting the protein from exposure to visible light having a wavelength of 500 nm or lower. Also provided herein are methods for inducing cleavage of a protein in a target region, the target region comprising an fGly amino acid. The methods may involve exposing the protein to visible light comprising a wavelength of 300 nm-500 nm in the presence of a flavin. Cleavage of the protein may be carried out in the presence of a molecule that is photoactivated to release singlet oxygen species. Cleavage of the protein may be carried out in the presence of a flavin.
1 . A method of reducing cleavage of a protein comprising a formylglycine (fGly) amino acid, the method comprising:
protecting the protein from exposure to visible light having a wavelength of 500 nm or lower.
2 . The method of claim 1 , wherein the method comprises culturing a cell, wherein the cell comprises the protein and wherein protecting the protein from exposure to visible light having a wavelength of 500 nm or lower comprises using visible light having a wavelength higher than 500 nm during the culturing.
3 . The method of claim 1 , comprising synthesizing the protein, wherein protecting the protein from exposure to visible light having a wavelength of 500 nm or lower comprises synthesizing the protein in visible light having a wavelength higher than 500 nm.
4 . The method of claim 1 , comprising purifying the protein, wherein protecting the protein from exposure to visible light having a wavelength of 500 nm or lower comprises purifying the protein in visible light having a wavelength higher than 500 nm.
5 . The method of claim 4 , wherein purifying comprises isolating the protein from a cell or a cell culture medium comprising the cell.
6 . The method of claim 2 , wherein the visible light having a wavelength higher than 500 nm comprises a wavelength higher than 500 nm and lower than 620 nm.
7 . The method of claim 2 , wherein the visible light having a wavelength higher than 500 nm is generated by a light source that produces visible light limited to green light, yellow light and/or orange light.
8 . The method of claim 2 , wherein the visible light having a wavelength higher than 500 nm is generated by passing visible light through a filter that significantly blocks transmission of visible light in the range of 380 nm to 500 nm.
9 . The method of claim 1 , wherein the method comprises culturing a cell, wherein the cell comprises the protein and protecting the protein from exposure to visible light having a wavelength of 500 nm or lower comprises culturing the cell in absence of visible light.
10 . The method of claim 1 , comprising synthesizing the protein, wherein protecting the protein from exposure to visible light having a wavelength of 500 nm or lower comprises synthesizing the protein in absence of visible light.
11 . The method of claim 1 , wherein the cleavage of the protein occurs at or adjacent the fGly amino acid.
12 . The method of claim 1 , wherein the fGly amino acid is generated in a formylglycine-generating enzyme (FGE) recognition site.
13 . The method of claim 12 , wherein the FGE recognition site comprises the consensus sequence X 1 C/SX 2 P/AX 3 R, wherein X 1 is present or absent and, when present, is any amino acid, with the proviso that when the FGE recognition site is at an N-terminus of the protein, X 1 is present; and X 2 and X 3 are each independently any amino acid.
14 . The method of claim 13 , wherein the FGE recognition site comprises the sequence LCTPSR.
15 . The method of claim 13 , wherein the FGE recognition site comprises the consensus sequence X 1 SX 2 PX 3 R.
16 . The method of claim 15 , wherein the FGE recognition site comprises the sequence LSTPSR.
17 . The method of claim 13 , wherein the FGE recognition site comprises the consensus sequence X 1 CX 2 AX 3 R.
18 . The method of claim 17 , wherein the FGE recognition site comprises the sequence LCTASR.
19 . The method of claim 13 , wherein the FGE recognition site comprises the sequence LCTASA.
20 . The method of claim 1 , wherein the protein is an antibody and/or a therapeutic protein.
21 . The method of claim 1 , wherein cleavage of the protein occurs in the presence of a molecule that is photoactivated to release singlet oxygen species.
22 . The method of claim 21 , wherein the molecule is photoactivated by exposure to visible light having a wavelength of 500 nm or lower.
23 . The method of claim 1 , wherein cleavage of the protein occurs in the presence of a flavin.
24 . The method of claim 23 , wherein the flavin is riboflavin.
25 . The method of claim 23 , wherein the flavin is flavin mononucleotide or flavin adenine dinucleotide.
26 . A method of inducing cleavage of a protein in a target region, the target region comprising a formylglycine (fGly) amino acid, the method comprising:
exposing the protein to light comprising a wavelength of 300 nm-500 nm.
27 .- 46 . (canceled)