Mutant DNase1L3 with improved serum half-life
The present invention includes a mutant Dnase1LS having at least about a 95% identity with a nucleic acid sequence encoding the protein of SEQ ID NO: 2-8 for a mutant Dnase1LS comprising at least one mutation for post-translational modification or attachment of a molecule to the mutant Dnase1LS to increase the serum half-life of the mutant Dnase1LS, nucleic acids encoding the same, host cells, and methods of making the mutant Dnase1LS.
1 . A mutant Dnase1L3 nucleotide sequence having at least about a 95% identity with a nucleic acid sequence encoding a mutant Dnase1L3 protein having SEQ ID NO: 2-8, wherein the mutant Dnase1L3 protein comprises at least one mutation for post-translational modification or attachment of a molecule to the mutant Dnase1L3 to increase a serum half-life of the mutant Dnase1L3, wherein the mutation to Dnase1L3 protein is selected from at least one of:
an S112C mutation, an S131C mutation, or an S279C mutation; or
at least two mutations selected from S91C, S112C, S131C, and S279C.
2 . The mutant Dnase1L3 nucleotide sequence of claim 1 , further comprising a nucleic acid sequence optimized for microbial expression.
3 . The mutant Dnase1L3 nucleotide sequence of claim 1 , wherein: the mutant Dnase1L3 protein further comprises at least one of S91C, an S131C or an S253C.
4 . The mutant Dnase1L3 nucleotide sequence of claim 1 , wherein the nucleotide sequence further comprises a nucleic acid segment encoding a leader sequence.
5 . The mutant Dnase1L3 nucleotide sequence of claim 1 , wherein said nucleotide sequence encodes a Dnase1L3 protein, wherein said protein is post-translationally modified with polyethylene glycol; or is post-translationally modified with a polyethylene glycol having a molecular mass from 5 kDa to 50 kDa.
6 . A mutant Dnase1L3 protein produced by a method comprising:
culturing a host cell transformed with an expression vector comprising a DNA sequence comprising a nucleotide sequence encoding the mutant Dnase1L3 protein having at least 95% identity with SEQ IDS NO: 2-8; expressing the mutant Dnase1L3 protein in the host cell; and isolating the mutant Dnase1L3 protein, wherein the mutation to Dnase1L3 protein is selected from at least one of:
an S112C mutation, an S131C mutation, or an S279C mutation; or
at least two mutations selected from S91C, S112C, S131C, and S279C.
7 . The mutant Dnase1L3 protein of claim 6 , wherein the mutant Dnase1L3 protein is post-translationally modified with polyethylene glycol; or the mutant Dnase1L3 protein is post-translationally modified with a polyethylene glycol having a molecular mass from 5 kDa to 50 kDa.
8 . The mutant Dnase1L3 protein of claim 6 , wherein the mutant Dnase1L3 protein further comprises at least one of: an S91C, an S131C, or an S253C mutation.
9 . The mutant Dnase1L3 protein of claim 6 , wherein the host cell encoding said protein comprises E. coli or Pichia pastoris.
10 . The mutant Dnase1L3 protein of claim 6 , wherein the host cell encoding said protein produces at least 0.25 or 7.5 mg/L mutant Dnase1L3 protein.
11 . A mutant Dnase1L3 protein comprising at least about a 95% identity with SEQ ID NO: 2-8, wherein the Dnase1L3 protein comprises at least one mutation selected from:
An S112C mutation, an S131C mutation, or an S279C mutation; or
At least two mutations selected from S91C, S112C, S131C and S279C.
12 . The mutant Dnase1L3 protein of claim 11 , wherein the mutant Dnase1L3 protein is post-translationally modified with polyethylene glycol; or the mutant Dnase1L3 protein is post-translationally modified with a polyethylene glycol having a molecular mass from 5 kDa to 50 kDa.
13 . The mutant Dnase1L3 protein of claim 11 , wherein the mutant Dnase1L3 protein further comprises at least one of: an S91C, an S131C, or an S253C mutation.