Modified Proteins and Methods of Making and Using Same
Methods, compositions, systems, apparatuses and kits comprising modified proteins, particularly modified nucleic acid-binding proteins with altered buffering properties are provided. For example, in some embodiments, methods of forming modified proteins including one or more amino acid modifications to achieve desired pKa values are described. Furthermore, the invention provides methods for using such modified proteins in ion-producing reactions, such as ion-based nucleic acid sequencing reactions.
1 .- 15 . (canceled)
16 . An apparatus for nucleotide polymerization, comprising:
(a) a field effect transistor (FET); and
(b) a modified polymerase including one or more amino acid substitutions that reduce the buffering capacity of the modified polymerase relative to the corresponding unmodified polymerase, wherein the polymerase is configured to catalyze nucleotide polymerization; and
wherein the FET is configured to detect the release of a chemical byproduct of nucleotide incorporation catalyzed by the modified polymerase.
17 . The apparatus of claim 16 , wherein the one or more amino acid substitutions of the modified polymerase include at least one substitution of an amino acid residue having a pKa within the range of about 7 to about 9 with any other amino acid residue.
18 . The apparatus of claim 17 , wherein the other amino acid residue is selected from the group consisting of: Ala Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).
19 . The apparatus of claim 16 , wherein at least one of the one or more amino acid substitutions includes a substitution of an amino acid residue having a pKa of between about 7 and about 9 with an amino acid residue having a pKa that is less than about 7 or greater than about 9.
20 . The apparatus of claim 16 , wherein the at least one of the one or more amino acid substitutions is a conservative amino acid substitution.
21 . The apparatus of claim 16 , wherein at least one of the one or more amino acid substitutions is selected from the group consisting of histidine to arginine, glutamic acid to glutamine, aspartic acid to asparagine, lysine to arginine, and tyrosine to phenylalanine.
22 . The apparatus of claim 16 , wherein at least one of the one or more amino acid substitutions is selected from the group consisting of: substitution of a surface histidine with an arginine.
23 . The apparatus of claim 16 , wherein the modified polymerase includes a modified Bst DNA polymerase.
24 . The apparatus of claim 16 , wherein the one or more amino acid substitutions in the modified Bst DNA polymerase are of one or more amino acid residues shown in Table 1.
25 . The apparatus of claim 16 , wherein the one or more amino acid substitutions in the modified Bst DNA polymerase are selected from the group consisting of H46R, H273R, H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acid residues being in accordance with that of SEQ ID NO: 1.
26 . The apparatus of claim 16 , wherein at least one of the one or more amino acid substitutions includes a substitution of an N terminal amino acid residue of the polymerase with any other different amino acid residue.
27 . The apparatus of claim 16 , wherein the modified polymerase includes the amino acid sequence of SEQ ID NO: 2 except for the N-terminal methionine residue of SEQ ID NO: 2, which is not included in the modified polymerase.
28 . The apparatus of claim 16 , wherein the FET includes an ion sensitive FET (ISFET).
29 . The apparatus of claim 28 , wherein the ISFET is configured to detect the release of hydrogen ions.