Molecules and methods for iterative polypeptide analysis and processing

Abstract

Reagents and methods for the digital analysis of proteins or peptides are provided. Specifically provided herein are proteins for identifying the N-terminal amino acid or N-terminal phosphorylated amino acid of a polypeptide. Also, an enzyme for use in the cleavage step of the Edman degradation reaction and a method for using this enzyme are described.

1 - 83 . (canceled) 
     
     
         84 . An isolated N-terminal amino acid binding protein (NAAB), comprising a modified, non-naturally occurring tRNA synthetase (RS) that selectively binds to a N-terminal amino acid residue of a polypeptide with at least about a 1.5:1 ratio of specific to non-specific binding. 
     
     
         85 . The isolated NAAB of  claim 84 , wherein the modified, non-naturally occurring aminoacyl tRNA synthetase is coupled with or bound to a fluorescent label. 
     
     
         86 . The isolated NAAB of  claim 85 , wherein the fluorescent label is covalently attached to the modified, non-naturally occurring RS. 
     
     
         87 . The isolated NAAB of  claim 84 , wherein the modified, non-naturally occurring RS selectively binds to N-terminal amino acid residue of a particular type. 
     
     
         88 . The isolated NAAB of  claim 87 , wherein the type of N-terminal amino acid residue is one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. 
     
     
         89 . The isolated NAAB of  claim 84 , wherein the modified, non-naturally occurring RS binds to an N-terminal amino acid residue with a post translational-modification. 
     
     
         90 . The isolated NAAB of  claim 89 , wherein the N-terminal amino acid residue with a post translational-modification is a phosphorylated N-terminal amino acid residue. 
     
     
         91 . The isolated NAAB of  claim 90 , wherein the NAAB binds to an N-terminal pTyr residue and is a modified Class I TyrRS from  Methanococcus janaschi  or related archaea. 
     
     
         92 . The isolated NAAB of  claim 91 , wherein the modified Class I TyrRS is modified at one or more of the following positions: Y32, L65, F108, Q109, D158, I59, and L162. 
     
     
         93 . The isolated NAAB of  claim 90 , wherein the NAAB binds to an N-terminal pSer residue and is a modified Class II SepRS from  Archaeoglobus fulgidus  or related methanogenic archaea. 
     
     
         94 . The isolated NAAB of  claim 90 , wherein the modified Class II SepRS is modified at one or more of the following positions: E412, E414, K417, P495, 1496 and F529. 
     
     
         95 . The isolated NAAB of  claim 84 , wherein the modified, non-naturally occurring RS selectively binds to methionine and comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 and also containing a serine residue at a position corresponding to position 10 of SEQ ID NO: 3; a leucine residue at a position corresponding to position 257 of SEQ ID NO: 3; a glycine residue at a position corresponding to position 293 of SEQ ID NO: 3; and/or a leucine residue at a position corresponding to position 298 of SEQ ID NO: 3. 
     
     
         96 . The isolated NAAB of  claim 95 , wherein the NAAB comprises the amino acid sequence of SEQ ID NO: 4. 
     
     
         97 . The isolated NAAB of  claim 84 , wherein the modified, non-naturally occurring RS selectively binds to phenylalanine and comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 6 and also containing an aspartate residue at a position corresponding to position 15 of SEQ ID NO: 6, an asparagine residue at a position corresponding to position 57 of SEQ ID NO: 6, a glycine residue at a position corresponding to position 58 of SEQ ID NO: 6, a valine residue at a position corresponding to position 67 of SEQ ID NO: 6, a glycine residue at a position corresponding to position 68 of SEQ ID NO: 6, a lysine residue at a position corresponding to position 69 of SEQ ID NO: 6, an aspartate residue at a position corresponding to position 80 of SEQ ID NO: 6, an alanine residue at a position corresponding to position 120 of SEQ ID NO: 6, an alanine residue at a position corresponding to position 127 of SEQ ID NO: 6, a valine residue at a position corresponding to position 143 of SEQ ID NO: 6, an asparagine residue at a position corresponding to position 144 of SEQ ID NO: 6, a glutamate residue at a position corresponding to position 145 of SEQ ID NO: 6, a glycine residue at a position corresponding to position 146 of SEQ ID NO: 6, an aspartate residue at a position corresponding to position 147 of SEQ ID NO: 6, a tyrosine residue at a position corresponding to position 149 of SEQ ID NO: 6, a threonine residue at a position corresponding to position 172 of SEQ ID NO: 6, a glycine residue at a position corresponding to position 202 of SEQ ID NO: 6, an asparagine residue at a position corresponding to position 204 of SEQ ID NO: 6, an aspartate residue at a position corresponding to position 218 of SEQ ID NO: 6, an alanine residue at a position corresponding to position 251 of SEQ ID NO: 6, a threonine residue at a position corresponding to position 253 of SEQ ID NO: 6, and/or a glycine residue at a position corresponding to position 255 of SEQ ID NO: 6. 
     
     
         98 . The isolated NAAB of  claim 97 , wherein the NAAB comprises the amino acid sequence of SEQ ID NO: 7. 
     
     
         99 . The isolated NAAB of  claim 84 , wherein the modified, non-naturally occurring RS selectively binds to histidine and comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9 and also containing an asparagine residue at a position corresponding to position 121 of SEQ ID NO: 9 and an alanine residue at a position corresponding to position 122 of SEQ ID NO: 9. 
     
     
         100 . The isolated NAAB of  claim 99 , wherein the NAAB comprises the amino acid sequence of SEQ ID NO: 10. 
     
     
         101 . The isolated NAAB of  claim 84 , comprising a modified, non-naturally occurring tRNA synthetase that selectively binds to a PITC-derivatized N-terminal amino acid residue of a polypeptide with at least about a 1.5:1 ratio of specific to non-specific binding. 
     
     
         102 . A method for making a N-terminal amino acid binding (NAAB) protein that selectively binds to a N-terminal amino acid residue of a polypeptide, the method comprising:
 identifying an amino acid binding domain of a tRNA synthetase (RS);   introducing one or more mutations into the amino acid binding domain to form a NAAB; and optionally   assaying the NAAB for specific binding to the N-terminal amino acid residue of a polypeptide.   
     
     
         103 . The method of  claim 102 , wherein the tRNA synthetase is a first class I tRNA synthetase and the identifying step comprises aligning an amino acid sequence of the first class I tRNA synthetase with an amino acid sequence of a second class I tRNA synthetase having a previously defined amino acid binding domain. 
     
     
         104 . The method of  claim 103 , wherein the identifying step comprises constructing a multiple sequence alignment that aligns the amino acid sequences of the first class I tRNA synthetase, the second class I tRNA synthetase, and at least one additional class I tRNA synthetase. 
     
     
         105 . The method of  claim 104 , wherein the multiple sequence alignment aligns the sequences of at least five class I tRNA synthetases. 
     
     
         106 . The method of  claim 105 , wherein the multiple sequence alignment aligns the amino acid sequence of full-length  E. coli  MetRS (SEQ ID NO: 5), or a fragment thereof which includes the amino acid binding domain, with the amino acid sequences of at least two other class I tRNA synthetases selected from the group consisting of arginine, cysteine, glutamate, glutamine, isoleucine, leucine, lysine, methionine, tyrosine, tryptophan, and valine tRNA synthetases. 
     
     
         107 . The method of  claim 102 , wherein the tRNA synthetase is a first class II tRNA synthetase and the identifying step comprises aligning an amino acid sequence of the first class II tRNA synthetase with an amino acid sequence of a second class II tRNA synthetase having a previously defined amino acid binding domain. 
     
     
         108 . The method of  claim 107 , wherein the identifying step comprises aligning the amino acid sequence of the monomeric fragment of  E. coli  HisRS with a corresponding domain of a class II tRNA synthetase selected from the group consisting of AlaRS, ProRS, SerRS, ThrRS, AspRS, AsnRS, LysRS, GlyRS, and PheRS. 
     
     
         109 . The method of  claim 108 , wherein the identifying step comprises constructing a multiple sequence alignment that aligns the amino acid sequences of the first class II tRNA synthetase, the second class II tRNA synthetase, and at least one additional class II tRNA synthetase. 
     
     
         110 . The method of  claim 109 , wherein the multiple sequence alignment aligns the sequences of at least five class II tRNA synthetases. 
     
     
         111 . A kit for analyzing or sequencing a polypeptide comprising:
 one or more N-terminal amino acid binding proteins (NAABs), wherein each of the one or more NAABs selectively binds to a N-terminal amino acid residue of a polypeptide;   an Edman degradation enzyme; and   instructions for using the NAABs and the Edman degradation enzyme for analyzing or sequencing a polypeptide.   
     
     
         112 . The kit of  claim 111 , wherein at least one of the NAABs comprises a modified, non-naturally occurring tRNA synthetase (RS) that selectively binds to a N-terminal amino acid residue of a polypeptide with at least about a 1.5:1 ratio of specific to non-specific binding. 
     
     
         113 . The kit of  claim 111 , wherein at least one of the NAABs comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 7; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; and SEQ ID NO: 28. 
     
     
         114 . The kit of  claim 111 , wherein the Edman degradation enzyme comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 29, and also containing a glycine residue at a position corresponding to position 25 of SEQ ID NO: 29; a serine residue at a position corresponding to position 65 of SEQ ID NO: 29; a cysteine residue at a position corresponding to position 138 of SEQ ID NO: 29; and/or a tryptophan residue at a position corresponding to position 160 of SEQ ID NO: 29.