Systems and methods for constructing frequency lookup tables for expression systems

Abstract

Methods for determining a property that affects expression of polynucleotides are provided. A plurality of polynucleotides each encoding a polypeptide sequence is constructed. A frequency that a sequence element is used in a first polynucleotide is different than in a second polynucleotide. Each polynucleotide is expressed in an expression system to obtain an expression property value thereby constructing a dataset that contains, for each respective polynucleotide, sequence element occurrence in the respective polynucleotide and the measured expression property value of the respective polynucleotide. A model is computed that describes variation in the measured expression property values as a function of a plurality of variables and weights. From the model, a property that affects expression of polynucleotides in the expression system is determined, where the property is an effect that the frequency of occurrence of one or more sequence elements has on the expression property of polynucleotides in the expression system.

1 . A method of evaluating polynucleotides in an expression system, the method comprising:
 (A) expressing each respective polynucleotide in a plurality of polynucleotides individually in the expression system, wherein
 (i) the plurality of polynucleotides comprises five or more polynucleotides, 
 (ii) each respective polynucleotide in the plurality of polynucleotides encodes a respective single polypeptide sequence the entirety of which is at least ninety-five percent identical to the entirety of the respective single polypeptide sequence encoded by at least one other polynucleotide in the plurality of polynucleotides 
 (iii) a first amino acid is encoded a first plurality of times in both a first polynucleotide and a second polynucleotide in the plurality of polynucleotides, 
 (iv) the first amino acid is encodable by a plurality of synonymous codons including a first codon, 
 (v) the first codon is present in the first polynucleotide with a first frequency relative to all other codons in the plurality of synonymous codons in the first polynucleotide, 
 (vi) the first codon is present in the second polynucleotide with a second frequency relative to all other codons in the plurality of synonymous codons in the second polynucleotide, and 
 (vii) the first frequency is different than the second frequency; and 
   (B) measuring an expression property value of each respective polynucleotide in the first plurality of polynucleotides in the expression system; and   (C) determining at least one property that affects an expression property of polynucleotides in the expression system, wherein   the at least one property is an effect that the frequency of (i) the first codon or (ii) the first codon and one or more other codons in a plurality of naturally occurring codons has on the protein expression property values of polynucleotides in the expression system; and   the expression property value of each respective polynucleotide in the plurality of polynucleotides in the expression system is (i) a total amount of protein encoded by the respective polynucleotide that is expressed in the expression system (ii) a total amount of active protein encoded by the respective polynucleotide that is expressed in the expression system, or (iii) a total amount of soluble protein encoded by the respective polynucleotide that is expressed in the expression system.   
     
     
         2 .- 33 . (canceled) 
     
     
         34 . The method of  claim 1 , wherein the expression property value of each respective polynucleotide in the plurality of polynucleotides in the expression system is a total amount of protein encoded by the respective polynucleotide that is expressed in the expression system in a predetermined period of time. 
     
     
         35 . The method of  claim 1 , wherein the expression property value of each respective polynucleotide in the expression system is a total amount of active protein encoded by the respective polynucleotide that is expressed in the expression system in a predetermined period of time. 
     
     
         36 . The method of  claim 1 , wherein the expression property value of each respective polynucleotide in the expression system is a total amount of soluble protein encoded by the respective polynucleotide that is expressed in the expression system in a predetermined period of time. 
     
     
         37 . The method of  claim 1  wherein, for each respective amino acid in a plurality of amino acids comprising five or more amino acids, a relative frequency of each of a plurality of synonymous codons for the respective amino acid is varied in a region of each of two or more of the polynucleotides in the plurality of polynucleotides. 
     
     
         38 . The method of  claim 1  wherein, for each respective amino acid in a plurality of amino acids comprising two or more amino acids, a relative frequency of each of a plurality of synonymous codons for the respective amino acid is varied in a region of each of five or more of the polynucleotides in the plurality of polynucleotides. 
     
     
         39 . The method of  claim 1 , wherein the plurality of polynucleotides comprises ten or more polynucleotides. 
     
     
         40 . The method of  claim 1 , wherein the plurality of polynucleotides comprises twenty or more polynucleotides. 
     
     
         41 . The method of  claim 1 , wherein the expressing (A) comprises:
 encoding the first polynucleotide using a first frequency lookup table, wherein the first frequency lookup table specifies a first target frequency or target frequency range for the use of the first codon relative to all other codons in the plurality of synonymous codons in a polynucleotide and wherein the first frequency of (A)(v) is within the first target frequency or target frequency range; and   encoding the second polynucleotide using a second frequency lookup table, wherein the second frequency lookup table specifies a second target frequency or target frequency range for the use of the first codon relative to all other codons in the plurality of synonymous codons in a polynucleotide and wherein the second frequency of (A)(vi) is within the second target frequency or target frequency range; and   wherein the first target frequency or target frequency range is different from the second target frequency or target frequency range.   
     
     
         42 . The method of  claim 1 , wherein the plurality of synonymous codons is three or more different codons. 
     
     
         43 . The method of  claim 1 , wherein
 a first frequency lookup table specifies a corresponding respective target frequency or frequency range for each codon in a first plurality of codons, each corresponding respective target frequency or target frequency range specifying a target frequency or target frequency range for a codon in the first plurality of codons to be used to encode a corresponding amino acid in an amino acid sequence relative to all other codons that are capable of encoding the corresponding amino acid, and wherein for each respective codon in the first frequency lookup table, the expressing (A) further comprises choosing a respective frequency that the respective codon is to be used to encode the amino acid encodable by the respective codon throughout an amino acid sequence encoded by said first polynucleotide in the plurality of polynucleotides relative to all other codons capable of encoding the amino acid, wherein the respective frequency is the frequency or is within the frequency range specified in the first frequency lookup table for the respective codon; and   a second frequency lookup table specifies a corresponding respective target frequency or frequency range for each codon in a second plurality of codons, each corresponding respective target frequency or target frequency range specifying a target frequency or target frequency range for a codon in the second plurality of codons to be used to encode a corresponding amino acid in an amino acid sequence relative to all other codons that are capable of encoding the corresponding amino acid, and wherein for each respective codon in the second frequency lookup table, the expressing (A) further comprises choosing a respective frequency that the respective codon is to be used to encode the amino acid encodable by the respective codon throughout an amino acid sequence encoded by said second polynucleotide in the plurality of polynucleotides relative to all other codons capable of encoding the amino acid, wherein the respective frequency is within the frequency or the frequency range specified in the second frequency lookup table for the respective codon; wherein   the first frequency of (A)(v) is a frequency or is within a frequency range specified for the first amino acid by the first frequency lookup table; and   the second frequency of (A)(vi) is a frequency or is within a frequency range specified for the first amino acid by the second frequency lookup table.   
     
     
         44 . The method of  claim 1 , wherein the expressing (A) comprises:
 encoding a first set of positions in the first polynucleotide using a first frequency lookup table, wherein the first frequency lookup table specifies a first target frequency or frequency range for the use of a predetermined codon, relative to all other codons that are synonymous to the predetermined codon, in the first set of positions in the first polynucleotide that encode a predetermined amino acid; and   encoding a second set of positions in the first polynucleotide using a second frequency lookup table, wherein the second frequency lookup table specifies a second target frequency or frequency range for the use of the predetermined codon, relative to all other codons that are synonymous to the predetermined codon, in the second set of positions in the first polynucleotide that encode the predetermined amino acid; wherein   the first set of positions do not include positions in the second set of positions; and   the second set of positions do not include positions in the first set of positions.   
     
     
         45 . The method of  claim 44 , wherein the predetermined amino acid can be encoded by three or more synonymous codons in the plurality of synonymous codons. 
     
     
         46 . The method of  claim 1 , wherein
 a first frequency lookup table specifies a corresponding respective first target frequency or frequency range for each codon in a first plurality of codons, each corresponding respective first target frequency or frequency range specifying a first target frequency or frequency range for a codon in the first plurality of codons to be used to encode a corresponding amino acid in an amino acid sequence relative to all other codons that are capable of encoding the corresponding amino acid, and wherein for each respective codon in the first frequency lookup table, the expressing (A) further comprises choosing a respective first frequency that the respective codon is to be used to encode the amino acid encodable by the respective codon in a predetermined first set of positions in the amino acid sequence encoded by said first polynucleotide in the plurality of polynucleotides relative to all other codons capable of encoding the amino acid, wherein the respective first frequency is within the first target frequency or frequency range specified in the first frequency lookup table for the respective codon; and   a second frequency lookup table specifies a corresponding respective second target frequency or frequency range for each codon in a second plurality of codons, each corresponding respective second target frequency or frequency range specifying a second target frequency or frequency range for a codon in the second plurality of codons to be used to encode a corresponding amino acid in an amino acid sequence relative to all other codons that are capable of encoding the corresponding amino acid, and wherein for each respective codon in the second frequency lookup table, the expressing (A) further comprises choosing a respective second frequency that the respective codon is to be used to encode the amino acid encodable by the respective codon in a predetermined second set of positions in the amino acid sequence encoded by said first polynucleotide in the plurality of polynucleotides relative to all other codons capable of encoding the amino acid, wherein the respective second frequency is within the second target frequency or frequency range specified in the second frequency lookup table for the respective codon.   
     
     
         47 . The method of  claim 1 , wherein the expression system is  E. coli , baculovirus, a mammalian tissue culture, yeast, or a plant. 
     
     
         48 . A method of determining at least one property that affects an expression property value of polynucleotides in an expression system, the method comprising:
 (A) defining a predetermined frequency or frequency range for incorporation of a first codon into a polynucleotide wherein the defining comprises defining a first frequency and a second frequency for the first codon based upon the predetermined frequency or frequency range;   (B) constructing a plurality of polynucleotides, wherein the plurality of polynucleotides comprises five or more polynucleotides, each respective polynucleotide in the five or more polynucleotides encoding a respective single polypeptide sequence the entirety of which is at least ninety-five percent identical to the entirety of the respective single polypeptide sequence encoded by another polynucleotide in the five or more polynucleotides wherein
 (i) a first amino acid is encoded a first plurality of times in a first polynucleotide and a second polynucleotide in the five or more polynucleotides, 
 (ii) the first amino acid is encodable by a plurality of synonymous codons including the first codon, 
 (iii) the first codon is present in the first polynucleotide with the first frequency, 
 (iv) the first codon is present in the second polynucleotide with the second frequency, and 
 (v) the first frequency is different than the second frequency, and 
   (C) expressing each respective polynucleotide in the plurality of polynucleotides in the expression system; and   (D) measuring an expression property value of each respective polynucleotide in the plurality of polynucleotides in the expression system thereby determining at least one property that affects an expression property value of polynucleotides in the expression system, wherein
 the at least one property is an effect that a frequency of use of one or more codons in a plurality of naturally occurring codons has on the expression property values of polynucleotides in the expression system; 
 the expression property value of each respective polynucleotide in the plurality of polynucleotides in the expression system is (i) a total amount of protein encoded by the respective polynucleotide that is expressed in the expression system, (ii) a total amount of active protein encoded by the respective polynucleotide that is expressed in the expression system, or (iii) a total amount of soluble protein encoded by the respective polynucleotide that is expressed in the expression system. 
   
     
     
         49 . The method of  claim 48 , wherein the first frequency and the second frequency are selected probabilistically based upon the predetermined frequency or frequency range. 
     
     
         50 . The method of  claim 1 , wherein the first frequency and the second frequency are selected using an experimental design technique. 
     
     
         51 . The method of  claim 1  wherein, the first frequency and the second frequency are selected so that they fit a multivariate space that can be searched using a global optimization algorithm. 
     
     
         52 . The method of  claim 1 , wherein each respective polynucleotide in the plurality of polynucleotides is expressed in the same expression vector in said expressing (A). 
     
     
         53 . The method of  claim 1 , wherein each respective polynucleotide in the plurality of polynucleotides is under the control of the same constitutive promoter in said expressing (A). 
     
     
         54 . The method of  claim 53 , wherein the constitutive promoter is a T7 promoter. 
     
     
         55 . The method of any one of  claims 34 - 36 , wherein the predetermined time is a period of time after the respective polynucleotide has been introduced into the expression system. 
     
     
         56 . The method of any one of  claims 34 - 36 , wherein the predetermined time is less than two days after the respective polynucleotide has been introduced into an expression system and the expression system is put under conditions that permit protein expression. 
     
     
         57 . The method of  claim 48 , wherein the expression system is  E. coli , baculovirus, yeast, mammalian tissue culture, or plants. 
     
     
         58 . A polynucleotide in the plurality of polynucleotides of  claim 1 , wherein the measuring (B) of  claim 1  demonstrates that the polynucleotide has an improved expression property relative to others in the plurality of polynucleotides in the expression system of  claim 1 , wherein
 the improved protein expression is an expression property value of the polynucleotide, and 
 the expression property value of the polynucleotide in an expression system is (i) a total amount of protein encoded by the respective polynucleotide that is expressed in the expression system, (ii) a total amount of active protein encoded by the respective polynucleotide that is expressed in the expression system, or (iii) a total amount of soluble protein encoded by the respective polynucleotide that is expressed in the expression system. 
 
     
     
         59 . The polynucleotide of  claim 58 , wherein the expression property value of the polynucleotide is the total amount of protein encoded by the polynucleotide that is expressed in the expression system in a predetermined period of time. 
     
     
         60 . The polynucleotide of  claim 58 , wherein the expression property value of each polynucleotide is the total amount of active protein encoded by the polynucleotide that is expressed in the expression system in a predetermined period of time. 
     
     
         61 . The polynucleotide of  claim 58 , wherein the expression property value of the polynucleotide is a total amount of soluble protein encoded by the polynucleotide that is expressed in the expression system in a predetermined period of time. 
     
     
         62 . The method of  claim 41 , wherein the first or second polynucleotide has an improved expression property. 
     
     
         63 . The method of  claim 1 , further comprising creating a codon frequency lookup table, wherein the codon frequency lookup table comprises a codon frequency or a codon frequency range for the first codon and wherein a value of the codon frequency for the first codon in the codon frequency table is selected based on the frequency of said first codon in a polynucleotide in the plurality of polynucleotides that has an improved expression property relative to other polynucleotides in the plurality of polynucleotides. 
     
     
         64 . the method of  claim 63 , wherein the codon frequency lookup table comprises a codon frequency for the first codon. 
     
     
         65 . the method of  claim 63 , wherein the codon frequency lookup table comprises a codon frequency range for the first codon. 
     
     
         66 . The method of  claim 1 , further comprising selecting a polynucleotide from said plurality of polynucleotides based on an expression property value of the polynucleotide relative to the expression property value of each other polynucleotide in said plurality of polynucleotides.