Patent ID: 7561973

Claim:
A method of determining at least one property that affects an expression property value of polynucleotides in an expression system, the method comprising: (A) 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 each other polynucleotide in the five or more polynucleotides, wherein (i) a first amino acid is encoded a first plurality of times in a first polynucleotide, a second polynucleotide, and a third polynucleotide in the five or more polynucleotides, (ii) the first amino acid is encodable by a plurality of synonymous codons including a first codon, (iii) 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, (iv) 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, (v) the first codon is present in the third polynucleotide with a third frequency relative to all other codons in the plurality of synonymous codons in the third polynucleotide; and (vi) the first frequency is different than the second frequency and the third frequency is between the first frequency and the second frequency; (B) expressing each respective polynucleotide in the plurality of polynucleotides individually in said expression system using the same vector and the same promoter; (C) measuring an expression property value of each respective polynucleotide in the plurality of polynucleotides in the expression system; and (D) 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 (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 in a predetermined period of time (ii) a total amount of active protein encoded by the respective polynucleotide that is expressed in the expression system in a predetermined period of time, or (iii) a total amount of soluble protein encoded by the respective polynucleotide that is expressed in the expression system in a predetermined period of time.