Recombinant proteins containing repeating units

A method for producing a recombinant protein containing repeating units comprising,

• a) providing a first pool of polynucleotides comprising at least two tandem repeats of sequences encoding said recombinant protein wherein said polynucleotides have an absence of any additional nucleotides coding for other than said recombinant protein between said tandem repeats and said tandem repeats contain different nucleotide sequences encoding said recombinant protein in accordance with the degeneracy of the genetic code;
• b) providing a second pool of polynucleotides at least some of which are complementary to the polynucleotides in said first pool of polynucleotides;;
• c) combining said first pool of polynucleotides and said second pool of polynucleotides under conditions whereby the polynucleotides will anneal;
• d) extending the 3′ ends of said annealed polynucleotides under conditions wherein said polynucleotides act as primers for their complements;
• e) denaturing the extended polynueleotides;
• f) repeating steps c-e at least once, whereby the products of step e provide the polynucleotides for annealing in step c of the next cycle;
• g) if necessary, adding at least one linker oligonucleotides to the end of the products of (f), said linker oligonucleotides containing at least one restriction enzyme cleavage site,
• h) inserting the products of (f) or (g) into a suitable vector;
• i) introducing said vector into a suitable host cell;
• and
• j) maintaining said host cell under conditions allowing for expression of said recombinant protein.

A method for producing a polynucleotide containing repeating units comprising,

• a) providing a first pool of polynucleotides comprising at least two tandem repeats of sequences encoding a recombinant protein wherein said tandem repeats contain degenerate nucleotide sequences in accordance with the degeneracy of the genetic code;
• b) providing a second pool of polynucleotides at least some of which are complementary to the polynucleotides in said first pool of polynucleotides;;
• c) combining said first and second pools of polynucleotides under conditions whereby the polynucleotides will anneal;
• d) extending the 3′ ends of said annealed polynucleotides under conditions wherein said polynucleotides act as primers for their complements;
• e) denaturing the extended polynucleotides;
• f) repeating steps c-e at least once, whereby the products of step e provide the polynucleotides for annealing in step c of the next cycle.

A method for producing a polynucleotide containing repeating units comprising:

• a) providing a first pool of polynucleotides comprising at least two tandem repeats of sequences encoding a recombinant protein wherein said polynucleotides have an absence of any additional nucleotides between said tandem repeats and said tandem repeats contain degenerate nucleotide sequences in accordance with the degeneracy of the genetic code;
• b) providing a second pool of polynucleotides at least some of which are complementary to the polynucleotides in said first pool of polynucleotides;
• c) combining said first and second pools of polynucleotides under conditions whereby the polynucleotides will anneal;
• d) extending the 3′ ends of said annealed polynucleotides under conditions wherein said polynucleotides act as primers for their complements;
• e) denaturing the extended polynucleotides;
• and
• f) repeating steps c-e at least once, whereby the products of step e provide the polynucleotides for step c in the next cycle.

A recombinant protein comprising, at least two tandem repeats of an amino acid sequence, produced by the process of:

• a) providing a first pool of polynucleotides, said polynucleotides comprising at least two tandem repeats of sequences encoding said recombinant protein, wherein said tandem repeats contain degenerate nucleotide sequences encoding said recombinant protein in accordance with the degeneracy of the genetic code;
• b) providing a second pool of polynucleotides at least some of which are complementary to the polynucleotides in said first pool of polynucleotides;
• c) combining said first pool of polynucleotides and second pool of polynucleotides under conditions whereby the polynucleotides will anneal;
• d) extending the 3′ ends of said annealed polynucleotides under conditions wherein said annealed polynucleotides act as primers for their complements;
• e) denaturing the extended polynucleotides;
• f) repeating steps c-e at least once, whereby the products of step e provide the polynucleotides for annealing in step c of the next cycle;
• g) if necessary, adding one or more linker oligonucleotides to the end of the products of (f), said linker oligonucleotides containing at least one restriction enzyme cleavage site;
• h) inserting the products of (f) or (g) into a suitable vector;
• i) introducing said vector into a suitable host cell, and
• j) maintaining said host cell under conditions allowing for expression of said recombinant protein.

A recombinant protein comprising, at least two tandem repeats of an amino acid sequence said repeats having an absence of amino acids between said repeats, produced by the process of:

• a) providing a first pool of polynucleotides comprising at least two tandem repeats of sequences encoding said recombinant protein wherein said polynucleotides have an absence of any additional nucleotides coding for other than said recombinant protein between said tandem repeats and said tandem repeats contain degenerate nucleotide sequences encoding said recombinant protein in accordance with the degeneracy of the genetic code;
• b) providing a second pool of polynucleotides at least some of which are complementary to the polynucleotides in said first pool of polynucleotides;
• c) combining said first pool of polynucleotides and second pool of polynucleotides under conditions whereby the polynucleotides will anneal;
• d) extending the 3′ ends of said annealed polynucleotides under conditions wherein said polynucleotides act as primers for their complements;
• e) denaturing the extended polynucleotides;
• f) repeating steps c-e at least once, whereby the products of step e provide the polynucleotides for annealing in step c of the next cycle;
• g) if necessary, adding at least one linker oligonucleotide to the end of the products of (f), said linker oligonucleotide containing at least one restriction enzyme cleavage site,
• h) inserting the products of (f) or (g) into a suitable vector;
• i) introducing said vector into a suitable host cell;
• and
• j) maintaining said host cell under conditions allowing for expression of said recombinant protein.

An isolated polynucleotide comprising repeating units, produced by the process of:

• a) providing a first pool of polynucleotides comprising at least two tandem repeats of sequences encoding a recombinant protein wherein said tandem repeats contain degenerate nucleotide sequences in accordance with the degeneracy of the genetic code;
• b) providing a second pool of polynucleotides at least some of which are complementary to the polynucleotides in said first pool of polynucleotides;
• c) combining said first and second pools of polynucleotides under conditions whereby the polynucleotides will anneal;
• d) extending the 3′ ends of said annealed polynucleotides under conditions wherein said polynucleotides act as primers for their complements;
• e) denaturing the extended polynucleotides;
• f) repeating steps c-e at least once, whereby the products of step e provide the polynucleotides for annealing in step c of the next cycle.

An isolated polynucleotide comprising repeating units, produced by the process of:

• a) providing a first pool of polynucleotides comprising at least two tandem repeats of sequences encoding a recombinant protein wherein said polynucleotides have an absence of any additional nucleotides between said tandem repeats and said tandem repeats contain degenerate nucleotide sequences in accordance with the degeneracy of the genetic code;
• b) providing a second pool of polynucleotides at least some of which are complementary to the polynucleotides in said first pool of polynucleotides;
• c) combining said first and second pools of polynucleotides under conditions whereby the polynucleotides will anneal;
• d) extending the 3′ ends of said annealed polynucleotides under conditions wherein said polynucleotides act as primers for their complements;
• e) denaturing the extended polynucleotides;
• and
• f) repeating steps c-e at least once, whereby the products of step e provide the polynucleotides for step c in the next cycle.

A isolated polynucleotide having the formula:

• Aw[LxSn]yBz
• or its complement where,
• A is a nucleotide sequence containing at least one restriction enzyme site;
• L is a nucleotide sequence containing at least one chemical or enzymatic cleavage site;
• S is a degenerate nucleotide sequence encoding one of the amino acid sequences selected from the group consisting of LKPNM, KPNM, VVYP, KPN, DKP,YKP EKP, DAP, EAP, HPP, VPP, LK, PN and NM such that Ss with different values of n comprise different nucleotide sequences, but encode the same amino acid sequence;
• B is a nucleotide sequence containing at least one restriction enzyme site, where B may or may not be the same as A;
• w is 0 or 1;
• x is 0 or 1;
• n varies randomly with each S, and is a whole number from 1 to the maximum number of possible nucleotide sequences encoding the amino acid sequence of S;
• y is at least 2;
• and
• z is 0 or 1.

A isolated polynucleotide having the formula:

• Aw[LxSnTm]yBz
• or its complement where,
• A is a nucleotide sequence containing at least one restriction enzyme site;
• L is a nucleotide sequence containing at least one chemical or enzymatic cleavage site;
• S is a degenerate nucleotide sequence encoding one of the amino acid sequences selected from the group consisting of LKPNM, KPNM, VVYP, KPN, DKP,YKP EKP, DAP, EAP, HPP, VPP, LK, PN and NM such that S's with different values of n comprise different nucleotide sequences, but encode the same amino acid sequence;
• T is a degenerate nucleotide sequence encoding one of the amino acid sequences selected from the group consisting of LKPNM, KPNM, VVYP, KPN, DKP,YKP EKP, DAP, EAP, HPP, VPP, LK, PN and NM such that the sequence of T encodes an amino acid sequence different from S and Ts with different values of m comprise different nucleotide sequences, but encode the same amino acid sequence;
• B is a nucleotide sequence containing at least one restriction enzyme site, where B may or may not be the same as A;
• w is 0 or 1;
• x is 0 or 1;
• n varies randomly with each S, and is a whole number from 1 to the maximum number of possible nucleotide sequences encoding the amino acid sequence of S;
• m varies randomly with each T, and is a whole number from 1 to the maximum number of possible nucleotide sequences encoding the amino acid sequence of T;
• y is at least 2;
• and
• z is 0 or 1.