Creation of DNA sequences encoding modified proinsulin precursors

Claim 2

A modified proinsulin precursor comprising proinsulin containing neither methionine nor aspartic acid in either the A or B chains and a. aspartic acid substituted for the amino acid at the amino terminus of The C chain, or b. methioni. substituted for the amino acid at the carboxyl terminus of the C chain; or c. aspartic acid substituted for the amino acid at the amino terminus of the C chain and methionine substituted for the amino acid at the carboxyl terminus of the C chain.

Claim 3

A modified proinsulin precursor comprising human proinsulin wherein: a. aspartic acid is substituted for arginine at amino acid positon 31; or b. methionine is substituted for arginine at amino acid position 65; or c. aspartic acid is substituted for arginine at amino acid position 31 and methionine is substituted for arginine at amino acid position 65.

Claim 4

A modified proinsulin precursor comprising human proinsulin wherein: a. aspartic acid is substituted for arginine at amino acid positon 31; or b. methionine is substituted for arginine at amino acid position 65; or c. aspartic acid is substituted for arginine at amino acid position 31 and methionine is substituted for arginine at amino acid position 65.

Claim 5

A DNA sequence encoding a modified proinsulin precursor comprising a DNA sequence encoding proinsulin wherein: a. a nucleotide triplet encoding aspartic acid is substituted for the nucleotide triplet encoding the amino acid at the amino terminus of the C chain; or b. a nucleotide triplet encoding methionine is substituted for the nucleotide triplet encoding the amino acid at the carboxyl terminus of the C chain: or c. a nucleotide triplet encoding aspartic acid is substituted for the nucleotide triplet encoding the amino acid at the amino terminus of the C chain and a nucleotide triplet encoding methionine is substituted for the nucleotide triplet encoding the amino acid at the carboxyl terminus of the C chain.

Claim 6

A DNA sequence encoding a modified proinsulin precursor comprising a DNA sequence encoding proinsulin wherein: a. a nucleotide triplet encoding aspartic acid is substituted for the nucleotide triplet encoding the amino acid at the amino terminus of the C chain; or b. a nucleotide triplet encoding methionine is substituted for the nucleotide triplet encoding the amino acid at the carboxyl terminus of the C chain: or c. a nucleotide triplet encoding aspartic acid is substituted for the nucleotide triplet encoding the amino acid at the amino terminus of the C chain and a nucleotide triplet encoding methionine is substituted for the nucleotide triplet encoding the amino acid at the carboxyl terminus of the C chain.

Claim 7

A DNA sequence as claimed in claim 3 wherein the nucleotide triplet encoding aspartic acid comprises a base sequence ATC.

Claim 8

A DNA sequence as claimed in claim 3 wherein the nucleotide triplet encoding aspartic acid comprises a base sequence GTC.

Claim 9

A DNA sequence as claimed in claim 3 wherein the nucleotide triplet encoding methionine comprises a base sequence CAT.

Claim 10

A DNA sequence encoding a modified proinsulin precursor comprising a DNA sequence encoding human proinsulin wherein: a. a nucleotide triplet comprising a base sequence selected from the group consisting of ATC and GTC is substituted for the nucleotide triplet encoding arginine at amino acid position 31 3 or b. a nucleotide triplet comprising a base sequence CAT is substituted for the nucleotide triplet encoding arginine at amino acid position 65; or c. a nucleotide triplet comprising a base sequence selected from the group consisting of ATC and GTC is substituted for the nucleotide triplet encoding arginine at amino acid position 31 and a nucleotide triplet comprising a base sequence CAT is substituted for the nucleotide triplet encoding arginine at amino acid position 65.

Claim 11

A DNA sequence encoding a modified proinsulin precursor comprising a DNA sequence encoding human proinsulin wherein: a. a nucleotide triplet comprising a base sequence selected from the group consisting of ATC and GTC is substituted for the nucleotide triplet encoding arginine at amino acid position 31 3 or b. a nucleotide triplet comprising a base sequence CAT is substituted for the nucleotide triplet encoding arginine at amino acid position 65; or c. a nucleotide triplet comprising a base sequence selected from the group consisting of ATC and GTC is substituted for the nucleotide triplet encoding arginine at amino acid position 31 and a nucleotide triplet comprising a base sequence CAT is substituted for the nucleotide triplet encoding arginine at amino acid position 65.

Claim 12

A process for the production of DNA sequences encoding modified proin:ulin precursor having aspartic acid at the amino terminus of the C chain comprising: a. preparing a primer comprising a nucleotide triplet encoding aspartic acid and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of a single-stranded, message-synonymous DNA sequence encoding a proinsulin precursor comprising proinsulin to enable the primer to be effectively annealed to the single-stranded DNA sequence at a position wherein the triplet encoding aspartic acid encodes the amino acid at the amino terminus of the C chain of the proinsulin precursory b. annealing the primer to a vector containing the single-stranded, message-synonymous DNA sequence encoding the proinsulin precursor; c. extending and ligating the primer to form a circular double-stranded vector; vector; and e. allowing the cells and the double-stranded vector to replicate, thereby forming at least a first group of vectors comprising unaltered DNA sequences encoding the proinsulin precursors and a second group of vectors comprising altered DNA sequences encoding modified proinsulin precursors containing aspartic acid at the amino terminus of the C chain.

Claim 13

A process for the production of DNA sequences encoding modified proin:ulin precursor having aspartic acid at the amino terminus of the C chain comprising: a. preparing a primer comprising a nucleotide triplet encoding aspartic acid and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of a single-stranded, message-synonymous DNA sequence encoding a proinsulin precursor comprising proinsulin to enable the primer to be effectively annealed to the single-stranded DNA sequence at a position wherein the triplet encoding aspartic acid encodes the amino acid at the amino terminus of the C chain of the proinsulin precursory b. annealing the primer to a vector containing the single-stranded, message-synonymous DNA sequence encoding the proinsulin precursor; c. extending and ligating the primer to form a circular double-stranded vector; vector; and e. allowing the cells and the double-stranded vector to replicate, thereby forming at least a first group of vectors comprising unaltered DNA sequences encoding the proinsulin precursors and a second group of vectors comprising altered DNA sequences encoding modified proinsulin precursors containing aspartic acid at the amino terminus of the C chain.

Claim 14

A process as claimed in claim 8 further comprising preparing and annealing a nucleotide sequence comprising a restriction enzyme recognition sequence to the single- stranded DNA sequence of the vector.

Claim 15

A process as claimed in claim 8 wherein the synthetic primer comprises a nucleotide sequence having a restriction enzyme recognition sequence.

Claim 16

A process as claimed in claim 8 wherein the nucleotide triplet encoding aspartic acid has a base sequence ot ATC.

Claim 17

A process as claimed in claim 8 wherein the proinsulin presurcors comprise human proinsulin and the primer comprises a nucleotide sequence having a base sequence CTCCCGATCGGTCTT.

Claim 18

A process as claimed in claim 8 further comprising: a. preparing a second primer comprising a nucleotide triplet encoding methionine and sufficient nucleotides on each side of the triplet that are complementary to the message-synonymous strand of the altered DNA sequence to enable the second primer to be effectively annealed to the message-synonymous strand of the altered DNA sequence at a position wherein the nucleotide triplet encoding methionine encodes the amino terminus of the C chain of the Proinsulin; b. providing from the second group of vectors, a vector comprisirg the message-synonymous strand of the altered DNA sequence; c. annealing the second primer to the message-synonymous strand of the altered DNA sequence; d extending and ligating the second primer to form a second circular douole-stranded vector; e. transforming cells with the second double stranded vector; and f. allowing the cells and the second double-stranded vector to replicate to thereby form at least a third group of vectors comprising the previously altered DNA sequence and a fourth group of vectors comprising a twice-altered DNA sequence encoding modified proinsulin precursors comprising human proinsulin having aspartic acid at the amino terminus of the C chain and methionine at the carboxyl terminus of the C chain.

Claim 19

A process as claimed in claim 13 wherein a nucleotide sequence comprising a second,restriction enzyme recognition sequence is prepared and annealed to the message-synonymous strand of the altered DNA sequence.

Claim 20

A process as claimed in claim 13 wherein the second primer comprises a nucleotide sequence having a restriction enzyme recognition sequence.

Claim 21

A process as claimed in claim 13 wherein the nucleotide triplet encoding methionine has a base sequence CAT.

Claim 22

A process as claimed in claim 13 wherein the proinsulin precursors comprise human proinsulin and the second primer comprises a nucleotide sequence having a base sequence AATGCCCATCTTCTG.

Claim 23

A process for the production of DNA sequences encoding modified proinsulin precursor having aspartic acid at the amino terminus of the C chain comprising: a. preparing a primer comprising a nucleotide triplet that is complementary to a nucleotide triplet encoding aspartic acid and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of a single-stranded coding DNA sequence encoding proinsulin precursors comprising proinsulin to enable the primer to be effectively annealed to the coding DNA sequence so that the triplet that is complementary to a triplet encoding aspartic acid is at the position of the complement of the triplet that encodes the amino acid at the amino terminus of the C chain of the proinsulin; b. annealing the primer to a vector containing the single-stranded coding DNA sequence encoding the proinsulin precursor; c. extending and ligating the primer to form a circular double-stranded vector; d. transforming cells with the double-stranded vector; and e. allowing the cells and the double-stranded vector to replicate, thereby forming at least a first group of vectors comprising unaltered DNA sequences encoding the proinsulin precursor and a second group of vectors comprising altered DNA sequences encoding modified proinsulin precursors containing aspartic acid at the amino terminus of the C chain.

Claim 24

A process for the production of DNA sequences encoding modified proinsulin precursor having aspartic acid at the amino terminus of the C chain comprising: a. preparing a primer comprising a nucleotide triplet that is complementary to a nucleotide triplet encoding aspartic acid and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of a single-stranded coding DNA sequence encoding proinsulin precursors comprising proinsulin to enable the primer to be effectively annealed to the coding DNA sequence so that the triplet that is complementary to a triplet encoding aspartic acid is at the position of the complement of the triplet that encodes the amino acid at the amino terminus of the C chain of the proinsulin; b. annealing the primer to a vector containing the single-stranded coding DNA sequence encoding the proinsulin precursor; c. extending and ligating the primer to form a circular double-stranded vector; d. transforming cells with the double-stranded vector; and e. allowing the cells and the double-stranded vector to replicate, thereby forming at least a first group of vectors comprising unaltered DNA sequences encoding the proinsulin precursor and a second group of vectors comprising altered DNA sequences encoding modified proinsulin precursors containing aspartic acid at the amino terminus of the C chain.

Claim 25

A process as claimed in claim 18 further comprising preparing and annealing a nucleotide sequence comprising a nucleotide sequence that is complementary to a restriction enzyme recognition sequence to the single-stranded coding DNA sequence.

Claim 26

A process as claimed in claim 18 wherein the synthetic primer comprises a nucleotide sequence that is complementary to a restriction enzyme recognition sequence.

Claim 27

A process as claimed in claim 18 wherein the nucleotide. triplet that is complementary to a nucleotide triplet encoding aspartic acid has a base sequence GATe

Claim 28

A process as claimed in claim 18 wherein the proinsulin precursors comprise human proinsulin and the primer comprises a nucleotide sequence having a base sequence AAGACCGATCGGGAG.

Claim 29

A process as claimed in claim 18 further comprising: a. preparing a second primer comprising a nucleotide triplet that is complementary to a nucleotide triplet encoding methionine and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of the coding strand of the altered DNA sequence to enable the second primer to be effectively annealed to the coding strand of the altered DNA sequence so that the nucleotide triplet that is complementary to a nucleotide triplet encoding methionine is at the position of the complement of the triplet that encodes the amino acid at the amino terminus of the C chain of the proinsulin; b. providing from the second group of vectors, a vector comprising the coding strand of the altered DNA sequence; c. annealing the second primer to the coding strand of the altered DNA sequence; d. extending and ligating the second primer to form a second circular double-stranded vector; e. transforming cells with the second double-stranded vector; and f. allowing the cells and the second double-stranded vector to replicate to thereby form at least a third group of vectors comprising the previously altered DNA sequence and a fourth group of.vectors comprising twice-altered DNA sequences encoding modified proinsulin precursors comprising human proinsulin having aspartic acid at the amino terminus of the C chain and methionine at the carboxyl terminus of the C clain from the fourth group of vectors.

Claim 30

A process as claimed in claim 23 wherein a nucleotide sequence comprising a nucleotide sequence that is complementary to a second restriction enzyme recognition sequence is prepared and annealed to the coding strand cf the altered DNA sequenceo

Claim 31

A process as claimed in claim 23 wherein the second primer comprises a nucleotide sequence that is complementary to a restriction enzyme recognition sequence.

Claim 32

A process as claimed in claim 23 wherein the nucleotide triplet that is complenentary to a nucleotide triplet encoding methionine has a base sequence ATG.

Claim 33

A process as claimed in claim 23 wherein the proinsulin precursors comprise human proinsulin and the second primer comprises a nucleotide sequence having a base sequence CAGAAGATGGGCATT.

Claim 34

A process for the production of a DNA sequence encoding a modified proinsulin precursor comprising: a. preparing a first primer comprising a nucleotide triplet encoding aspartic acid and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of a single-stranded, message-synonymous DNA sequence encoding proinsulin precursors comprising a proinsulin to enable the first primer to be effectively annealed to the single-stranded DNA sequence at a position wherein the triplet encoding aspartic acid encodes the amino acid at the amino terminus of the C chain of the proinsulin; b. preparing a second primer comprising a nucleotide triplet encoding methionine and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of the single-stranded, message-synonymous DNA sequence to enable the second primer to be effectively annealed to the single-stranded DNA sequence at a position wherein the triplet encoding methionine encodes the amino acid at the carboxyl terminus of the C chain of the proinsulin; c. annealing the first primer and second primer to a vector containing the single-stranded, message-synonymous DNA sequence encoding the proinsulin precursory d. extending and ligating the primers to form a circular double-stranded vector; e. transforming a cell with the double-stranded vector; and f. allowing the cell and the double-stranded vector to replicate to thereby form at least a first group of vectors comprising unaltered DNA sequences encoding proinsulin precursors and a second group of vectors comprising altered DNA sequences encoding modified proinsulin precursors containing aspartic acid at the amino terminus of the C chain and methionine at the carboxyl terminus of the C chain.

Claim 35

A process for the production of a DNA sequence encoding a modified proinsulin precursor comprising: a. preparing a first primer comprising a nucleotide triplet encoding aspartic acid and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of a single-stranded, message-synonymous DNA sequence encoding proinsulin precursors comprising a proinsulin to enable the first primer to be effectively annealed to the single-stranded DNA sequence at a position wherein the triplet encoding aspartic acid encodes the amino acid at the amino terminus of the C chain of the proinsulin; b. preparing a second primer comprising a nucleotide triplet encoding methionine and sufficient nucleotides on each side of the triplet that are complementary to nucleotides of the single-stranded, message-synonymous DNA sequence to enable the second primer to be effectively annealed to the single-stranded DNA sequence at a position wherein the triplet encoding methionine encodes the amino acid at the carboxyl terminus of the C chain of the proinsulin; c. annealing the first primer and second primer to a vector containing the single-stranded, message-synonymous DNA sequence encoding the proinsulin precursory d. extending and ligating the primers to form a circular double-stranded vector; e. transforming a cell with the double-stranded vector; and f. allowing the cell and the double-stranded vector to replicate to thereby form at least a first group of vectors comprising unaltered DNA sequences encoding proinsulin precursors and a second group of vectors comprising altered DNA sequences encoding modified proinsulin precursors containing aspartic acid at the amino terminus of the C chain and methionine at the carboxyl terminus of the C chain.

Claim 36

A process as claimed in claim 28 wherein at least one nucleotide sequence comprising a restriction enzyme recognition sequence is also prepared and annealed to the single-stranded DNA sequence encoding the proinsulin.

Claim 37

A process as claimed in claim 28 wherein the firut primer comprises a nucleotide sequence having a restriction enzyme recognition sequence.

Claim 38

A process as claimed in claim 28 wherein the second primer comprises a nucleotide sequence having a restriction enzyme recognition sequence.

Claim 39

A process as claimed in claim 28 wherein the first primer and the second primer each comprise a nucleotide sequence having a restriction enzyme recognition sequence.

Claim 40

A process as claimed in claim 28 wherein the nucleotide triplet encoding aspartic acid has a base sequence ATC,

Claim 41

A process as claimed in claim 28 wherein the proinsulin precursors comprise human proinsulin and the first primer comprises a nucleotide sequence having a base sequence CTCCCGATCGGTCTT.

Claim 42

A process as claimed in clain 28 wherein the nucleotide triplet encoding methionine has a base sequence CAT.

Claim 43

A process as claimed in claim 28 wherein the proinsulin precursors comprise hunan proinsulin and the second primer comprises a nucleotide sequence having a base sequence AATGCCCATCTTCTG.

Claim 44

A process for the production of a DNA sequence encoding a modified proinsulin precursor comprising: a. preparing a first primer comprising a nucleotide triplet that is complementary to a nucleotide triplet encoding aspartic acid and sufficient nucleotides on each side of the nucleotide triplet that are complementary to nucleotides of the coding strand of a DNA sequence encoding proinsulin precursors comprising proinsulin to enable the first primer to be effectively annealed to the coding strand of the DNA sequence so that the nucleotide triplet that is complementary to a nucleotide triplet encoding aspartic acid is at the position of the complement of the nucleotide triplet that encodes the amino acid at the amino terminus of the C chain of the proinsulin; b. preparing a second primer comprising a nucleotide triplet that is complementary to a nucleotide triplet encoding methionine and sufficient nucleotides on each side of the nucleotide triplet that are complementary to nucleotides of the coding strand of the DNA sequence encoding the proinsulin precursors to enable the second primer to be effectively annealed to the coding strand of the DNA sequence so that the nucleotide triplet that is complementary to a nucleotide triplet encoding methionine is at position of the complement of the nucleotide triplet that encodes the amino acid at the carboxyl terminus of the C chain of the proinsulin; c. annealing the first primer and the seccnd primer to a vector containing the coding strand of the DNA sequence encoding the proinsulin precursors; d. extending and ligating the primers to form a circular double-stranded vector; e. transforming a cell with the double-stranded vector; and f. allowing the cell and the double-stranded vector to replicate to thereby form at least a first group of vectors comprising unaltered DNA sequences encoding proinsulin precursors and a second group of vectors comprising altered DNA sequences encoding modified proinsulin precursors containing aspartic acid at the amino terminus of the C chain and methionine at the carboxyl terminus of the C chain.

Claim 45

A process for the production of a DNA sequence encoding a modified proinsulin precursor comprising: a. preparing a first primer comprising a nucleotide triplet that is complementary to a nucleotide triplet encoding aspartic acid and sufficient nucleotides on each side of the nucleotide triplet that are complementary to nucleotides of the coding strand of a DNA sequence encoding proinsulin precursors comprising proinsulin to enable the first primer to be effectively annealed to the coding strand of the DNA sequence so that the nucleotide triplet that is complementary to a nucleotide triplet encoding aspartic acid is at the position of the complement of the nucleotide triplet that encodes the amino acid at the amino terminus of the C chain of the proinsulin; b. preparing a second primer comprising a nucleotide triplet that is complementary to a nucleotide triplet encoding methionine and sufficient nucleotides on each side of the nucleotide triplet that are complementary to nucleotides of the coding strand of the DNA sequence encoding the proinsulin precursors to enable the second primer to be effectively annealed to the coding strand of the DNA sequence so that the nucleotide triplet that is complementary to a nucleotide triplet encoding methionine is at position of the complement of the nucleotide triplet that encodes the amino acid at the carboxyl terminus of the C chain of the proinsulin; c. annealing the first primer and the seccnd primer to a vector containing the coding strand of the DNA sequence encoding the proinsulin precursors; d. extending and ligating the primers to form a circular double-stranded vector; e. transforming a cell with the double-stranded vector; and f. allowing the cell and the double-stranded vector to replicate to thereby form at least a first group of vectors comprising unaltered DNA sequences encoding proinsulin precursors and a second group of vectors comprising altered DNA sequences encoding modified proinsulin precursors containing aspartic acid at the amino terminus of the C chain and methionine at the carboxyl terminus of the C chain.

Claim 46

A process as claimed in claim 37 wherein at least one nucleotide sequence comprising a nucleotide sequence that is complementary to a restriction enzyme recognition sequence is also prepared and annealed to the coding strand.of the DNA sequence.

Claim 47

A process as claimed in claim 37 wherein the first primer comprises a nucleotide sequence that is complementary to restriction enzyme recognition sequence.

Claim 48

A process as claimed in claim 37 wherein the second primer comprises a nucleotide sequence that is complementary to a restriction enzyme recognition sequence.

Claim 49

A process as claimed in claim 37 wherein the first primer and the second primer each comprise a nucleotide sequence that is complementary to a restriction e zyme recognition sequence.

Claim 50

A process as claimed in claim 37 wherein the nucleotide triplet that is complementary to a nucleotide triplet encoding aspartic acid has a base sequence GAT.

Claim 51

A process as claimed in claim 37 wherein the proinsulin precursors comprise human proinsulin and the first primer comprises a nucleotide sequence having a base sequence AAGACCGATCGGGAG.

Claim 52

A,process as claimed in claim 37 wherein the second primer comprises a nucleotide triplet having a base sequence ATG.

Claim 53

A process as claimed in claim 18 wherein the proinsulin precursors comprise human proinsulin and the second primer comprises a nucleotide sequence having a base sequence CAGAAGATGGGCATT.

Claim 54

A process for the preparation of insuline comprising: a. preparing a DNA sequence encloding a modified proinsulin precursor as described in claim 3; b. inserting the DNA sequence into a first vector; c. transforming a cell with the first vector; d. isolating modified proinsulin precursors elaborated by the cell; and e. cleaving the modified proinsulin precursors to yield insulin.

Claim 55

A process as claimed in claim 46 wherein the modified proinsulin precursors comprise aspartic acid at the amino terminus of the C chain and are cleaved between the carboxyl terminus of the B chain and the amino terminus of the C chain by digestion with a sufficient amount of P. fragi mutant Me 1 enzyme.

Claim 56

A process as claimed in claim 46 wherein the modified proinsulin precursors comprise aspartic acid at the amino terminus of the C chain and at the carboxyl terminus of an amino acid chain following the A chain and are cleaved between the carboxyl terminus of the B chain and the amino terminus of the A chain and between the carboxyl terminus of the amino acid chain following the A chain and the amino terminus of the A chain by digestion with a sufficient amount of P. fragi mutant Me 1 enzyme.

Claim 57

A process as claimed in claim 47 to 48 wherein the modified proinsulin precursors further comprise methionine at the carboxyl terminus of the C chain and are cleaved between the C chain and the A chain by digestion with a sufficient amount of trypsin.

Claim 58

A process as claimed in claim 46 wherein the modified proinsulin precursors comprise methionine at the carboxyl terminus of the C chain and are cleaved between the amino terminus of the C chain and the carboxyl terminus of the A chain by treatment with an aqueous solution comprising cyanogen bromide in a concentration sufficient to thus cleave the modified proinsulin precursors.

Claim 59

A process as claimed in claim 46 wherein the modififed proinsulin presursors comprise methionine at the carboxyl terminus of the C chain and at the carboxyl terminus of an amino acid sequence attached to the anino terminus of the B chain and are cleaved between the amino terminus of the B chain and the carboxyl terminus of the amino acid sequence attached to the amino terminus of the B chain by treatment with cyanogen bromide in a concentration sufficient to thus cleave the modifired proinsulin precursors.