Method for expression and secretion in bacillus

Claim 3

The method of claim 1 wherein said vector is a plasmid.

Claim 4

The method of claim 2 wherein the sequence that codes for a polypeptide is selected from the group consisting of sequences that code for human proinsulin, human insulin A-chain, human insulin B-chain, human pre-proinsulin, human growth hormone, bovine growth hormone, porcine growth hormone, growth hormone releasing factor, interferon, interleukin II, an enzyme, and a hormone.

Claim 5

The method of claim 3 in which the sequence that codes for a polypeptide codes for human proinsulin.

Claim 6

The method of claim 3 in which the sequence that codes for a polypeptide codes for human pre-proinsulin.

Claim 7

The method of claim 3 in which the sequence that codes for a polypeptide codes for human growth hormone.

Claim 8

The method of claim 3 in which the sequence that codes for a polypeptide codes for bovine growth hormone.

Claim 9

The method of claim 3 in which the sequence that codes for a polypeptide codes for growth hormone releasing factor.

Claim 10

The method of claim 3 in which the sequence that codes for a polypeptide codes for an enzyme.

Claim 11

The method of claim 3 in which the sequence that codes for a polypeptide codes for a hormone.

Claim 12

The method of claim 3 in which the sequence that codes for a polypeptide codes for human insulin B-chain.

Claim 13

The method of claim 1 wherein a) said recombinant DNA expression vector further comprises a signal peptide coding sequence, and b) said transformed cells are cultured under conditions suitable for expression and secretion of said polypeptide, subject to the limitation 1) that said signal peptide coding sequence is exclusive of the nucleotide triplet that codes for the N-terminal amino acid of the encoded signal peptide when said amino acid is methionine and that said signal peptide coding sequence is positioned for expression immediately adjacent, downstream and in the translational reading frame of said transcriptional and translational activating sequence 2) that the nucleotide triplet coding for the C-terminus of the signal peptide encoded by said signal peptide coding sequence is immediately adjacent, upstream and in the translational reading frame of said cloned DNA sequence that codes for said polypeptide, and 3) that said cloned DNA sequence that codes for said polypeptide is inclusive of the nucleotide triplet that codes for the N-terminal amino acid of said polypeptide.

Claim 14

The method of claim 12 wherein said vector is a plasmid.

Claim 15

The method of claim 13 wherein the transscriptional and translational activating sequence, the signal peptide coding sequence and the sequence that codes for a polypeptide comprise the ˜1.4 kb BamHI fragment of plasmid pOW440.

Claim 16

The method of claim 13 wherein the transscriptional and translational activating sequence and the signal peptide coding sequence comprise the ˜.65 kb XhoII fragment of plasmid pOW440.

Claim 17

The method of claim 12 wherein the transscriptional and translational activating sequence and signal peptide coding sequence comprise the ˜.65 kb XhoII-BglII fragment of plasmid pOW650.

Claim 18

The method of claim 12 wherein the sequence that codes for a polypeptide is selected from the group consisting of sequences that code for human insulin, human insulin A-chain, human insulin B-chain, human proinsulin, human growth hormone, bovine growth hormone, porcine growth hormone, interferon, growth hormone releasing factor, interleukin II, an enzyme, and a hormone.

Claim 19

The method of claim 17 in which the sequence that codes for a polypeptide codes for human insulin A-chain.

Claim 20

The method of claim 17 in which the sequence that codes for a polypeptide codes for human growth hormone.

Claim 21

The method of claim 17 in which the sequence that codes for a polypeptide codes for growth hormone releasing factor.

Claim 22

The method of claim 17 in which the sequence that codes for a polypeptide codes for bovine growth hormone.

Claim 23

The method of claim 17 in which the sequence that codes for a polypeptide codes for human proinsulin.

Claim 24

The method of claim 17 in which the sequence that codes for a polypeptide codes for a human hormone.

Claim 25

A recombinant DNA expression vector which is selectable and capable of replication in a Bacillus host cell, said vector comprising 1) the transcriptional and translational activating sequence of the Staphylococcus aureus nuclease gene and 2) a cloned DNA sequence that codes for a polypeptide, subject to the limitation 1) that said cloned DNA sequence that codes for said polypeptide and said transcriptional and translational activating sequence are immediately adjacent, in translational reading frame and positioned for expression of said polypeptide and 2) that said cloned DNA sequence that codes for said polypeptide is exclusive of the nucleotide triplet that codes for the N-terminal amino acid of said polypeptide when said amino acid is methionine.

Claim 26

A recombinant DNA expression vector which is selectable and capable of replication in a Bacillus host cell, said vector comprising 1) the transcriptional and translational activating sequence of the Staphylococcus aureus nuclease gene and 2) a cloned DNA sequence that codes for a polypeptide, subject to the limitation 1) that said cloned DNA sequence that codes for said polypeptide and said transcriptional and translational activating sequence are immediately adjacent, in translational reading frame and positioned for expression of said polypeptide and 2) that said cloned DNA sequence that codes for said polypeptide is exclusive of the nucleotide triplet that codes for the N-terminal amino acid of said polypeptide when said amino acid is methionine.

Claim 27

The recombinant DNA expression vector of claim 24 which is a plasmid.

Claim 28

The recombinant DNA expression vector of claim 24 wherein the cloned DNA sequence that codes for said polypeptide is selected from the group consisting of sequences that code for human proinsulin, human insulin A-chain, human insulin B-chain, human pre-proinsulin, human growth hormone, bovine growth hormone, procine growth hormone, growth hormone releasing factor, interferon, interleukin II, an enzyme, and a hormone.

Claim 29

The recombinant DNA expression vector of claim 26 wherein the sequence that codes for a polypeptide codes for human growth hormone.

Claim 30

The recombinant DNA expression vector of claim 26 wherein the sequence that codes for a polypeptide codes for human proinsulin.

Claim 31

The recombinant DNA expression vector of claim 26 wherein the sequence that codes for a polypeptide codes for human insulin B-chain.

Claim 32

The recombinant DNA expression vector of claim 26 wherein the sequence that codes for a polypeptide codes for bovine growth hormone.

Claim 33

The recombinant DNA expression vector of claim 26 wherein the sequence that codes for a polypeptide codes for growth hormone releasing factor.

Claim 34

The recombinant DNA expression vector of claim 26 wherein the sequence that codes for a polypeptide codes for an enzyme.

Claim 35

The recombinant DNA expression vector of claim 24 which further comprises a signal peptide coding sequence, and subject to the limitation 1) that said signal peptide coding sequence is exclusive of the nucleotide triplet that codes for the N-terminal amino acid of the encoded signal peptide when said amino acid is methionine and that said signal peptide coding sequence is positioned for expression immediately adjacent, downstream and in the translational reading frame of said transcriptional and translational activating sequence, 2) that the nucleotide triplet coding for the C-terminus of the signal peptide encoded by said signal peptide coding sequence is immediately adjacent, upstream and in the translational reading frame of said cloned DNA sequence that codes for said polypeptide, and 3) that said cloned DNA sequence that codes for said polypeptide is inclusive of the nucleotide triplet that codes for the N-terminal amino acid of said polypeptide.

Claim 36

The recombinant DNA expression vector of claim 33 which is a plasmid.

Claim 37

The recombinant DNA expression vector of claim 34 which is plasmid pOW440.

Claim 38

The recombinant DNA expression vector of claim 34 which is plasmid pOW448.

Claim 39

A transformed Bacillus host cell which is rendered competent for transformation with a recombinant DNA expression vector which is selectable and capable of replication in said host cell said vector comprising 1) the transcriptional and translational activating sequence of the Staphylococcus aureus nuclease gene and 2) a cloned DNA sequence that codes for a polypeptide, and 3) culturing said transformed cell under conditions suitable for expression of said polypeptide, subject to the limitation 1) that said cloned DNA sequence that codes for said polypeptide and said transcriptional and translational activating sequence are immediately adjacent, in translational reading frame and positioned for expression of said polypeptide and 2) that said cloned DNA sequence that codes for said polypeptide is exclusive of the nucleotide triplet that codes for the N-terminal amino acid of said polypeptide when said amino acid is methionine.

Claim 40

A transformed Bacillus host cell which is rendered competent for transformation with a recombinant DNA expression vector which is selectable and capable of replication in said host cell said vector comprising 1) the transcriptional and translational activating sequence of the Staphylococcus aureus nuclease gene and 2) a cloned DNA sequence that codes for a polypeptide, and 3) culturing said transformed cell under conditions suitable for expression of said polypeptide, subject to the limitation 1) that said cloned DNA sequence that codes for said polypeptide and said transcriptional and translational activating sequence are immediately adjacent, in translational reading frame and positioned for expression of said polypeptide and 2) that said cloned DNA sequence that codes for said polypeptide is exclusive of the nucleotide triplet that codes for the N-terminal amino acid of said polypeptide when said amino acid is methionine.

Claim 41

The transformed Bacillus of claim 37 which is Bacillus subtilis.

Claim 42

The transformed Bacillus subtilis of claim 38 which is Bacillus subtilis SR22/pOW440.

Claim 43

The transformed Bacillus host cell of claim 37 which further comprises a signal peptide coding sequence, and subject to the limitation 1) that said signal peptide coding sequence is exclusive of the nucleotide triplet that codes for the N-terminal amino acid of the encoded signal peptide when said amino acid is methionine and that said signal peptide coding sequence is positioned for expression immediately adjacent, downstream and in the translational reading frame of said transcriptional and translational activating sequence, 2) that the nucleotide triplet coding for the C-terminus of the signal peptide encoded by said signal peptide coding sequence is immediately adjacent, upstream and in the translational reading frame of said cloned DNA sequence that codes for said polypeptide, and 3) that said cloned DNA sequence that codes for said polypeptide is inclusive of the nucleotide triplet that codes for the N-terminal amino acid of said polypeptide.

Claim 44

The transformed Bacillus of claim 40 which is Bacillus subtilis.

Claim 45

The transformed Bacillus of claim 41 which is Bacillus subtilis MI112/pOW440.

Claim 46

The transformed Bacillus of claim 41 which is Bacillus subtilis SR22/pOW448.