Recombinant plasmids encoded for human preproinsulin and their use in producing human insulin

Claim 2

A cloning vector for producing human preproinsulin through expression of DNA by a transformed host organism comprising a plasmid capable of transforming a host organism and having a preproinsulin coding DNA sequence therein, said preproinsulin coding DNA sequence including a functional transport coding DNA presequence fused to a structural gene coding for human proinsulin, said preproinsulin coding DNA sequence being under the control of an operator, promoter and ribosome binding site sequence and being positioned in the vector reading frame with respect to the initiation codon for the functional transport coding DNA presequence so that a preproinsulin product is expressed by a host organism transformed by said cloning vector.

Claim 3

Plasmid pJW2172 (ATCC 31891) and its progeny.

Claim 4

Plasmid pJW2172 (ATCC 31891) and its progeny.

Claim 5

A host organism transformed by the cloning vector of claim 1 or the plasmid of claim 2 or a transformant culture cloned from at least one said host organism.'

Claim 6

A host organism or culture according to claim 3 which is Escherichia coli.

Claim 7

A host organism or culture according to claim 4 which is E. coli K12.

Claim 8

Preproinsulin which has been produced by a host organism or culture according to any one of claims 3 to 5.

Claim 9

A host organism or culture according to claim 3 which is capable of correctly processing expressed preproinsulin to human proinsulin in vivo and transporting the human proinsulin across the host's cell membrane.

Claim 10

Human proinsulin which has been produced by a host organism or culture according to claim 7 or which has been produced by in vitro conversion of preproinsulin according to claim 6.

Claim 11

Human insulin which has been produced by in vitro conversion of human proinsulin according to claim 8.

Claim 12

A process either (a) for preparing a cloning vector for producing human preproinsulin, or (b) for preparing an organism or culture capable of expressing a preoro- insulin peptide product, or (c) for preparing preproinsulin or human proinsulin, or (d) for preparing human insulin; which process comprises: (a) providing a human preproinsulin c DNA sequence (including a functional transport presequence fused to a structural gene coding for human proinsulin) in a parent vector capable of becoming a functional part of a host organisms genetic material, said preproinsulin sequence being positioned to be under the control of an operator, promoter and ribosome binding site sequence and in correct reading frame with respect to the initiation codon for the functional transport presequence of said sequence; and, optionally, (b) transforming, with said cloning vector, the host organism and optionally cloning said organism to produce a transformant culture; and, optionally, (c) growing said transformed host organism or transformant culture under conditions suitable,for expression of a DNA sequence coding for a preproinsulin peptide product and recovering the expressed preproinsulin peptide from the host cells and optionally converting said peptide to human proinsulin, or, if the organism is capable of correctly processing expressed preproinsulin to human proinsulin in vivo and transporting the resulting human proinsulin across the host's cell membrane, allowing said transformed host organism or transformant culture to process the expressed preproinsulin peptide to human proinsulin in vivo, allowing human proinsulin to accumulate outside the host's cell membrane and recovering accumulated human proinsulin; and, optionally, (d) converting said human proinsulin to human insulin in vitro and purifying said insulin.

Claim 13

A process either (a) for preparing a cloning vector for producing human preproinsulin, or (b) for preparing an organism or culture capable of expressing a preoro- insulin peptide product, or (c) for preparing preproinsulin or human proinsulin, or (d) for preparing human insulin; which process comprises: (a) providing a human preproinsulin c DNA sequence (including a functional transport presequence fused to a structural gene coding for human proinsulin) in a parent vector capable of becoming a functional part of a host organisms genetic material, said preproinsulin sequence being positioned to be under the control of an operator, promoter and ribosome binding site sequence and in correct reading frame with respect to the initiation codon for the functional transport presequence of said sequence; and, optionally, (b) transforming, with said cloning vector, the host organism and optionally cloning said organism to produce a transformant culture; and, optionally, (c) growing said transformed host organism or transformant culture under conditions suitable,for expression of a DNA sequence coding for a preproinsulin peptide product and recovering the expressed preproinsulin peptide from the host cells and optionally converting said peptide to human proinsulin, or, if the organism is capable of correctly processing expressed preproinsulin to human proinsulin in vivo and transporting the resulting human proinsulin across the host's cell membrane, allowing said transformed host organism or transformant culture to process the expressed preproinsulin peptide to human proinsulin in vivo, allowing human proinsulin to accumulate outside the host's cell membrane and recovering accumulated human proinsulin; and, optionally, (d) converting said human proinsulin to human insulin in vitro and purifying said insulin.