US5013653A — Product and process for introduction of a hinge r…

Claim 1

Independent Claim 218 words

A method of producing a selected target polypeptide expressed by a host cell culture, said method comprising the steps of: a. expressing a fused polypeptide by culturing said host cell culture, the cells of said culture containing and being capable of expressing a recombinant DNA encoding said fused polypeptide, said fused polypeptide comprising: a first sequence of amino acids defining a leader sequence, a hinge region, and at least one amino acid defining a cleavage site recognizable and cleavable by a selected cleavage agent, said hinge region being a cysteine-free flexible amino acid sequence not normally associated with said leader sequence or said target polypeptide and comprising at least two amino acids defining a secondary structure which can promote cleavage by said cleavage agent at said cleavage site; and a second sequence of amino acids linked to said first sequence defining a said selected target polypeptide; b. exposing said fused polypeptide to said selected cleavage agent in an environment wherein said target polypeptide is disposed in its three dimensional conformation and said selected cleavage agent is rendered preferentially accessible to said cleavage site by said hinge region thereby to cleave said target polypeptide from the remainder of said fused polypeptide preferentially at said cleavage site; and c. separating said target polypeptide from the remainder of said fused polypeptide.

Claim 2

Independent Claim 218 words

A method of producing a selected target polypeptide expressed by a host cell culture, said method comprising the steps of: a. expressing a fused polypeptide by culturing said host cell culture, the cells of said culture containing and being capable of expressing a recombinant DNA encoding said fused polypeptide, said fused polypeptide comprising: a first sequence of amino acids defining a leader sequence, a hinge region, and at least one amino acid defining a cleavage site recognizable and cleavable by a selected cleavage agent, said hinge region being a cysteine-free flexible amino acid sequence not normally associated with said leader sequence or said target polypeptide and comprising at least two amino acids defining a secondary structure which can promote cleavage by said cleavage agent at said cleavage site; and a second sequence of amino acids linked to said first sequence defining a said selected target polypeptide; b. exposing said fused polypeptide to said selected cleavage agent in an environment wherein said target polypeptide is disposed in its three dimensional conformation and said selected cleavage agent is rendered preferentially accessible to said cleavage site by said hinge region thereby to cleave said target polypeptide from the remainder of said fused polypeptide preferentially at said cleavage site; and c. separating said target polypeptide from the remainder of said fused polypeptide.

Claim 3

Dependent Claim 45 words

The method of claim 1 wherein said first sequence of amino acids imparts a preselected property to said fused polypeptide operable to facilitate concentration of said fused polypeptide, and wherein prior to step b, said fused polypeptide is concentrated by exploitation of said selected property.

Claim 4

Dependent Claim 45 words

The method of claim 2 wherein said sequence of amino acids which imparts a preselected property comprises an amphiphilic helix, said method comprising the additional step of permitting said fused polypeptide to form intracellular inclusion bodies and collecting said inclusion bodies prior to step b.

Claim 5

Dependent Claim 15 words

The method of claim 1 wherein said cleavage site is immediately adjacent said target polypeptide.

Claim 6

Dependent Claim 22 words

The method of claim 1 wherein said cleavage site comprises one or a sequence of amino acids absent from said target polypeptide.

Claim 7

Dependent Claim 22 words

The method of claim 1 wherein said cleavage site comprises a unique one or sequence of amino acids in said fused polypeptide.

Claim 8

Dependent Claim 15 words

The method of claim 1 wherein said hinge region comprises at least one proline residue.

Claim 9

Dependent Claim 28 words

The method of claim 1 wherein said hinge region comprises an amino acid sequence which forms a random coil when said fused polypeptides is disposed in aqueous solution.

Claim 10

Dependent Claim 40 words

The method of claim 1 wherein said hinge region includes a member selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, serine, proline, and combinations thereof, in amounts sufficient to render said hinge region soluble in water.

Claim 11

Dependent Claim 23 words

The method of claim 1 wherein said cleavage site is a glutamic acid residue and said cleavage agent is S. aureus V-8 protease.

Claim 12

Dependent Claim 80 words

The method of claim 1 wherein said second sequence of amino acids comprises an amino acid or an amino acid sequence cleavable by said cleavage agent at a rate less than the rate at which said cleavage site is cleaved, said method comprising the additional steps of: stopping the cleavage reactions of step b prior to its completion; separating cleaved target polypeptide from the remainder of the reaction mixture; and repeating step b with the remainder of said reaction mixture.

Claim 13

Dependent Claim 32 words

The method of claim 1 wherein said cleavage agent and said cleavage site are selected from the group consisting of the cleavage agents and cleavage site pairings set forth in Table 1.

Claim 14

Dependent Claim 51 words

The method of claim 1 wherein said target polypeptide comprises at least two cysteine residues, said method comprising the additional step of subjecting said fused polypeptide prior to step b to oxidizing conditions so that said selected target polypeptide is held in its tertiary conformation by at least on disulfide bond.

Claim 15

Dependent Claim 33 words

The method of claim 1 wherein said target polypeptide is selected from the group consisting of growth factors, hormones, lymphokines, enzymes, antibody binding sites, viral proteins, non-enzymatically active procaryotic proteins, and analogs thereof.

Claim 16

Dependent Claim 171 words

A recombinant DNa encoding and capsule of expressing a precursor fused polypeptide including a selected target polypeptide in a procaryotic or eucaryotic organism, said DNA comprising: a first DNA segment encoding a sequence of amino acids comprising a leader sequence, a hinge region, and at least one amino acid defining a cleavage site recognizable and cleavable by a selected cleavage agent, said hinge region being a cysteine-free flexible amino acid sequence not normally associated with said leader sequence or said target polypeptide and comprising at least two amino acids defining a secondary structure which can promote cleavage by said cleavage agent at said cleavage site; and a second DNA segment linked to said first segment encoding a sequence of amino acids defining a said selected target polypeptide, whereby said cleavage site is a favored site for cleavage upon treatment of said fused polypeptide with said cleavage agent when said fused polypeptide is disposed in solution and said amino acid sequence defining a target polypeptide is disposed in its three dimensional conformation.

Claim 17

Dependent Claim 171 words

A recombinant DNa encoding and capsule of expressing a precursor fused polypeptide including a selected target polypeptide in a procaryotic or eucaryotic organism, said DNA comprising: a first DNA segment encoding a sequence of amino acids comprising a leader sequence, a hinge region, and at least one amino acid defining a cleavage site recognizable and cleavable by a selected cleavage agent, said hinge region being a cysteine-free flexible amino acid sequence not normally associated with said leader sequence or said target polypeptide and comprising at least two amino acids defining a secondary structure which can promote cleavage by said cleavage agent at said cleavage site; and a second DNA segment linked to said first segment encoding a sequence of amino acids defining a said selected target polypeptide, whereby said cleavage site is a favored site for cleavage upon treatment of said fused polypeptide with said cleavage agent when said fused polypeptide is disposed in solution and said amino acid sequence defining a target polypeptide is disposed in its three dimensional conformation.

Claim 18

Dependent Claim 39 words

The recombinant DNA of claim 15 wherein said first DNA segment comprises DNA encoding a leader sequence comprising an amino acid sequence which imparts a preselected property to said fused polypeptide operable to facilitate concentration of said fused polypeptide.

Claim 19

Dependent Claim 21 words

The recombinant DNA of claim 16 wherein said sequence of amino acids which imparts a selected property comprises an amphiphilic helix.

Claim 20

Dependent Claim 16 words

The recombinant DNA of claim 15 wherein said cleavage site is immediately adjacent said target polypeptide.

Claim 21

Dependent Claim 26 words

The recombinant DNA of claim 15 wherein said cleavage site comprises one or a sequence of amino acids absent from said sequence defining said target polypeptide.

Claim 22

Dependent Claim 23 words

The recombinant DNA of claim 15 wherein said cleavage site comprises a unique one or sequence of amino acids in said fused polypeptide.

Claim 23

Dependent Claim 27 words

The recombinant DNA of claim 15 wherein said cleavage site comprises a member selected from the group consisting of the cleavage site set forth in Table 1.

Claim 24

Dependent Claim 14 words

The recombinant DNA of claim 15 wherein said cleavage site comprises a Glu residue.

Claim 25

Dependent Claim 16 words

The recombinant DNA of claim 15 wherein said hinge region comprises at least one proline residue.

Claim 26

Dependent Claim 29 words

The recombinant DNA of claim 15 wherein said hinge region comprises an amino acid sequence which forms a random coil when said fused protein is disposed in aqueous solution.

Claim 27

Dependent Claim 41 words

The recombinant DNA of claim 15 wherein said hinge region includes a member selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, serine, proline, and combinations thereof, in amounts sufficient to render said hinge region soluble in water.

Claim 28

Dependent Claim 37 words

The recombinant DNA of claim 15 wherein said second segment encodes a sequence of amino acids defining a target polypeptide, growth factors, hormones, lymphokines, enzymes, antibody binding sites, viral proteins, non-enzymatically active procaryotic proteins, and analogs thereof.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1994-05-06	1994-11-05	1995-05-07	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1998-05-06	1998-11-05	1999-05-07	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2002-05-06	2002-11-05	2003-05-07	$7700.00	Window Closed / Lapsed

Product and process for introduction of a hinge region into a fusion protein to facilitate cleavage

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