Production of GAD65 in methylotrophic yeast
AssigneeZymoGenetics, Inc.
InventorsChristopher K. Raymond, Thomas R. Bukowski, Paul D. Bishop
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Abstract
Methylotrophic yeast are used for high-level expression of GAD65 that makes the production of GAD65 feasible on an industrial scale. A methanol-inducible promoter from, for example, an alcohol oxidase gene, such as Pichia pastoris AOX1, can be used to regulate GAD65 expression. The recombinant GAD65 has high specific activity and retains antigenic characteristics of the native molecule that are essential to immunological assays and therapeutic protocols.
Claims56
A methylotrophic yeast which grows on methanol as a carbon and energy source, transformed with a DNA construct comprising the following operatively linked elements:
- a) a methanol-inducible transcriptional promoter;
- b) a DNA segment encoding a GAD65 polypeptide;
- c) a transcriptional terminator;
- and
- d) a selectable marker,
- which yeast when cultured in a fermentor expresses said GAD65 polypeptide at a concentration of at least 500 mg/L.
A method for producing a GAD65 polypeptide comprising the steps of:
- culturing a methylotrophic yeast which grows on methanol as a carbon and energy source, transformed with a DNA construct which comprises the following operatively linked elements:
- a) a methanol-inducible transcriptional promoter;
- b) a DNA segment encoding a GAD65 islet cell polypeptide;
- c) a transcriptional terminator;
- and
- d) a selectable marker,
- in a culture medium,, whereby said GAD65 polypeptide is produced at a concentration of at least 500 mg/L in said culture medium.
A method for purifying GAD65 expressed by a culture of methylotrophic yeast cells, which comprises the steps of:
- isolating a GAD65-containing cell fraction from the yeast cell culture in a buffer containing a reducing agent and a detergent;
- phase-partitioning the GAD65-containing cell fraction into a GAD65-containing detergent phase and an aqueous phase;
- separating the GAD65 from the GAD65-containing detergent phase by a first anion exchange chromatography in a buffer containing a reducing agent and a detergent to produce a first GAD65 anion exchange fraction;
- applying the first GAD65 anion exchange fraction to a column containing a cation exchange medium at a slightly acidic pH and adjusting the GAD65-containing fraction therefrom to an alkaline pH;
- loading the GAD65 cation exchange fraction on a second anion exchange column at an alkaline pH in a buffer containing a reducing agent and a detergent, eluting the GAD65 in an alkaline to acid pH gradient, and adjusting the pH of the GAD65 eluate to about neutral;
- and
- purifying the GAD65 anion exchange eluate by hydroxyapatite chromatography in a buffer containing a reducing agent and a detergent, and obtaining purified GAD65.
A. A method for purifying GAD65 expressed by a culture of methylotrophic yeast cells, which comprises the steps of:
- isolating a GAD65-containing cell fraction from the yeast cell culture in a buffer containing a reducing agent and a detergent;
- applying the GAD65-containing cell fraction to a first anion exchange column in a buffer containing a reducing agent and a detergent to produce a first GAD65 anion exchange fraction;
- phase-partitioning the first GAD65 anion exchange fraction into a GAD65-containing detergent phase and an aqueous phase;
- loading the GAD65-containing detergent phase on a second anion exchange column at an alkaline pH in a buffer containing a reducing agent and a detergent, eluting the GAD65 in an alkaline to acid pH gradient, and adjusting the pH of the GAD65 eluate to about neutral;
- and
- purifying the GAD65 anion exchange eluate by hydroxyapatite chromatography in a buffer containing a reducing agent and a detergent, and obtaining purified GAD65.
A method of preparing a methylotrophic yeast strain that produces a GAD65 polypeptide comprising the steps of:
- a) transforming a methylotrophic yeast host with a DNA construct which comprises the operatively linked elements of (i) a methanol-inducible transcriptional promoter, (ii) a DNA segment encoding a GAD65 polypeptide, (iii) a transcriptional terminator, and (iv) a selectable marker;
- b) culturing the transformed cells from step (a) under conditions wherein the DNA segment is expressed and GAD65 polypeptide is produced;
- c) assaying the level of GAD65 polypeptide produced by isolates of the transformed cells;
- and
- d) selectively culturing isolates that produce levels of GAD65 polypeptide of at least 500 mg/L.
A Pichia methanolica yeast strain transformed with a DNA construct comprising the following operatively linked elements:
- a) a methanol-inducible transcriptional promoter;
- b) a DNA segment encoding a GAD65 polypeptide;
- c) a transcriptional terminator;
- and
- d) a selectable marker.
A method for producing a GAD65 polypeptide comprising the steps of:
- culturing a Pichia methanolica yeast strain transformed with a DNA construct which comprises the following operatively linked elements:
- a) a methanol-inducible transcriptional promoter;
- b) a DNA segment encoding a GAD65 islet cell polypeptide;
- c) a transcriptional terminator;
- and
- d) a selectable marker
- in a culture medium,, whereby said GAD65 polypeptide is produced in said culture medium.
Dependencies are inferred from the claim text.