Fusion products containing insoluble proteinaceous tag

Details Fusion products containing insoluble proteinaceous tag Fusion products containing insoluble proteinaceous tag

2001-01-24

2003-09-02

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S191000

Reexamination Certificate

active

06613548

ABSTRACT:

FIELD OF INVENTION
The present invention relates to fusion products prepared by recombinant DNA procedures. The products are comprised of a soluble protein of interest and an insoluble proteinaceous tag. More particularly, the invention relates to the separation of such products from a cellular host in which the products have been expressed by utilization of the insolubility of the tag.
BACKGROUND OF THE INVENTION
Fusion techniques are popular for the preparation and recovery of recombinantly produced proteins of interest which are soluble in the host cell in which they are expressed. The proteins, which term herein also includes peptides, are of interest for research, diagnostic, or pharmaceutical use because of actual or supposed biological activity.
Preparation of fusion products involves ligating a fragment containing the DNA sequence coding for the protein of interest and a fragment containing the DNA sequence coding for a second protein commonly termed the “tag” into a vector. The vector is then introduced into a host cell, such as an insect cell, and the fusion product (protein of interest fused to the tag) is expressed. After expression, the cell is lysed thereby releasing the fusion product and other cellular proteins into the lysate solution. The fusion product is then separated from the lysate solution and subjected to further manipulation which can include cleavage and separation of the protein of interest from the tag.
A function of the tag in a fusion technique as above set forth is to facilitate separation of the fusion product from the host cell lysate solution. Separation techniques based on the well known principals of chromatography, in particular affinity chromatography, have commonly been employed; with affinity chromatography the tag functioning as a receptor having specific attraction for a ligand which typically is immobilized on a solid matrix. Contacting the lysate solution containing the fusion product with the solid matrix results in selective adsorption of the product onto the matrix. After washing the matrix to remove non-binding substances, the fusion product can be recovered by various elution techniques such as those based on pH, ionic strength or ligand competition.
Descriptions of the preparation of fusion products and the recovery and separation thereof by chromatographic techniques from host cell lysate solution are illustrated in the following U.S. patents and other publications, the disclosures of which are hereby incorporated by reference: 5,643,758; 5,654,176; 5,179,007; 4,879,236, 4,745,051, Biochem. J. (1986) 240, 1-12, Methods in Enzymology, Vol. 182, Guide to Protein Purification, and Methods in Molecular Biology, Vol. 59, Protein Purification Protocols. U.S. Pat. No. 5,496,934 is also so referenced as illustrating an affinity separation procedure where the tag (cellulose binding domain) binds directly to the matrix (cellulose), thus avoiding the use of a separate ligand bound to a matrix.
A problem associated with chromatographic techniques for the separation of fusion products from their cellular lysate solutions is that they are time consuming, involving as they do multiple steps including binding to a matrix, washing to remove non-specifically bound cellular components and subsequent elution. Also, the degree of binding of the fusion product to a matrix can be interfered with by various factors such as pH, ion concentration detergents and the like which can reduce the yield of fusion product.
SUMMARY OF THE INVENTION
One aspect of the present invention provides an improvement in those recognized processes for the preparation of a recombinantly expressed fusion product comprised of a proteinaceous tag and a soluble protein of interest and the subsequent separation of the fusion product from the host cell in which it is expressed. The process to which the improvement described herein pertains involves (1) preparing a vector containing the DNA sequence coding for the protein of interest and the DNA sequence coding for the tag, (2) introducing the vector into a host cell and expressing the fusion product, (3) lysing the host cell to liberate cellular proteins and then (4) utilizing the characteristics of the tag to separate the fusion product from the lysate solution. The improvement to this process provided by the present invention is the use, as the tag, of a protein which is insoluble in a normal lysate solution, which typically is at a pH of about 7-8. Accordingly, the fusion product containing the tag precipitates from the lysate solution and the precipitated fusion product can then be separated from the lysate solution by centrifugation or filtration.
Advantages accompanying the use of the process described herein include the fact that the separation of fusion product from lysate solution is a simple, one-step centrifugation or filtration procedure and that the expressed fusion product can be recovered in high yield from the host cell. An additional and particularly surprising advantage of the present process is that the level of expression or the fusion product in the host cell is increased above what would normally be expected.
An additional aspect of the present invention resides in providing a new and improved vector useful in practicing the foregoing process. Yet a further feature of this invention is to provide a recombinantly expressed fusion product comprised of a soluble protein of interest and an insoluble proteinaceous tag, the insolubility of the tag permitting the fusion product to precipitate from the host cell in which it is expressed.
A still further aspect of this invention resides in providing a easy process for cleaving the fusion product to separate the tag from the protein of interest and the subsequent recovery of the protein. In this respect, after the insoluble, precipitated fusion product is centrifuged or filtered from the lysate solution, it is dissolved in an aqueous medium at a basic pH of about 9-11, thus solubilizing the product. The tag can then be released from the protein of interest by cleaving with a site specific protease such as enterokinase. The pH of the solution containing the tag and protein is then reduced to about 7-8 by addition of an acidic buffer, thus causing precipitation of the tag which can then again be removed from solution by centrifugation or filtration; the protein of interest remaining in solution.


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