Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-12-27
2002-12-24
Wehbe′, Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C424S184100, C424S185100, C530S350000, C530S412000, C530S413000
Reexamination Certificate
active
06498020
ABSTRACT:
The present invention relates to novel fusion partners for recombinantly-produced proteins. In particular, the invention relates to a polypeptide which is resistant to proteolytic degradation and capable of acting as a carrier to increase the immunogenicity of a protein fused to it.
Recombinant DNA technology has allowed industry to produce many proteins of commercial importance. Proteins are produced in a wide variety of expression systems which are based on, for example, bacterial, yeast, insect, plant and mammalian cells. one of the problems associated with the production of proteins by recombinant means is that host cells contain enzymes which degrade proteins and the presence of such enzymes present particular difficulties in the production of small polypeptides.
One approach to overcoming such difficulties is to express a recombinant protein of interest in the form of a fusion protein. DNA encoding the protein of interest is fused in-frame to a fusion partner protein and the resulting fusion is expressed. Often, a linker sequence encoding a protease cleavage sited between the two parts of the fusion is included to allow cleavage of the fusion after it has been recovered from its host cell.
The fusion partner protein is often one which may be recovered and purified by some form of highly specific affinity purification means. Examples of such proteins are well known in the art and include, for example, glutathione-S-transferase, maltose binding protein and &bgr;-lactamase.
However these fusion partner proteins are all relatively large and thus have a number of disadvantages. For example, it is essential to remove them before any meaningful procedure may be carried out on the protein of interest, since they are too large to enable it to function with any degree of independence. Many small polypeptides are still thus made by chemical synthesis.
The present invention arose in the course of our investigations into the structure of a bovine ATPase inhibitor protein, IFS. This is a small—84 amino acid—protein which helps to regulate the activity of ATP synthase in mitochondria.
SUMMARY OF THE INVENTION
We have expressed in
E. coli
a number of fusion proteins which comprise the foregoing region of bovine IF
1
ATPase inhibitor protein and a short sequence of a second protein. We have found that these fusion proteins can be expressed at high levels and isolated in intact form, from which the second protein may efficiently be recovered. The use of the C-terminal 40 amino acids as a fusion partner protein in a recombinant expression system thus allows expression of a desired polypeptide in a manner which protects the polypeptide from degradation in the host cell.
Thus the present invention provides a fusion protein comprising:
a) a first region comprising the sequence of the C-terminal 40 amino acids of bovine IF
1
ATPase inhibitor protein, or a derivative thereof; and
b) a second region not naturally associated with the first region comprising a polypeptide sequence of interest.
The first region may be located either N-terminal to or C-terminal to the second region. However it is preferred that it is located N-terminal to the second region, and most preferably it is at the N-terminus of the fusion protein. Preferably, the fusion protein further comprises a cleavable linker region between the first and second regions.
The invention further comprises a nucleic acid encoding the fusion protein of the invention, and preferably the nucleic acid forms part of an expression vector comprising the nucleic acid operably linked to a promoter.
The invention further comprises a host cell carrying the expression vector of the invention, and a method of preparing the fusion protein of the invention comprising (i) culturing the host cell under conditions which provide for the expression of the fusion protein from the expression vector within the host cell; and (ii) recovering the fusion protein from the cell.
In cases where the fusion protein further comprises a protease cleavable linker region between the first and second regions the method optionally further comprises cleaving the protein at the protease cleavable linker and recovering the second region.
DETAILED DESCRIPTION OF THE INVENTION
A: First Region
The first region of the fusion protein of the invention may comprise any natural or synthetic polypeptide comprising the sequence of bovine IF
1
ATPase inhibitor or a derivative thereof. Preferably, the first region has the sequence of the C-terminal 40 amino acids of bovine IF
1
ATPase inhibitor or a derivative thereof. A derivative, as defined herein, is a fragment, mutant or other derivative which retains a common structural determinant of the C-terminal 40 amino acids of bovine IF
1
ATPase inhibitor.
“Common structural determinant” means that the derivative in question at least one structural feature of the C-terminal 40 amino acids of bovine IF
1
ATPase inhibitor. In the following, references to “bovine IF
1
ATPase inhibitor” are to be understood as encompassing references to the C-terminal 40 amino acids of this protein, unless otherwise provided for.
Structural features includes possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured bovine IF
1
ATPase inhibitor polypeptide or a fragment thereof, possession of amino acid sequence identity with bovine IF
1
ATPase inhibitor and features having common a structure/function relationship. Thus the fusion partner provided by the present invention includes splice variants encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutant s, glycosylation variants and other co valent derivatives of bovine IF
1
ATPase inhibitor which retain the physiological and/or physical properties of bovine IF
1
ATPase inhibitor. Exemplary derivatives include molecules wherein the protein of the invention is covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Such a moiety may be a detectable moiety such as an enzyme or a radioisotope. Further included are naturally occurring variants of bovine IF
1
ATPase inhibitor found with a particular species, preferably a mammal. Such a variant may be encoded by a related gene of the same gene family, by an allelic variant of a particular gene, or represent an alternative splicing variant of the bovine IF
1
ATPase inhibitor gene.
Derivatives which retain common structural features can be fragments of bovine IF
1
ATPase inhibitor. Fragments of bovine IF
1
ATPase inhibitor comprise individual domains thereof, as well as smaller polypeptides derived from the domains. Preferably, smaller polypeptides derived from bovine IF
1
ATPase inhibitor according to the invention define a single feature which is characteristic of bovine IF
1
ATPase inhibitor. Fragments may in theory be almost any size, as long as they retain the advantageous feature of bovine IF
1
ATPase inhibitor described herein.
Derivatives of bovine IF
1
ATPase inhibitor also comprise mutants thereof, which may contain amino acid deletions, additions or substitutions, subject to the requirement to maintain the characteristic feature of bovine IF
1
ATPase inhibitor described herein. Thus, conservative amino acid substitutions may be made substantially without altering the nature of bovine IF
1
ATPase inhibitor, as may truncations from the 5′ or 3′ ends. Deletions and substitutions may moreover be made to the fragments of bovine IF
1
ATPase inhibitor comprised by the invention. bovine IF
1
ATPase inhibitor mutants may be produced from a DNA encoding bovine IF
1
ATPase inhibitor which has been subjected to in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids. For example, substitutional, deletional or insertional variants of bovine IF
1
ATPase inhibitor can be prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of bovine IF
1
ATPase in
Miroux Bruno
Walker John
Li Q. Janice
Medical Research Council
Palmar & Dodge, LLP
Wehbe′ Anne M.
Williams Kathleen M.
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