Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1994-06-07
2001-03-20
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06203976
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Promoters Useful for Recombinant Protein Expression
Relatively large amounts of substantially pure protein is often necessary (1) to carry out in vitro or in vivo experimentation or (2) for protein structure determination. Often the tissue or cell source harboring the protein of interest may be in short supply or contain insufficient endogenous quantities. Further complicating the ability to obtain adequate amounts of protein is the need to devise often lengthy and multi-stop purification procedures which must usually be specifically designed for a particular protein to be isolated and can ultimately result in drastic losses of product with each successive step being carried out as well as the potential loss of critical protein function due to the inclusion of reagents in the protocol which may be necessary for protein isolation but detrimental to its activity. As a means to circumvent these and other problems encountered when attempting to isolate large amounts of functional protein, investigators have turned to the use of a variety of different expression systems with which to produce proteins.
Four major expression systems common to investigators include bacterial, yeast, insect and mammalian cells (1). The method of choice is usually dictated by a number of criteria, including size of protein being produced, whether or not the protein is secreted, the presence of host modifying enzymes which can effect the recombinant produced protein structure or function, and for what purpose for which the expressed protein will be used. With these and other parameters in mind, a number of different expression vectors with which to insert the appropriate genetic material has been developed. These often include segments from viral or yeast promoter regions which, when orientated in the proper context to the gene, or cDNA to be expressed, can be recognized by the host cell's transcriptional machinery and result in the production of sufficient protein. The next section of this introduction will deal with some of the more common viral and yeast promoters used for this purpose.
Viral Promoters
A number of viral vectors have been described and include retroviruses, non-defective and defective viral vectors and factors made from various promoters and other regulatory elements derived from DNA and RNA virus sources (2). Promoters consist of short arrays of nucleic acid sequences that interact specifically with cellular proteins involved in transcription. The combination of different recognition sequences and the amounts of the cognate transcription factors determine the efficiency with which a gene is transcribed in a particular cell type (3). Below is a brief description of some viral vectors and their promoters which are used for expressing recombinant proteins.
1. Papovovirusus: These are small, non-enveloped DNA containing viruses, SV-40 and polyoma being two of the best studied examples (4). The viral genome of SV-40 is a covalently closed circular double-stranded DNA molecule of 5243 bp. The genome is divided into early and late regions which are transcribed from the two DNA strands in opposite directions. Various plasmid-based expression vectors contain specific regulatory regions derived from SV-40, the most commonly used being a 300 bp segment which lies between the viral early and late transcription units and containing a number of different controlling cis elements, including the DNA origin of replications and promoters, and sites of initiation of transcription of early and late mRNAs (5). Use of the SV-40 regulatory region can result in high levels of expression in transfected host cells.
2. Cytomegalovirus (CMV): The human CMV immediate early promoters serves as an efficient transcription element with which to express foreign proteins. In combination with the CMV enhancer element, the CMV promoter's transcriptional activity can be increased to 10 to 100-fold. The human CMV enhancers are also active in a wide variety of cells from many species.
3. Mouse Mammary Tumor Virus (MMTV): The long terminal repeat (LTR) promoter region of MMTV is probably the best studied example of glucocorticoid-inducible promoters. The glucocorticoid-responsive element (GRE) behaves as an enhancer element and has been localized between −100 and −200 of the MMTV LTR (6). High level protein expression, using the MMTV LTR in combination with other viral promoters/enhancers, can occur in glucocorticoid-response cells.
4. Baculovirus: High level expression of foreign proteins in insect cells has been demonstrated using the Baculovirus expression vectors (7, 8). The baculovirus vector utilizes the highly expressed and regulated
Autographa californica
nuclear polyhedrosis virus polyhedron promoter which has been modified for the insertion of foreign genes. The viral genome consists of double-stranded circular, supercoiled DNA 128 kilobases long. Most transfer vectors contain the promoter of the polyhedron gene (which is non-essential for replication or production of extracellular virus in cultured cells). The foreign gene sequences in the recombinant plasmid can be transferred to the wild type virus by homologous recombination within a cell transfected with both the plasmid and wild-type virus DNAs.
Yeast Promoters
Protein expression in yeast offers certain advantages over bacterial expression systems. Yeast, being eukaryotic, possess much of the complex cell biology typical of multicellular organisms, including a highly compartmentalized intracellular organization and an elaborate secretory pathway which mediates the secretion and modification of many host proteins (9). Using a yeast expression system thus affords a broader range of potential applications then is possible with bacterial expression systems. A number of yeast promoters are available for high level protein expression. Below is a brief description of the now commonly used systems.
1. Galactose-inducible Promoters (GAL): The Saccharomyces galactose-inducible promoters GAL1, GAL7 and GAL10 have been used for high-level protein production in yeast (10). These promoters can be switched from a near-zero off state to a very high on state by the addition of galactose to a nonglycose-containing medium. At least 28 GAL promoter-containing plasmids are available for protein expression in yeast.
2. Copper Metallothionine Promoter: Induction of the yeast metallothionine (MT) promoter results in good expression of recombinant genes. The MT promoter, originating upstream of the CUPI coding sequence, is rapidly induced during addition of copper ions to the media. The promoter has been described as approximately a 450 bp fragment which contains the metal regulatory sequences, the mRNA cap site, the TATA box and associated transcription signals (11).
3. CYC1: The CYC1 gene of Saccharomyces cerevisiae encoded the cytochrome C protein. Two independent upstream activation sequenced (UAS) are found in the CYC1 gene, which appears to function as regulatory sites. A limited number of CYC1 promoter-containing vector have been reported.
4. Alcohol dehydrogenase 2 (ADH2) promoter: The ADH2 gene is regulated by glucose repression. When grown on glucose, ADN2 transcription is undetectable; however, derepression to a level of about 1% of soluble cellular proteins occurs when yeast are grown on a non-fermentable carbon source (12). Analysis of the ADH2 promoter reveals two cis-acting regulatory components (upstream activation sequences) which mediates derepression (13,14), both of which act synergistically to confer maximum expression on the promote
Foulkes J. Gordon
Leichtfried Franz E.
Pieler Christian
Stephenson John R.
Cooper & Dunham LLP
McKelvey Terry
OSI Pharmaceuticals, Inc.
White John P.
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