Expression vector for the production of dead proteins

Details Expression vector for the production of dead proteins Expression vector for the production of dead proteins

2000-09-29

2003-06-24

Guzo, David (Department: 1636)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C424S093100, C424S093600, C435S320100, C435S455000, C435S456000, C435S457000, C435S069100, C435S325000, C435S348000, C536S023100, C536S023200, C536S023500, C536S024100

Reexamination Certificate

active

06582691

ABSTRACT:

The present invention relates to an insect cell vector for the production of proteins from the DEAD protein family.
The modulation of the RNA structure plays an essential role in cellular processes, such as, for example, in pre-mRNA splicing, in RNA transport or in protein translation, as the cellular RNA is present in the cell in different secondary and tertiary structures and, in addition, a large number of RNA-binding proteins provides for further structuring of the RNA. Proteins from the family of the so-called DEAD box proteins, inter alia, are involved in these modulation processes. The members of this protein superfamily, which as a characteristic contain a number of homologous protein sequences, so-called “protein boxes”, are named after the highly conserved tetrapeptide Asp-Glu-Ala-Asp (residues 21-24 of SEQ ID NO: 23) in the single-letter code D-E-A-D, as a motif. This protein superfamily also includes a number of RNA and DNA helicases.
The characteristic protein sequences of the DEAD proteins are highly conserved in evolution. A schematic representation of the proteins from the DEAD superfamily and its subfamilies as in
FIG. 1
shows the similarity between the individual family members (see also Schmid, S. R. & Linder, P. (1992) Molecular Microbiology, 6, 283, No. 3; Fuller-Pace F. V. (1994) Trends in Cell Biology, 4, 271). It is recognized that the DEAD superfamily is divided into various subfamilies, which according to their sequence motif are called DEAH, DEXH or DEAH* subfamily. All family members have an ATP-binding and RNA-binding function and also an ATP hydrolysis and RNA helicase function.
EP-A-0778347 now describes a novel ATP- and nucleic acid-binding protein having putative helicase and ATPase properties, which is assigned to the DEAH subfamily. In addition to the properties mentioned, the RNA helicase described is also connected with the tolerance of certain cells to leflunomide and related compounds and is thus suitable for the production of cell lines which are helpful in cancer, inflammation and apoptosis research and also in the elucidation of mechanisms of action of pharmaceuticals. A further possibility of application of this helicase is the identification of already known substances with respect to possible pharmaceutical properties such as, for example, an anticarcinogenic or antiviral action in a test or assay system. Sufficient amounts of the protein, however, are necessary for the desired types of use of the RNA helicase.
Interestingly, it has not been possible to date to homologously or heterologously express proteins from the DEAD protein superfamily in adequate amounts functionally. In addition to the size of the proteins, many representatives of this family have a molecular mass of 100 kD and above, certain structural motifs appear to inhibit the expression in foreign organisms. In particular, it is suspected of the so-called RS domain, a region of between 50 and 200 amino acids in size, which exhibits a greatly increased number of argenine-serine repetitions (single-letter code RS), that it directly or indirectly complicates protein expression. A direct effect can be caused, for example, by incorrect phosphorylation of the serine residues in this region. Indirectly, overexpression of proteins with this domain can cause toxic effects in the cell, as specific protein-protein interactions are mediated via this protein domain. In the case of heterologous protein overexpression, the native interaction can thus be disturbed or inhibited via RS domains.
The family of RS proteins is a “subfamily” of proteins which is defined by the possession of the RS domain. These proteins are involved in the most different of processes of pre-mRNA splicing. RS domains can mediate protein-protein interactions, influence RNA binding, modulate RNA-RNA annealing and function as subcellular location signals. The relationship between the DEAD box and the RS proteins consists in the fact that both are involved in the modulation of RNA structure and function and therefore many proteins are to be assigned to the protein families.
The RS domain in human RNA helicase according to SEQ ID No. 7 is in the range from about 131 to about 253 and in particular in the range from about 175 to about 216 based on the amino acid position.
It was therefore the object of the present invention to make available a process which makes possible the production by genetic engineering of proteins from the DEAD protein superfamily in large amounts.
It has now surprisingly been found that, in contrast to expression in
E. coli
or yeast, expression in insect cells is possible in an advantageous manner.
One subject of the present invention is therefore an insect cell vector comprising a nucleic acid coding for a protein from the DEAD protein superfamily. The term “nucleic acid” is understood according to the present invention as meaning preferably single- or double-stranded DNA or RNA, in particular double-stranded DNA.
In a preferred embodiment, the coding nucleic acid at the 3′ end of the coding region additionally contains a native 3′-noncoding region, which in preferred embodiments is at least about 50, preferably about 50 to about 450, in particular about 50 to about 400, nucleotides long.
“Native” within the meaning of the present invention designates 3′-noncoding nucleic acid regions which originates from the same organism, preferably from the same gene as the coding nucleic acid. If, for example, the nucleic acid codes for a human RNA helicase according to EP-A-0778347, the 3′-noncoding region according to the preferred embodiment likewise originates from human cells, in particular from the gene coding for the designated RNA helicase. The 3′-noncoding region according to SEQ ID No. 10 is preferred.
It is known that the 3′-noncoding region of genes can bind various regulatory proteins or factors. Thus, for example, the so-called “Cleavage and Polyadenylation Specificity Factor” (CPSF) binds to the noncoding RNA sequence AAUAAA. The CPSF protein consists of a complex of subunits with molecular weights of 160, 100, 73 and 30 kD. A further RNA binding protein is the so-called “Cleavage Stimulation Factor” (CstF). This protein is a heterotrimer of three subunits of 77, 64 and 50 kD. In addition, there are further RNA binding proteins such as the so-called “Cleavage Factors” CF I and CF II and also a poly(A) polymerase. The poly(A) polymerase is a polypeptide with a molecular mass of 83 kD. The polymerase is involved both in the poly(A) tail synthesis and in its cleavage. The extension of the poly(A) tail is strongly stimulated by the so-called “poly(A) binding protein II” (PABII). Further information and literature references are found in Wahle, E. (1995) Biochemica at Biophysica Acta, 1261, 183. Thus in addition to the AAUAAA binding sequence, Wahle, E. (1995), for example, also describes further consensus motifs such as a GU-rich region having the proposed consensus sequence YGUGUUYY and U-rich elements (see also Proudfoot, N. (1991) Cell, 64, 671-674).
In a preferred embodiment, the present invention therefore relates to 3′-noncoding regions which contains a binding site for the CPSF protein, the CstF protein, the CF I protein, the CF II protein, the poly(A) polymerase and/or the poly(A)-binding protein II (PABII), such as, for example, an AATAAA binding site, ATTAAA binding site, a GT-rich element, in particular a YGTGTTYY element, and/or a T-rich element designated in the form of its cDNA form.
A protein from the DEAD protein superfamily is understood according to the present invention as meaning proteins which have conserved motifs, under which a conserved motif contains the amino acid sequence DEAD, DEAH or DEXH. The proteins preferably contain sequence motifs which are responsible for a nucleic acid-binding activity, a helicase activity and/or an ATPase activity. The proteins in particular contain an RNA helicase and ATPase activity. FIG.
1
and
FIG. 2
shows examples of the conserved motifs for the DEAD protein superfamily and the DEAH,

Affiliated with

Also associated with

Rating

Expression vector for the production of dead proteins does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Expression vector for the production of dead proteins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Expression vector for the production of dead proteins will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3154022