Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1999-07-23
2001-10-16
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S192100, C435S320100, C435S325000, C435S235100, C435S069100, C435S069700
Reexamination Certificate
active
06303128
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an improved method for protein expression, particularly in bacteria such as
Escherichia coli
. In particular, this invention relates to an improved expression vector system and to the use of this system to obtain expression of foreign proteins or polypeptides in bacteria such as
E. coli
. By way of example, the improved expression vector system may be used to obtain expression of non-transforming variants of human papilloma virus (HPV) antigens as disclosed in detail in International Patent Application No. PCT/AU95/00868.
BACKGROUND OF THE INVENTION
In International Patent Application No. PCT/AU88/00164, it is disclosed that a fusion protein having a foreign protein or polypeptide component fused to the enzyme glutathione-S-transferase (GST), (E.C. 2.5.1.18), preferably to the carboxy-terminal of the enzyme, avoids several of the difficulties associated with known fusion proteins, for instance fusions wherein the foreign protein or polypeptide is expressed as a fusion with
E. coli
&bgr;-galactosidase, in that the GST fusion proteins are generally soluble and can be purified from bacterial lysates under non-denaturing conditions, for example by affinity chromatography on a column of immobilised glutathione. The GST enzyme in the fusion protein may be derived from the parasite helminth
Schistosoma japonicum
, or it may be derived from other species including humans and other mammals.
The GST fusion proteins disclosed in International Patent Application No. PCT/AU88/00164 may be used as such, since the foreign protein or polypeptide component thereof often retains its antigenicity and functional activity. Alternatively, the fusion protein may be cleaved to provide the foreign protein or polypeptide as a synthesis product, and when the production of such a synthetic protein or polypeptide is desired a cleavable link may be provided in the fusion protein between the glutathione-S-transferase component and the foreign protein or polypeptide component. The cleavable link is preferably one which can be cleaved by a site-specific protease such as thrombin, blood coagulation Factor Xa, or the like.
Expression vectors for use in the expression of such GST fusion proteins are now available commercially and are known as “pGEX vectors”. A pGEX vector is an expression vector, more particularly a bacterial plasmid for use in the production of a foreign protein or polypeptide, wherein the vector has inserted therein a nucleotide sequence capable of being expressed as the glutathione-S-transferase enzyme followed by at least one restriction endonuclease recognition site for insertion of a nucleotide sequence capable of being expressed as a foreign protein or polypeptide fused with the COOH-terminus of the glutathione-S-transferase enzyme, optionally with a sequence capable of being expressed as a cleavable link between the enzyme and the foreign protein or polypeptide.
Expression of the GST fusion proteins by the pGEX expression vectors is under the control of the tac promoter which enables inducible, high-level production of these fusion proteins. The pGEX expression vectors also contain the lac Iq gene, so that they can be used in any
E. coli
strain.
Polypeptides expressed in
E. coli
as fusions with GST have proven useful for the analysis of protein-DNA and protein-protein interactions. Part of the reason for this is that, in contrast to many other expression systems, the purification of GST fusion proteins involves non-denaturing conditions so that the expressed polypeptide is recovered in a relatively native state and retains at least some of its normal properties.
The present invention provides an improved expression vector system based on the pGEX expression vectors described above, which retains the high expression characteristics of this system but yields a protein product that is not a GST fusion. Thus, the system of this invention has an advantage over the existing pGEX technology in that a high yield of a “native” protein can be obtained without the necessity to cleave away the GST enzyme moiety of a GST fusion protein. This is especially useful where it is demonstrated that GST does not function as a purification aid.
SUMMARY OF THE INVENTION
In accordance with this invention, there is provided an expression vector having inserted therein a recombinant nucleotide sequence operatively linked to an expression control sequence, said recombinant nucleotide sequence comprising, in the 5′ to 3′ direction:
(i) a first nucleotide sequence which encodes a glutathione-S-transferase (GST) enzyme, said first nucleotide sequence including a termination codon in frame with the GST initiation codon to truncate the protein or polypeptide expressed by said first nucleotide sequence; and
(ii) a second nucleotide sequence comprising at least one restriction endonuclease recognition site for insertion of a further nucleotide sequence capable of being expressed as a desired protein or polypeptide.
In another aspect, the invention provides an expression vector as broadly described above, wherein the recombinant nucleotide sequence further comprises:
(iii) a third nucleotide sequence comprising a ribosome binding site (RBS) sequence, said third nucleotide sequence being located between said first nucleotide sequence and said restriction endonuclease recognition site.
In yet another aspect, the present invention provides an expression vector as broadly described above, wherein the recombinant nucleotide sequence further comprises:
(iv) a further nucleotide sequence capable of being expressed as a desired protein or polypeptide inserted into said restriction endonuclease recognition site.
In yet another aspect, there is provided a host cell, particularly a prokaryotic host cell such as
E. coli
, transformed with an expression vector as broadly described above.
The present invention also extends to a method for production of a desired protein or polypeptide, which comprises the step of culturing host cells as broadly described above under conditions such that the desired protein or polypeptide is expressed in recoverable quantity, and optionally the further step of recovering the desired protein or polypeptide from the cell culture.
The GST enzyme expressed by the first nucleotide sequence may be derived from
Schistosoma japonicum
, or it may be derived from other species including humans and other mammals.
The precise nature of the desired protein or polypeptide which may be expressed in accordance with this invention is not essential. Accordingly, the present invention extends to the production of any polypeptide or protein of interest as the desired protein or polypeptide. By way of example, this polypeptide or protein of interest may be a particular antigen to be used for diagnostic or therapeutic purposes in the human or veterinary fields.
The RBS sequence which is provided “upstream” of the restriction endonuclease recognition site(s) in the expression vector of this invention may be any known ribosome binding site. By way of example, the ribosome binding site sequence may comprise the AGGAG sequence, or the consensus sequence TAAGGAGG required for binding to 16
S
ribosomal RNA.
Preferably, the expression control sequences in the expression vectors of the present invention are the same as in the pGEX expression vectors.
DETAILED DESCRIPTION OF THE INVENTION
GST fusion proteins derived from pGEX expression vectors have been in widespread use since 1988. The pGEX expression system has proved to be a reliable way of producing certain proteins in quantity and the presence of GST in the fusion protein provides a means of purification using glutathione affinity chromatography. However, in a number of cases GST fusion proteins have proven to be difficult to purify using this method. This has led researchers to investigate alternative means of purifying these fusion proteins. As well, even in the case where purification on glutathione is practical, the protein product retains a GST moiety which in many cases is undesirable. In order to o
Edwards Stirling John
Webb Elizabeth Ann
CSL Limited
Foley & Lardner
Salimi Ali R.
LandOfFree
Method for protein expression does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for protein expression, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for protein expression will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2574449