Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-01-14
2003-01-14
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S006120, C435S320100, C435S252300, C435S252800, C435S252100
Reexamination Certificate
active
06506590
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the construction and use of expression plasmids in the manufacture of recombinant interleukin-4 (IL-4) and interleukin-4 muteins.
BACKGROUND OF THE INVENTION
Mature human interleukin-4 (IL-4) is composed of 129 amino acids with 50% homology to mouse IL-4. IL-4 is the only cytokine known to direct the differentiation of T helper cells to a TH
H2
phenotype (Mosmann and Sad, Immunol. Today 17, 138-146, 1996). IL-4 signals on lymphocytes and other cells through a heterodimeric complex of two cytokine receptors, the IL4R&agr; and the common &ggr;-chain (&ggr;c). Antagonistic IL-4 mutants have been described (Kruse et al., EMBO J. 11, 3237-3244, 1992). Three amino acids close to the C-terminus (R121, Y124 and S125) are important for binding to the &ggr;c-chain. The introduction of Asp (D) into these positions blocks receptor dimerization and transmembrane signaling.
The interleukin-4 double mutein (IL-4 DM) is an IL-4 variant with 2 amino acid changes in position 121 and 124 termed IL-4 R121D Y124D. IL-4 DM is able to block both IL-4 and IL-13 activities. In contrast to all single site mutants no residual agonistic activity has ever been found for this mutein. It is believed that these antagonistic properties of IL-4 DM are useful for the treatment of diseases which involve T
H2
development and/or IgE production (Ryan, Allergy Clin. Immunol. 99, 1-5, 1997).
As described in various publications, procaryotic organisms can be used to produce recombinant IL-4 and IL-4 muteins. Unfortunately, the described systems have a number of drawbacks (low expression level, low stability of the expression vector) which make large scale production of IL-4 and IL-4 muteins impossible or economically unfeasible.
The main criteria for an efficient and safe expression system are:
high product yield
regulatable stable expression
stability of the expression vector
Several features of an expression plasmid are important for the criteria listed above (Hannig et al., TIBTECH. 16, 54-60, 1998). These are:
Promoter
Ribosomal binding site (rbs)
Codon usage of the corresponding gene
Transcriptional terminator
Resistance gene
Regulation of expression
Origin of replication (ori)
SUMMARY OF THE INVENTION
Expression plasmids for IL-4 and IL-4 muteins with modifications in all of the relevant elements for an efficient and safe expression system were generated. The quality and suitability of the corresponding expression system was ranked mainly according to the following criteria:
Yield of IL-4 and IL-4 muteins
Plasmid stability
Maintenance of induction capability
The object of the present invention is, therefore, to make available a process for the construction and use of expression plasmids in the large scale manufacture of recombinant interleukin-4 (IL-4) and interleukin-4 muteins. In addition the newly developed host/vector system should be well suited for the expression of other proteins (cytokines, growth factors, soluble receptors, antibodies etc.).
Surprisingly, it has been found that bacteria transformed with plasmids according to the present invention give expression rates, plasmid and expression stability values many times higher than those observed after transforming the identical hosts with plasmids known in the art. Therefore, the plasmids of this invention are far more useful for the preparation of recombinant interleukin-4 and interleukin-4 muteins than all plasmids previously known.
The newly developed vector system contains the following elements:
T5 Promoter
The
E. coli
phage T5 promoter together with two lac operator sequences is derived from the pQE30 plasmid (Qiagen) belonging to the pDS family of plasmids (Bujard et al., Methods Enzymol. 155, 416-433, 1987 and Stüber et al., Immunological Methods, I. Lefkovits and B. Pernis, eds., Academic Press, Inc., Vol. IV, 121-152, 1990).
T7 g10 Ribosomal Binding Site
The ribosomal binding site (rbs) is derived from the region upstream from gene 10 of the phage T7 (T7 g 10 leader). Gene 10 of phage T7 codes for the coat protein, which is the major protein expressed after T7 infection. The T7 g10 rbs was obtained from the vector pET-9a (Studier et al., Methods Enzymol. 185, 60-89, 1990). The T7 g10 leader spans a region of about 100 bp (Olins et al., Gene 227-235, 1988). In the final expression construct the region upstream of the XbaI site is deleted. The T7 g10 leader sequence now spans 42 bp and harbours one base exchange from G to A in position 3638 of the preferred plasmid.
Codon Usage of the Natural IL-4 Sequence
As an effective measure of synonymous codon usage bias, the codon adaptation index (CAI) can be useful for predicting the level of expression of a given gene (Sharp et al., Nucleic Acids Res. 15, 1281-1295, 1987 and Apeler et al., Eur. J. Biochem. 247, 890-895, 1997). The CAI is calculated as the geometric mean of relative synonymous codon usage (RSCU) values corresponding to each of the codons used in a gene, divided by the maximum possible CAI for a gene of the same amino acid composition. RSCU values for each codon are calculated from very highly expressed genes of a particular organism, e.g.
E. coli
, and represent the observed frequency of a codon divided by the frequency expected under the assumption of equal usage of the synonymous codons for an amino acid. Highly expressed genes, e.g. genes encoding ribosomal proteins, have generally high CAI values≧0.46. Poorly expressed genes like lad and trpR in
E. coli
have low CAI values≦0.3.
The calculated
E. coli
CAI value for the natural IL-4 sequence is 0.733. This means that the natural gene should be well-suited for high level expression in
E. coli.
Nevetheless a synthetic gene with optimal
E. coli
codon usage (CAI value=1) has the potential to further increase the expression level. Therefore synthetic IL-4 and IL-4 mutein genes were designed and cloned.
Transcriptional Terminator
A T7 DNA fragment containing the transcription terminator T&phgr; is derived from the vector pET-9a (Studier et al., Methods Enzymol. 185, 60-89, 1990). Transcriptional terminators determine the points where the mRNA-RNA polymerase-DNA complex dissociates, thereby ending transcription. The presence of a transcriptional terminator at the end of a highly expressed gene has several advantages: they minimize sequestering of RNA polymerase that might be engaged in unnecessary transcription, they restrict the mRNA length to the minimal, thus limiting energy expense, as strong transcription may interfere with the origin of replication, a transcriptional terminator increases plasmid stability due to copy number maintenance (Balbas and Bolivar, Methods Enzymol. 185, 14-37, 1990).
Resistance Gene
The kan resistance gene is derived from the vector pET-9a (Studier et al., Methods Enzymol. 185, 60-89, 1990). Originally, this is the kan gene of Tn903 from the vector pUC4KISS (Barany, Gene 37, 111-123, 1985). In the preferred plasmid the kan gene and the IL-4 and IL-4 mutein gene have opposite orientations, so there should not be an increase in kan gene product after induction due to read-through transcription from the T5 promoter. Kanamycin was chosen as selective marker because it is the preferred antibiotic for GMP-purposes. In addition, kan gene based vectors are more stable than ampicillin resistant (bla) plasmids. Ampicillin selection tends to be lost in cultures as the drug is degraded by the secreted &bgr;-lactamase enzyme. The mode of bacterial resistance to kanamycin relies upon an aminogly-coside phosphotransferase that inactivates the antibiotic.
Regulation of Expression
Controlled gene expression is absolutely necessary for the set-up of a stable plasmid system, particularly if the protein of interest is deleterious to the host cell. The preferred plasmid uses a lac-based inducible system consisting of a lac repressor gene (lacI) and two synthetic lac operator sequences fused downstream to the
E. coli
phage T5 promoter. The lacI
q
promoter and the lacI structural gene were isolated from the vector pTrc99A (Amann et al., Gene 69, 301
Apeler Heiner
Wehlmann Hermann
Bayer Aktiengesellschaft
McGarry Sean
Pellegrino Susan M.
LandOfFree
Plasmids, their construction and their use in the... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Plasmids, their construction and their use in the..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Plasmids, their construction and their use in the... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3017048