Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
2001-10-03
2004-10-19
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S320100, C536S024100
Reexamination Certificate
active
06806066
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to improved expression vectors having a ColE1 origin of replication system, for the production of recombinant proteins and plasmid DNA.
2. Related Art
The use of fermentation processes with genetically modified microorganisms (GMO) for the production of recombinant proteins of interest or for producing plasmid DNA has become widespread in industry.
When optimizing a fermentation process the major goal is to obtain as much product as possible, with good quality, in a cost-effective way. To achieve this, the volumetric productivity, defined as units of product formed per volume and time, needs to be optimized. Factors with great impact on the optimization process are the biomass per volume, i.e., the amount of cells capable of producing the product, and the quantity of protein each cell can produce. To a certain limit, the production capacity per cell is proportional to the plasmid copy number (PCN), the number of plasmids in the cell carrying the gene coding for the recombinant protein. Furthermore, the strength of the transcription system for the recombinant protein is important. While some promoters are weak and do not take full advantage of the metabolic potential, many promoters are too strong and lead to overexpression of the recombinant protein. Since the metabolic resources have to be shared between the expression of the recombinant protein and the host protein, an expression system which is too strong, will soon lead to a depletion of the metabolic resources, which results in cell death.
Recently, the use of plasmid DNA in the field of gene therapy has become the focus of a whole new industry. Therefore, sufficient amounts of high quality plasmid DNA are required. In plasmid production processes, no recombinant protein is produced; instead, the cell factory is exploited for plasmid DNA production. Extremely high plasmid replication rates are necessary in order to achieve this goal, whereby the host cell has to accomplish tasks that differ from recombinant protein production.
For bacterial fermentation processes, ColE1 plasmids have been suggested mainly because high plasmid copy numbers can be obtained using this system.
ColE1 plasmids have been extensively described previously (Chan, P. T., et al.,
J. Biol. Chem.
260:8925-35 (1985)), and the replication mechanism of ColE1 origin of replication has been well studied (Cesareni, G., et al.,
Trends Genet.
7:230-5 (1991)). Replication from a ColE1 plasmid starts with the transcription of the preprimer RNAII, 555 bp upstream of the replication origin by the host's RNA polymerase (Tomizawa, J.,
Cell
40:527-535 (1985)). RNAII folds into specific structures during elongation and after polymerization of about 550 nucleotides begins to form a hybrid with the template DNA. The preprimer transcription terminates heterogeneously and after hybrid formation the RNAII preprimer is cleaved by RNase H to form the active primer with a free 3′ OH terminus, which is accessible for DNA polymerase I (Tomizawa, J.,
J. Mol. Biol.
212:683-694 (1990); Lin-Chao, S. and Cohen, S.,
Cell
65:1233-1242 (1991); Merlin, A. and Polisky, B.,
J. Mol. Biol.
248:211-219 (1995)).
The ColE1 region contains two promoters. RNAI is an antisense RNA molecule of 108 nucleotides, which is transcribed from the second promoter on the opposite strand and is complementary to the 5′ end of RNAII. RNAI is transcribed from 445 bp upstream from the replication origin, to about where the transcription of RNAII starts (Merlin, A. and Polisky, B.,
J. Mol. Biol.
248:211-219 (1995); Tomizawa, J.,
J. Mol. Biol.
212:683-694 (1990)).
For regulation of plasmid copy number in ColE1 plasmids, the kinetics is more important than the equilibrium features. For example, some mutant strains with mutations in the RNAII, although not influencing the regions complementary to RNAI, result in decreased inhibition by RNAI. This is probably due to affecting the half-life of intermediate RNA structures, decreasing the time for RNAI susceptibility, and hence resulting in increased plasmid copy numbers. This finding suggests the importance of intermediate RNAII structures and kinetics of RNAII folding pathway (Gultyaev, A., et al,
Nucleic Acids Res.
23:3718-25 (1995)).
It has been observed that starvation of amino acids results in large amounts of tRNAs that are not charged with the specific amino acid. (In the following, these tRNAs are termed “uncharged tRNAs”.) This phenomenon can be compared with the situation at the time after induction of recombinant protein expression, when the metabolic resources are depleted, as discussed above.
Wróbel, B. and Wegrzyn, G.,
Plasmid
39:48-62 (1998) tested a strategy of selectively inducing starvation of five different amino acids. It was found that there is a positive correlation between the homology of the anticodon loops of the tRNAs corresponding to the particular deprived amino acids, and particular loops in RNAI and RNAII. It was assumed that most of the charged tRNAs are captured by the translation mechanism, but that the uncharged tRNAs could instead have a chance to interact with other molecules, the RNAI and RNAII. Interaction between tRNA and RNAI or RNAII would most probably interfere with the interaction between RNAI and RNAII, resulting in a higher RNAII-DNA hybridization frequency (supposed that the tRNA interacting with RNAII does not change the RNAII structure in any drastic way). The latter would imply a higher replication frequency and thus a higher PCN.
Zavachev, L. and Ivanov, I.,
J. Theor. Biol.
131:235-241 (1988) compared the homology between all 21 tRNAs and RNAI/RNAII. Of these, 11 showed a homology greater than 40% to either RNAI or RNAII. They divided these into three categories: a) tRNAs homologous to RNAI: Arg, His, Leu, Lys, Phe and Thr, b) tRNAs homologous to RNAII: f-Met, Try and Gly and c) tRNAs homologous to both RNAI and RNAII: Met and Val. All tRNAs have anticodon loops of 7 nucleotides (Hjalt, T., et al.,
Nucl. Acids Res.
24:6723-32 (1992)). In the case with tRNAs homologous to RNAI, the highest homology was found in the region of loop 2, while most showed less homology in the 5′ end of RNAI.
Starvation and cellular stress lead to increased pools of uncharged tRNAs, which interact with the origin of replication of ColE1 plasmids. This interaction occurs due to the tRNAs' sequence homology to three RNA-loop structures, present in RNAI and RNAII of the origin of replication, which leads to interference with the PCN control mechanism of the system. Thus, PCN increases rapidly and causes a breakdown of the fermentation process.
To overcome these problems, International Appl. No. WO 89/07141 suggests an expression vector having a ColE1 replication system, comprising a mutation in the RNAII gene and/or the rop gene with the goal to increase expression. This was achieved without substantially increasing plasmid copy number.
Since a bacterial fermentation process is only efficient when the system can be maintained over an extended period of time and since an increased plasmid copy number is one of the main factors that cause collapse of the expression system, it was an object of the invention to provide an improved expression system with a prolonged bacterial viability during fermentation.
SUMMARY OF THE INVENTION
The present invention relates to an expression vector having a ColE1 replication system, in which the homology of the RNAI and RNAII of the ColE1 origin of replication to uncharged tRNAs is modified by one or more mutations in the coding region of the RNAI gene and one or more corresponding mutations in the RNAII gene, said mutation(s) resulting in one or more base exchanges in loop 1 and/or loop 2 and/or loop 3 of RNAI and RNAII.
REFERENCES:
patent: WO 89/07141 (1989-08-01), None
Dooley et al. Isolation and characterization of mutants affecting functional domains of ColE1 RNAI. J Mol Biol. Nov. 5, 1985;186(1):87-96.*
Cesareni, G. et al., “Control of ColE1 plasmid replication by antisen
Bayer Karl
Grabherr Reingard
Nilsson Erik
Striedner Gerald
Boehringer Ingelheim International GmbH
Falk Anne-Marie
Sterne Kessler Goldstein & Fox P.L.L.C.
Sullivan Daniel M.
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