Mutant E. coli strains, and their use for producing...

Details Mutant E. coli strains, and their use for producing... Mutant E. coli strains, and their use for producing...

2001-05-17

2003-10-14

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S199000, C435S252330

Reexamination Certificate

active

06632639

ABSTRACT:

The invention concerns certain mutant
E. coli
strains, and their use for performing processes for producing recombinant polypeptides.
Genomic study of higher organisms, micro-organisms, and viruses almost invariably requires, in addition to the cloning of their genes, large-scale production of their products (proteins), so as for example to obtain antibodies or to perform biochemical or crystallographic studies.
From the applications viewpoint, the utilization in the medical field of numerous human peptides and proteins also requires expression of corresponding genes in heterologous organisms.
Although expression systems have been established in various eukaryotic hosts (especially in yeasts, insects and primate cells), the most widely used host for these expression strategies remains the bacteria
Escherichia coli
(
E. coli
). The list of proteins of biotechnological or pharmacological interest that are produced in
E. coli
is extensive; classic examples include human insulin and human growth hormone.
The most well-known expression system in, prokaryotes was developed in the USA by the Studier and Richardson groups, during the 1980's (Tabor and Richardson, 1985; Studier and Moffat, 1986). It is based on exploiting the properties of T7 RNA polymerase (namely RNA polymerase encoded by the T7 bacteriophage). That enzyme, which can be expressed in
E. coli
cells without toxicity, recognizes a very specific promoter. Any gene of interest (target gene) may be transcribed very efficiently, upon placing it downstream of this promoter and introducing it into an
E. coli
cell expressing T7 polymerase.
Nevertheless, in terms of expression, the results remain uncertain. Some target genes may be duly overexpressed, whereas others are expressed only moderately or not at all.
Previous work by the inventors revealed that one of the principal causes of these setbacks resides in the specific instability of the m-RNA synthesized by T7 RNA polymerase, which causes a decrease in the number of polypeptides synthesized by messaging (Lopez et al., 1994; Iost and Dreyfus, 1994, 1995). This instability is the consequence of the high speed of elongation of T7 RNA polymerase (Makarova et al., 1995). Specifically, the elongation speed of T7 polymerase, in contrast to that of bacterial RNA polymerase, is much greater than the translation speed of m-RNA by ribosomes. Nascent m-RNA is therefore exposed over most of its length, and is therefore readily attacked by nucleases, and in
E. coli
especially by the E-type ribonuclease (or RNase E), whose amino acid sequence is described by Casaregola et al. (Casaregola et al., 1992, 1994).
RNase E is an essential enzyme of
E. coli
; it is involved both in the degradation of m-RNA as well as in the maturation of ribosomal RNA (r-RNA). Mutations in the catalytic region (that is, in the N-terminal portion of RNase E) affect these two functions at the same time, and slow down or even arrest the growth of
E. coli
(Cohen and McDowall, 1997).
On the other hand, deletions in the C-terminal portion of RNase E do not affect the viability of
E. coli
. Specifically, by researching revertants of mutations in a protein (MukB) necessary for the segregation of chromosomes after replication, Kido et al. obtained various viable mutations in the rne gene, coding RNase E in
E. coli
, which cause synthesis of an RNase E that is truncated in its C-terminal portion (Kido et al., 1996). These authors concluded from these experiments that the C-terminal portion of RNase E is not essential for viability of
E coli
. They moreover formed the hypothesis that suppression of the mukB mutations by truncating of the RNase E, reflects the fact that truncated RNase E is less effective than the wild-type enzyme for degrading mukB m-RNA. Thus stabilized, a stronger synthesis of the mutant MukB protein could be achieved, thereby correcting the phenotype associated with the mutation. However, this stabilization of the mukB messenger was not demonstrated, and other authors proposed an entirely different interpretation to explain the suppressive effect of the truncating of RNase E on mukB mutations (Cohen and McDowall, 1997). These authors postulate in particular a direct interaction between RNase E and MukB. The basis for that idea is the fact that RNase E has a very substantial similarity with eukaryotic myosin (Casaregola et al., 1992: McDowall et al., 1993), which suggests that aside from its own RNase activity, it could, like MukB, play a structural role.
The present invention arises from the demonstration by the inventors of the fact that the truncating of RNase E causes an overall stabilization of cellular m-RNA, considered as a whole, as well as of the majority of individual m-RNAs that were examined, without significantly impeding the maturation of the r-RNAs (Lopez et al., 1999).
In that regard, the effect of the deletion is very different from that of a mutation in the N-terminal region, such as the ams mutation (Ono and Kuwano, 1979), renamed rne1 (Babitzke and Kushner, 1991), which confers thermosensitive activity to RNase E. For example, at 37° C., this latter mutation causes a moderate increase in the lifespan of the m-RNAs (1.5 times each on average; the lifespan of the m-RNAs is here defined as the time during which they serve as a matrix for protein synthesis (Mudd et al., 1990a)), but it also causes a significant slowdown in maturation of the r-RNAs (estimated by the “Northern” method; see Lopez et al., 1994) and it retards the growth by a factor of 2. On the contrary, deletion of the C-terminal portion of RNase E, especially of amino acids 586 to 1061 of this latter, causes a more significant stabilization of the m-RNA (two times on average), without causing a slowdown in the maturation of the r-RNA and without retarding growth. Thus, in hindsight, it is likely that the lack of growth that was observed with N-terminal mutations of RNase E, is due solely to the inability of the cells to mature r-RNA.
In summary, deletions in the C-terminal portion of RNase E have no effect on the activity of the catalytic domain, judging from the rapid maturation of the r-RNA. That rapid maturation explains why the cells containing such a deletion are viable. On the other hand, the deletion stabilizes the m-RNA as a whole, perhaps because it inhibits the association of the RNase E with other enzymes within a multi-protein structure, the so-called “degradosome”, which might be necessary for degradation of the m-RNA (Carpousis et al., 1994; Miczack et al, 1996; Py et al., 1996; Kido et al., 1996; Cohen and McDowall, 1997). The important point from the perspective of the invention is that, by virtue of these deletions, it is possible to obtain
E. coli
strains having enhanced m-RNA stability, while preserving normal growth.
The inventors have also shown that the stabilization of m-RNA due to the deletion of the C-terminal portion of RNase E, is not uniform, but rather is more pronounced for less stable m-RNA. As is known, this is often the case for the m-RNA of “target” genes in expression systems. The contribution of this m-RNA to the overall protein synthesis is therefore enhanced by the presence of the deletion.
E. coli
strains comprising such a deletion therefore express recombinant exogenous polypeptides with sharply higher yields (in particular about 3 to 25 times higher) with respect to the expression yields of those recombinant polypeptides by
E. Coli
strains not comprising that mutation, especially when the expression of the said recombinant polypeptides is placed under the control of T7 RNA polymerase.
The present invention therefore has as an object to provide novel processes for producing recombinant proteins or polypeptides from
E. coli
, especially those of pharmaceutical or biological interest, at production yields substantially greater than those of the processes described up to now.
The present invention also has as an object to provide novel
E. coli
strains for practicing the above-mentioned processes, as well as methods for preparing such strains.
The present i

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