Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1991-06-27
2001-09-04
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
Reexamination Certificate
active
06284484
ABSTRACT:
The present invention is concerned with a process for the expression of recombinant DNA which is under the control of an inducible promoter in a host cell.
By means of recombinant DNA technology, many problems in the production of proteins can be solved or considerably simplified. Briefly, the DNA sequence (gene) coding for the desired protein is integrated into an expression vector and expression of the protein is controlled via regulatory sequences, especially via promoters.
Especially preferred promotors, with which high yields of homologous and heterologous proteins can be obtained, are those which, as a rule, show both high and controllable activity. Such promotors include natural, hybrid and bacteriophage promotors, for example the lac, lacuv5, trp, tac, trc, rac, phoA, mgl, &lgr;-P
L
, &lgr;-P
R
, T
5
, T
7
and SP6 promoters, when the host cell is
Escherichia coli.
The activity of these promotors can be controlled, for example, by inducers (substrates, substrate analogues), by the temperature, or by the amount of available bacteriophage RNA polymerase. Using these, one can ensure, e.g., that heterologous protein is expressed only during particular periods of cultivation. For example, one can control expression so that it does not take place at the beginning of cultivation of the micro-organism used as host. Such control is especially advantageous when the heterologous protein is toxic for the host cell. When the expression of toxic heterologous proteins in hosts, such as
Escherichia coli,
is desired, it is usual to proceed in such a manner that, after a culturing phase in which the promotor is present in a repressed state, an appropriate inducer is added for the complete activation of the promotor. The promotor and thus the expression of the foreign protein is often induced in the late logarithmic to early stationary growth phase of the host cell after the achievement of an appropriate high cell density (biomass) (Mitzukamie et al., Biotech. Lett., 8, 611-614/1986). In the case of the use of the lac, lacuv5, tac, trc and rac promotor, as inducer there is added, for example, isopropyl-&bgr;-D-thiogalactopyranoside (IPTG) in a concentration of about 1 mM (Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1972). The expression of protein which is under lac repressor synthesis (lacI
q
) increases with increasing inducer concentration (IPTG: 0, 2 and 20 mM) (Luck et al., DNA, 5, 21-28/1986). The repression of the trp promotor by tryptophan is removed by removal of the co-repressor tryptophan (tryptophan-free medium) and/or by induction with 3,&bgr;-indolylacrylic acid (IAA) in a concentration of 20 to 50 mg/l. (Smith et al., Gene, 32, 321-327/1984; Mosteller et al., Symp. Quant. Biol., 35, 461-466/1970).
However, it has been shown that this process does not: lead for all homologous and heterologous proteins to satisfactory yields of proteins of the desired properties,, such as solubility, natural conformation and possibly biological activity. Whereas for some enzymatically-active proteins, for example &bgr;-galactosidase, &bgr;-lactamase, chloramphenicol acetyltransferase (CAT), creatinase and penicillin acylase, solubility and high enzyme activity are achieved after expression in
Escherichia coli,
with the use of the same conditions and of the same promotors, in the case of other proteins only small amounts of soluble proteins or no soluble protein at all or even biologically inactive product are obtained. Such effects are observed, for example, in the case of &agr;-glucosidase, prochymosin, the heavy and light chains of antibodies, immunoglobulins, urokinase, tPA, interferon, growth hormone, fusion proteins from &bgr;-galactosidase and HIV antigens and human insulin polypeptides (Marston, Biochem. J., 240, 1-12/1986).
However, it has been shown that these proteins are certainly present in large amounts in the appropriate cell extracts but are insoluble and/or display no biological activity.
As the cause for the low expression of soluble and especially of active protein in
Escherichia coli,
there has been discussed, for example, the denaturing of the foreign protein to give refractile bodies (inclusion bodies), i.e. to give insoluble protein aggregates, as well as proteolysis of the foreign gene product by the cell's own proteases (Carriers et al., Trends in Biotechnology, 1, 109-113/1983). In order here to provide some help, it has been proposed by Mitzukamie et al. (Biotech. Lett., 8, 605-610/1986), that for the expression of beta interferon (IFN-&bgr;) in
Escherichia coli
under the control of the trp promotor, to carry out the fermentation at 20° C. At this temperature, the yield of active IFN-&bgr; increases. However, it is a disadvantage of this process that fermentation times of at least 4 days are needed in order to obtain activities for which the working up is worthwhile.
Furthermore, it has been suggested to use host cells from which the proteolytic system responsible for the cleavage of the gene product is absent. Thus, for example, a lon mutant of
Escherichia coli
can be used as host cell. Such mutants are deficient with regard to a protease occurring in the wild type. Since, however, at least 7 further proteases are present in
Escherichia coli
(Swamy et al., J. Bacteriol., 149, 1027-1033/1982), the process is then only appropriate when the protein to be expressed is not cleaved by these other proteases. Furthermore, the choice of appropriate host cells is very limited.
Furthermore, it has been suggested to increase the expression by increasing the copy number of the expression vector to such an extent that the proteases are inundated by large amounts of protein formed and, consequently, only inactivate a comparatively small percentage of the protein. However, a disadvantage of this method is that the expression must be increased immensely within only 1 or 2 generations and cannot be carried out for a comparatively long period of time (Trends in Biotechnology, 1, 109-113/1983).
Therefore, it is an object of the present invention to avoid the above-described disadvantages of the prior art and to make possible the production of soluble and biologically-active proteins.
Thus, according to the present invention, there is provided a process for the expression in appropriate host cells of recombinant DNA which is under the control of an inducible promotor, wherein, for the production in soluble form of proteins which, under usual gene-technological production conditions, are obtained in insoluble form, the induction of the promotor is limited to less than 10% of the maximum induction in comparison with a standard system , which results a limited transcription rate of the DNA coding for the protein and a reduced rate of the synthesis of said protein.
By the limitation of the induction of the promotor, it is possible to limit the formation of refractile bodies and, instead, to produce soluble and active protein in large amounts. Without such a limitation of the speed of expression, complete induction admittedly leads to very rapid formation of protein. These proteins, however are present in the form of refractile bodies or as inactive proteins or protein fragments.
In the case of the process according to the present invention, the induction of the promotor is preferably limited to less than 5% and an especially preferred embodiment limits it to less than 1% of the maximum induction in comparison with a standard system.
As standard system, one may use a vector in which the foreign gene to be expressed has been replaced by another heterologous or homologous gene which, in this system (equal to the host cell), expresses soluble, active protein, in which case this “standard gene” must then, of course, be under the control of the same promotor as the foreign gene. Examples of standard gene includes &bgr;-lactamase, CAT and creatinase. As another standard system, the host cell itself (without vector) could also be used when it already contains, chromosomally integrated, a standard gene under comparable cont
Kopetzki Erhard
Schumacher Gunther
Fulbright & Jaworski LLP
Leffers, Jr. Gerald G.
McKelvey Terry
Roche Diagnostics GmbH
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