Recombinant DNA, process for the production thereof and the use thereof

The present invention provides a recombinant DNA, wherein it contains DNA sequences coding for the N-terminal membrane-penetrating domain of the E-protein of the phage .phi.X 174 and DNA sequences coding for the C-terminal membrane-penetrating domain of the L-protein of the phage MS2 and the DNA sequences of both phages are connected by a DNA sequence coding for a hydrophilic flexible amino acid sequence. The present invention also provides a process for the production of this recombinant DNA. Furthermore, the present invention provides for the use of the recombinant DNA and of a plasmid containing it for obtaining eukaryotic and prokaryotic metabolic products and gene-technologically produced proteins.

The present invention is concerned with recombinant DNA, processes for the 
preparation thereof and the use thereof for obtaining eukaryotic and 
prokaryotic metabolic products and gene-technologically produced proteins. 
The importance of the gene-technological production of metabolic products 
and proteins has increased continuously in recent years. Thus, in this 
way, it is possible to produce on a large scale substances which hitherto 
could only be isolated very laboriously and in small amounts and which 
were consequently expensive. For example interferons, interleukins, 
insulin and the like, making their use in medicaments, e.g. possible. 
However, after the production of such substances in the cell line 
employed, the isolation thereof is, in some cases, very laborious since 
the cells must first be broken up in order to liberate the substance 
formed, whereafter purification from the medium is necessary in which all 
cell fragments and components must be separated off. This breaking up of 
the cells, which is called lysis, can be carried out in several ways. 
Thus, it is possible to use a lytic enzyme, for example lysozyme, or the 
cells can be digested by the application of osmotic pressure or with the 
help of ultrasonics or other biochemical or physical methods. 
However, specific lysis of particular membranes is desirable as is 
secretion of the substances through membranes made partly permeable 
thereto. Continuous production of substances is made possible thereby. 
For obtaining gene-technologically produced products by secretion or by 
lysis of the cells, it would, be advantageous to have available proteins 
which are as lytically effective as possible and which, also, display 
specificity for certain membranes in order to make specific cell membranes 
permeable for the appropriate product. It would also be favourable not to 
have to supply such lysing proteins from outside of the cell but rather to 
be able to form the proteins in the cell at a desired point of time. 
It is, therefore, an object of the present invention to provide a 
recombinant DNA coding for a protein which satisfies these requirements, 
the use of which simplifies the obtaining of gene-technologically produced 
products. 
Thus, according to the present invention, there is provided a recombinant 
DNA which contains DNA sequences coding for the N-terminal, 
membrane-penetrating domain of the E-protein of the phage .phi.X1714 and 
DNA sequences coding for the C-terminal, membrane-penetrating domain of 
the L-protein of the phage MS2 wherein the DNA sequences of both phages 
are connected by a DNA sequence coding for a hydrophilic, flexible amino 
acid sequence. 
The single-stranded DNA phage .phi.X174 and the single-stranded RNA phage 
MS2 each contain a phage-coding, lytic protein, namely the E-protein 
(.phi.X174) and the L-protein (MS2). These two lytic proteins are 
relatively small and their total amino acid sequences are known. These 
amino acid sequences are shown in FIG. 2 of the accompanying drawings. It 
can be seen therefrom that the E-protein has a length of 91 amino acids 
and the L-protein a length of 75 amino acids. However, both 
lytically-active proteins do not contain any enzymatic activity. 
Therefore, it is to be assumed that they bring about the lysis of the 
membranes by destruction of the membrane structure in combination with 
autolytic processes. The lytic activity for the E-protein was ascribed by 
Blasi and Lubitz (J. Gen. Virol., 66, 1209-1213/1985) and Schuller et al. 
(Nucl. Acids Res., 13, 4143-4153/1985) to the membrane-penetrating 
N-terminal region. In addition, for the functionality, the presence of an 
oligomerizing structure in the C-terminal region of the protein appears to 
be necessary, as was ascertained by Maratea et al. (Gen., 40, 39- 64/1985) 
and Buckley and Hayashi (Mol. Gen. Genet., 204, 120-125/1986). From 
protein structure predictions obtained from computer calculations for the 
L-protein, a counter functional orientation is assumed. Consequently, the 
domain which brings about the lytic activity is limited to the C-terminal 
region of the L-protein (Berkhout et al., Gen., 37, 171-179/1985). 
The recombinant DNA according to the present invention makes it possible to 
produce a fusion protein from the N-terminal region of the protein E, 
which alone is not lytically active, and of the C-terminal, 
membrane-penetrating part of the L-protein, which is also alone not 
lytically active, but which, as a fusion protein in which the two phage 
DNA sequences are connected by a hydrophilic, flexible amino acid 
sequence, displays significantly greater lytic activity than either of the 
starting proteins themselves. 
A preferred recombinant DNA contains the DNA sequence which codes for the 
amino acids 1 to 54 or the protein E of the phage .phi.X174 and the DNA 
sequence which codes for amino acids 21 to 75 of the protein L of the 
phage MS2, these DNA sequences being connected by a further DNA sequence 
which codes for 5 linker amino acids. 
An especially preferred recombinant DNA contains DNA sequence which codes 
for an amino acid chain is illustrated in FIG. 1 of the accompanying 
drawings. 
Recombinant DNA as described can be incorporated into a vector, the vector 
preferably being a plasmid or a phage genome. It is preferred that a 
promoter which controls the expression of the recombinant DNA be 
positioned before the DNA sequence. As promoters, prokaryotic or 
eukaryotic promoters and especially regulatable promoters are preferred, 
especially a lambda promoter, the lac promoter and the Ga110 promoter. 
The present invention also provides a recombinant DNA which, before the 
phage sequences, additionally contains a DNA sequence coding for a signal 
sequence. Due to the presence of a signal sequence in the expressed 
protein, it is possible to transfer a specificity for particular membranes 
to the expressed protein. 
The present invention also provides the plasmid pRM17, DSM 4092P, which 
contains recombinant DNA coding for an amino acid sequence as is 
illustrated in FIG. 1 of the accompanying drawings. 
Furthermore, the present invention provides a process for the production of 
the recombinant DNA according to the present invention wherein, according 
to known methods, the corresponding DNA sequences are isolated from the 
double-stranded DNA form of the phage .phi.X174 and from the DNA copy of 
the phage MS2 RNA and ligated with one another in the desired sequence. 
The recombinant DNA produced in this way can then be inserted into a 
vector, the vector preferably being a plasmid or phage genome. It is 
preferred to insert into the vector, a eukaryotic or prokaryotic promoter 
before the phage DNA sequence. As especially preferred embodiment uses a 
vector which already contains a promoter under the control of which a 
foreign gene is expressed. 
It is preferred to insert a DNA sequence coding for a signal sequence 
before the phage DNA sequence. 
The plasmid pRM17, DSM 4092P, can be produced by inserting the 
oligonucleotide according to FIG. 4 of the accompanying drawings into the 
plasmid pSU729 (Nucl. Acids Res., 13, 4145-4153/1985), cleaved with 
XbaI/Hind III, the XbaI position of the plasmid pSU729 thereby being 
previously filled with DNA polymerase I (Klenow fragment). 
The present invention is also concerned with the use of a recombinant DNA 
or of the plasmid pRM17, DSM 4092P, for obtaining eukaryotic and 
prokaryotic metabolic products and gene-technologically produced proteins 
by partial or complete lysis of membranes or of the cell walls of the 
cells or micro-organisms used for the expression of the lytic protein 
coded by the recombinant DNA. 
The expression of the lytic protein is thereby preferably inducably 
controlled by a regulatable promoter at a desired point of time. This 
makes it possible first to allow cells to grow to a particular growth 
phase and to carry out the production of the desired metabolic product or 
of the gene-technologically produced product in the most effective way 
possible before the cell wall is lysed partly or completely by induction 
of the expression of the lytic protein. Following lysis, the desired 
products thereby passing into the medium from which they can be isolated 
without problems. 
As promoters, all inducable (regulatable) promoters can be used. Examples 
of these include the promoter .lambda..sub.PL and .lambda..sub.PR with cI 
857 as repressor (Gene, 5, 59/1979) and the promoter-operator region Lac 
PO with Lac I.sup.q or Lac I.sup.ql as repressor (Mol. Gen. Genet., 185, 
493-497/1982). 
Furthermore, due to the presence of a signal sequence in the lytic protein, 
the specific lysis of particular membranes can be achieved. In particular, 
the lysis of organelle membranes can be brought about, such as the 
membranes, of lysosomes, mitochondria and chloroplasts. A known signal 
sequence which is specific for a particular membrane is attached before 
the N-terminal part of the lytic fusion protein and brings about 
incorportion of the fusion protein into this membrane. 
Via partial lysis of particular specific membranes, the secretion of the 
metabolic products or proteins is achieved, continuous production thereby 
being made possible without needing to destroy the producing cells. The 
metabolic products produced can then be simply isolated from the growth 
medium, laborious separation of the cell components no longer being 
necessary.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
The following Examples are given for the purpose of illustrating the 
present invention: 
EXAMPLE 1 
Production of pRM 17 
The chimeric gene E-L of the plasmid pRM 17 is obtained by the insertion of 
the oligonucleotide sequence (gene L partial sequence, FIG. 4), which 
corresponds to the amino acid codon 21-75 of the gene L of the phage MS2 
(Beremand and Blumenthal, Cell, 18, 257-266/1979), into the filled XbaI 
position of the plasmid pSU729. 5'G of the sequence corresponds to 
nucleotide 1736 and 3'A to the nucleotide 1905 of the sequence of the 
bacteriophage MS2 (Fiers et al., Nature, 260, 500-507/1976). This plasmid 
(pSU729) was constructed by insertion of the PhiX174 partial sequence nt 
447-729 into the EcoRI/SmaI position of the plasmid pSU1 (Schuller et al., 
Nucl. Acids Res., 13, 4143-4153/1985). The orientation of the 
oligonucleotide sequence in pRM174 is 5'-3', corresponding to the 
orientation of the PHiX174 gene E partial sequence (codon 1-54) which is 
contained on this plasmid. The polylinker sequence between the gene E 
partial sequence and the gene L partial sequence corresponds to 
##STR1## 
EXAMPLE 2 
Production of pRM18 
Plasmid pRM18 is obtained by the insertion of the oligonucleotide sequence 
(gene L partial sequence, FIG. 4) into the filled BamHI position of the 
plasmid pPLcAT10 (Stanssens et al., Gene, 36, 211-223/1985). The 
orientation of the oligonucleotide in pRM18 is 5'-3', corresponding to the 
ribosome binding point and of the ATG start codon contained on the plasmid 
on the 5'-side of the BamHI position. 
Plasmid sequences which contribute to the shortened protein L and lie 
before the oligonucleotide sequence of the gene L partial fragment are 
##STR2## 
EXAMPLE 3 
Lysis of Escherichia coli Cells by Plasmid-coded E-, L- and E-L-protein 
The lysis of Escherichia coli cells by the lytically-active proteins E and 
L and the E-L-protein produced from recombinant DNA according to the 
present invention was investigated in the Escherichia coli strain K 12 PC 
2479, DSM 4089. Apart from the plasmid pRM17, DSM 4092P, the plasmid 
pSB12, DSM 4091P, which contains the .phi.X174 gene E (J. Gen. Microbiol., 
131, 1107-1114/1985), the plasmid pMS1.17, DSM 4092P, which contains the 
MS2 gene L (Nature, 305, 741-743/1983), the plasmid pSU730-1, DSM 4095P, 
which contains the E subunit of the recombinant gene according to the 
present invention (Nucl. Acids Res., 13, 4143-4153/1985) and the plasmid 
pRM18, DSM 4093P, which contains the L-DNA subunit of the recombinant E-L 
gene were used. The expression of the plasmid-coded genes was controlled 
via controlling lambda-P .sub.L promoter (cI 857 as repressor, cf. Gene, 
5, 59/1979). In order to induce the expression of the plasmids, the growth 
temperature of the exponentially growing cultures was increased from 
28.degree. C. to 42.degree. C. The growth of the cultures was monitored by 
spectrophotometry at 580 nm. The result of this experiment is illustrated 
in FIG. 3. The time point 0 minutes thereby indicates the increasing of 
the temperature from 28.degree. C. to 42.degree. C.