AU617999B2 - A genetic engineering process for the preparation of angiogenins - Google Patents
A genetic engineering process for the preparation of angiogenins Download PDFInfo
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- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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Description
COMMONWEALTH OF AUSTRALIA I 7 PATENTS ACT 1952-69 6 1 1 '9 COMPLETE SPECIFICAFICJN
(ORIGINAL)
Class mnt. Class Application Number: Lodged: e,:bmplete Specification Lodged: Accepted! Published: Priolity: Related Art Name o Applicant I HOECHST AKTI ENGESELLSCHAFT Adiiress of Applicant Actual Inventor: Address for Service Bruningstrasse, D-6230 Frankfurt/Main Federal Republic of Germany EDWD, WATERS SONSt 50 QUEt N STREET, MELBOURNE, AUSTRALIA, 3000.
Complete Specification for the invention entitled-, A GENETIC ENGINEERING PROCESS FOR THE PREPARATION OF
ANGIOGENINS
The following statement Is a full description of this invention, including the best method of performing It known to I. us i i n i C I i 3-1 i i I HOECHST AKTIENGESELLSCHAFT HOE 87/F 139 Dr.KL/fe Specification A genetic engineering process for the preparation of angiogen ins The observation that vascularization is a prerequisite for the growth of most solid tumors Led to the isolation and characterization of an angiogenesis factor caLLed angiogenin from the supernatant of cultivated HT29 adenocarcinoma cells (Fett et al., Biochemistry 24 (1985) 5480-5486; Strydom et al., Biochemistry 24 (1985) 5486-5494). Angiogenin comprises a non-glycosylated polypeptide chain with a molecular mass of 14,200 D and an isoelectric point 9.5. Based on protein sequencing and by use of specific probes it was possible to isolate an angiogenin cDNA and a genomic angiogenin clone (Kurachi et al., Biochemistry 24 (1985) 5494-5499). Angiogenin is 35% homologous with ribonuclease A (RNase with 3 of the 4 disulfide bridges of RNase A being retained in exactly the same positions, just as are the amino acid residues His-12, Lys-41 and His- S 119 which are essential for the catalytic activity of RNase A. Further studies subsequently showed that angiogenin also has RNase activity, but this is more specific than that of RNase A (Shapiro et al., Biochemistry 25 (1986) 3527-2532).
The oossible therapeutic uses of angiogenin are the elimination of local blood-flow disturbances, uses under consideration being for coronary blood-flow disturbances and for wound healing, especially of bone and ligament injuries and for skin transplantations. The second therapeutic principle comprises inhibition of angiogenesis, for example by antibodies against angiogenin. Given the homology of angiogenin with RNase A, it would also be possible to think of synthetic Low molecular weight inhibitors. This priniciple might be used in the diagnosis and therapy of solid tumors and their metastases (synergism with chemoi i I~ '-UL3 2 therapy), and for rheumatoid arthritis, Late sequelae of diabetes (retinopathy) and other pathological states.
However, in order to exploit a.L these therapeutic possibilities, it is first of all necessary to isolate biologically active angiogenin in an advantageous way and in amounts sufficient to permit further studies.
In the genetic engineering preparation of polypeptides in bacteria, the structural gene for the desired polypeptide is frequently coupled in the reading frame to the gene for the polypeptide p-gaLactosidase which is intrinsic to the bacterium. The bacterium then produces a fusion protein in which the desired poLypeptide is bonded via the amino terminus to the carboxyl terminus of the B-galactosidase.
S It is also known in this process not to make use of the gene for the complete 8-galactosidase (EP-A 0,001,930 and 0,012,494). However, only an extremely short fragment or else essentially the complete sequence of B-gaLactosidase have been used for this.
A process for the preparation of a genetically encodable polypeptide has already been proposed d uL a Pl ee I Fl with the structural gene for this poLypeptide being coupled in the correct reading frame via the gene for -galactosidase or a fragment of 6-gaLactosidase to a reguLator region, this gene structure being introduced into a bacterium, an insoluble fusion protein being expressed therein, the latter being isolated after a cell disruption, and che desired polypeptide being obtained by chemical or enzymatic cleavage, which process comprises codons for methionine and/or arginine and/or cysteine in the gene for B-galactosidass or for the fragment of B-galactosidase being wholly or partially replaced by codons for other amino acids.
It has now been found that angiogenin and its derivatives can be obtained particularly advantageously by this process.
35 Advantageous embodiments of the invention are explained in 1I 4 I *II 1 3 detail hereinafter tl v\en\on \o \rc.es sec.iC can\ofgenis dep.v tas as race, ba o."a process The figures illustrate the invention as follows: Fig. 1 shows constructions of shortened -galactosidase sequences without (A 1. and with (B 1. linkers.
Fig. 2 shows the construction of the plasmid pWZ RI (a pBR 322 derivative with B-galactosidase fragments from pUC 9 and pUR 270).
Fig. 3 shows the construction of the plasmid pLZA.
Fig. 4 shows the construction of the plasmid pLZP RI dMdC from pWZP dMdC.
Fig. 5 shows the construction of the plasmids pLZPWB1 dMdC and pLZPWB2 dMdC.
Fig. 6 shows diagrammatically the ligation reaction which results in a synthetic angiogenin, with R denoting purification, L denoting ligation, and the circular symbol denoting the 5'-phosphate group.
The figures are not true to scale.
In the first place, details of the process of Australia Patent Application 14469/89 for the preparation of the fusion proteins will be explained: The 3-galactosidase portion in the fusion proteins advantageously has more than 250 amino acids but significantly less than the complete -galactosidase sequence. A-Galactosidase fragments of about 300 to about 800, preferably about 320 to about 650, amino acids, which expediently contain an amino-terminal and/or carboxyl-terminal portion of B-galact:.sidase, have proved useful. This 0-galactosidase gene fragment is linked in the correct reading frame to a regulator region and, if necessary, via an adaptor,
@"V
1d\~ l .1 ,i I: 3o.
*a to the structural gene for angiogenin.
The terms used herein to describe the modified 0-galactosidase portion in the instant invention have .he same meaning and are described in Australian Patent Application No. 14469/88. The term amino terminal part sequence refers to a portion of the amino terminal B-galactosidase gene. Similarly "carboxy terminal part sequence" refers to a portion of the carboxy terminus of the p-galactosidase gene.
4 t v-o-t u a ene-, a.n, an- This adaptor, which can also be emitted where appropriate, advantageously codes for one or more amimio acids which are located in front of the amino terminus of angiogenin and allow it to be easily separated chemically or enzymatically from the B-galactosidase fragment. If, for example, the desired angiogenin derivative contains no methionine, or has been modified by genetic engineering in such a way that it contains no methionine, it is advantageous to choose methionine as the amino acid in front of the amino terminus of the desired polypeptide, after which it is possible to separate the desired polypeptide from the B-galactosidase portion by cleavage with cyanogen chloride or cyanogen bromide.
15 Th' process used according to the invention has the advantage that the fusion protein which is formed is insoluble and thus can easily be isolated after a cell disruption.
On the other hand, in the preferred embodiments, by reason of the relatively small B-galactosidase 20 portion, the fusion protein will accumulate in relatively Large aiounts in the bacterium. In addition, because of the portion of protein which is intrinsic to the bacterium, the fusion protein is not degraded to a noteworthy extent by the bacterium and thus correspondingly longer induction periods, and hence a higher yield, are possible.
Thus, an angiogenin can be obtained according to the invention by coupling the structural gene for this polypeptide in the correct reading frame, if necessary via an adaptor, with the gene for B-galactosidase, or a 8-gaLactosid'~se fragment of preferably more than 250 amino acids but significantly Less than the complete 8-galactosidase sequence, to a regulator region, introducing this gene structure into a bacterium, expressing an insoluble fusion protein therein, isolating the Latter after a cell disruption, and obtaining the desired polypeptide by chemical or enzymatic cLeavage.
-L -i L i i L 1 r The regulator region can be natural, in particular intrinsic to the bacterium, synthesized chemically or a hybrid region, for example the fusion promoter tac. The regulator regions additionally contain an operator, for example the lac operator, and a ribosome birding site 6 to 14 nucleotides in front of the methionine codon of the B-galactosidase fragments.
The 0-galactosidase fragment preferably comprises a fusion of an amino-terminal and carboxyl-terminal part-sequence.
This considerably reduces the B-galactosidase portion in the fusion protein. In addition, this const:ruction allows the replacement of various regulator systems without special effort. However, it is also possible to use exclusively amino-terminal or predominantly carboxyl-terminal '15 sequences of 6-galactosidase. It is possible to make use of natural restriction enzyme cleavage sites for these truncated B-galactosidase constructions. However, it is also possible to use constructions with chemically synthesized Linkers or adaptors which ensure a correct reading framefree of stop codons and contain, where appropriate, an ATG start codon. Figure 1 A shows B-galactosidase gene fragments in which use has been made of natural restriction enzyme cleavage sites, and Figure 1 B shows some in which a Linker is used.
In specific cases, further modifications, singly or in c'unbination, of the 8-galactosidase fragment may prove advantageous.
Purification of polypeptides which are to be searated from the B-galactosidase fragment by cleavage with cyanogen chloride or cyanogen bromide is faciLitated if targeted in vitro mutagenesis has been carried out to replace some, or advantageously all, of the codons for the interfering methionine residues by codons for other amino acids, preferably leucine or isoleucine.
Besides the desired polypeptide, cleavage of a fusion 1; :I i__li 1 1 6 protein which has been modified in this way results in a smaller number of B-galactosidase cleavage fragments, which can be chosen so that they can easily be separated from the desired polypeptide on the basis of their size and/or charge.
In the case of polypeptides which are to be separated from the B-ga.actosidase fragment by acidic cleavage of the peptide bond between aspartic acid and proline, it may be advantageous to modify the corresponding codons in the B-galactosidase gene fragment by targeted in vitro mutagenesis in such a way that acidic cleavage is no longer possible at these sites, preferably by converting the codon for aspartic acid into a codon for gLutamic acid.
In the case of polypeptides for which the formation of di- 15 sulfide bridges is important for their activity, it gene- S rally proves beneficial to convert, by targeted in vitro mutagenesis, the codons for cysteine which are present in the 8-galactos!dase fragment into codons for other amino acids, preferably serine, in order thus to avoid the forma tion of incorrect disulfidc! bridges between the 0-galactosidase fragment and the polypeptide.
The adaptor between the 0-galactosidase fragment and the structural gene for the desired polypeptide, which can be omitted in favorable cases, codes immediately in front of the amino terminus of the desired polypeptide for an amino acid or a sequence of amino acids which allow the desired polypeptide to be easily separated from the 8-galactosidase portion. As already mentioned, this amino acid can be methionine, which allows simple cyanogen bromide cLeavage, as long as the desired polypeptirt does not contain methionine or the corresponding codons have undergone genetic engineering modification. An example of an adaptor of this type has the following nu-leotide sequence: AAT TAT GAA TTC GCA ATG (Eco RI) TA CTT AAG CGT TAC 7 -7- The various modalities of cleavage off chemical or enzymatic can be additionally facilitated by steric separation of the desired polypeptide from the a-galactosidase portion. For this, the codons for a poly(amino acid) are inserted, via a special chemically synthesized adaptor, between the 0-galactosidase portion and the polypeptide.
In general, it is possible from the aspect of varying the structure of this poly(amino acid) arm to use as amino acids all genetically encodable amino acids, for example small uncharged amino acids such as glycine, alanine, serine or proline, or charged ones such as aspartic acid and glutamic acid on the one hand, or Lysine and arginine o**o on the other hand. The poly(amino acid) chain expediently comprises 5 to 30, preferably 10 to 24, in particular to 20, amino acids. It is possible, depending on the Schoice of the poly(amino acids), to achieve direct folding back of the desired gene product in conjuction with the B8-galactosidase portion.
The structural gene for this desired angiogenin can be |120 chenieatly synthesized in a manner known per se. Chemically synthesized genes harmonized to the specific codon usage in the host cell are advantageous, such as have been S described, for example, in German Offenlegungsschriften 3,327,007 (derivatives of growth hormone releasing factor), 3,328,793 (deriva'tive of secretin), 3,409,966 (human yinterferon), 3,414,831 (derivatives of human y-interferon), 3,419,995 (interleukin-2 and derivatives) and 3,429,430 (hirudin derivatives) or have been proposed in German Patent Application 3,632,037 (calcitonin).
For this purpose, a gene for an angiogenin which has several advantages is prepared by a chemical synthetic route. The preferences of E. coli are taken into account in the synthetic gene (Tab. A number of unique recognition sequences for restriction endonucleases is incorporated within the structural gene, and these provide access to part-sequences of angiogenin and facilitate the modification of the gene by mutations. In addition, the -8 codon for the only methionine which is cont' in natural angiogenin has been converted into a leucir eucine or valine codon, which permits, without loss of bioLogical activity, advantageous separation of the angiogenin from the -gaLactosidase fragment of the fusion protein by cleavage with cyanogen bromide.
The incorporation of the gene structure composed of regulator region, 0-galactosidase fragment, adaptor where appropriate, and structural gene into a suitable vector, the insertion of the hybrid vector obtained in this way into a suitable host cell, the cultivation of the host celLs, the cell disruption, the isolation and cleavage of the fusion protein, and the isoLation of the desired polypeptide are generally known. Reference may be made for this to the wideLy used textbooks and handbooks.
Preferred vectors are pLasmids, especially the pLasmids which are compatible with E. coLi, such as pOR 322, pBR 325, pUC 8 and pUC 9, as well as other commercially available or generally accessible plasmids. The preferred bao- P0 terial host is E. coLi.
The invention is explained in detail in the examples which foLlow.
Example 1 pg of the commercially available plasmid pUC 9 (cf.
Vieira et al, Gene 19 (1982) 259 268; The MoLecular BioLogy Catalogue, Pharmacia P-L BochemicaLs, 1984, Appendix, page 40) are subjected to double-digestion with the restriction endonucleases Eco RI and Pvu 1, and a DNA fragment 123 base pairs (Bp) in Length is separated out by gel electrophoresis. This fragment embraces part of the amino-terminaL coding sequence of a-gaLactosidase.
to isotate the carboxyL-terinaL portion of the O-gallCtd* sidase gene up to the natural Ecd RI cleavage site, a 9 of the plasmid pUR 270 (RUther and M'u er-Hill, EMBO J. 2 (1983) 1791-1794) are initiaLLy digested with Eco RI and then subjected to a r tial digestion with the enzyme Pvu I.
A DNA fragment of 2895 Bp is separated out and isolated by eLectrophoresis or a 5% poLyacrylamide geL.
The amino-terminal and carboxyL-terminal DNA fragm;ents of the p-galactosidase gene are ligated together at 16 0 C over the course of 6 hours, and the ligation product is precipitated with ethanol. The precipitated and resuspended DNA is cut with Eco RI and once again fractionated on a polyacrylamide geL. ine DNA fragment 3018 Bp in Length is isolated from the gel by electroelution and Ligated into the Eco RI cleavage site of the plasmid pBR 322. The hybrid plasmid obtained in this way is called pWZ RI.
The reaction steps descr oed above are depicted in Figure 2.
The individual measures were carried out in a known manner (Maniati et aL., Molecular Cloning, Cold Spring Harbor 1982).
The plasmid pWZ RI is transformed into E. coLi, ampLified there and reisolated. It is possible by digestion with Eco RI to cleave out the shortened B-gaLactosidase aminoand carboxyl-terminal gene ragment and to isoLate it preparatively. The known restriction enzyme cLeavage sites can be used for further shortened constructions, and these can be inserted into suitable expression plasmids. Figure I A shows shortened examples.
Figure 1 B shows constructions using a chemically synthesized Linker.
Both the constructions in Figure 1 A and those in Figure 1 B are chosen so that the reading frame for the shortened 0-gaLactosidase is continuous with the reading frame for the desired carboxyL-terminal polypeptide. The chemically synthesized linkers in Figure 1 B can have any desired structure but, of course, must guarantee a reading frame i i_ i 10 free of stop codons and, where appropriate, guarantee an ATG start codon, and have the desired restriction enzyme cleavage sites.
Example 2 An advantageous shortening and modification of the B-galactosidase gene fragment obtainable from pWZ RI by Eco RI cleavage can be obtained as follows: 4424 10 ,i 4 44 4 44 4 4f 4 i 15 pg of the plasmid pWZ RI (Fig. 2) are cut with the restriction enzymes Eco RI and Pvu I, and fractionation on a 7.5% polyacrylamide gel is car'ied out. DNA fragments 123 and 1222 Bp in length are isolated. Equimolar amounts of the two DNA fragments are then ligated together at 10 0 C for 6 hours, followed by digestion with Eco RI. The Ligation mixture obtained in this way is fractionated on a 7.5% polyacrylamide gel, ard the DNA band with a length of 1345 Bp is isolated preparatively. This DNA fragment is Ligated into the vector pBR 322 which has been opened with Eco RI and then dephosphor)lated. The plasm:d obtained in this way is called pWZP RI.
To remove the 8 codons for methionine (MI-M8) contained in pWZP RI and the 6 codons for cysteine (C1-C6) present in this plasmid, 1 pg of DNA from pWZP RI is cleaved with Eco RI. The fragment 1345 Bp in size is ligated into the phage vector M'3mpl9am (Patschinsky et al., J. Virol. 59 (1986) 341-353) which has been opened with Eco RI and then dephospn rylated. The phage obtained after transfection of E. coli JM101 is called MWZPam. As the first step in the removal of the codons for mtithionine,. a targeted in vitro mutagenesis is carried out by the gapped duplex method (Kramer et al., Nucl. Acids Res. 12 (1984) 9441-9456) with the 4 oligonucleotides dM5 dM8 (Tab. 2) being used, because of the high efficiency of this method (70% on average), as mutagenic primers. The ssDNA of 12 of the resulting phages is sequenced, with dM7 and dC5 (Tab. 2) being used as primers for the sequencing in addition to the normal 17mer 11 primer. 2 of the 12 DNAs have aLL 4 desired mutations, i.e.
the codons for M5-M8 have been changed into codons for isoleucine. These phages are called MWZP dM5,8. To remove the remaining methionine codons, the RF DNA of MWZP dM5,8 is cleaved with Eco RI, and the DNA fragment which is 1345 Bp in size is cloned into dephosphorylated M13mpl9am. The phage obtained in this way is called IWZP dM5,8am. The ssDNA of this phage is subjected to a further in vitro mutagenesis with dMl-dM4 as mutagenic primers. The ssDNA of 12 of the resulting phages is sequenced, aad 3 of the 12 phages have all the desired mutations, i.e. the codons for M1-M3 have been changed into codons for leucine, and the codon for M4 has been changed to a codon for isoleucine.
These phages are called MWZPdM. Cloning at the 1345 dp Eco RI fragment from the RF form of these phages into the de- S phosphorylated vector pBR 322 results in the plasmid pWZP dM. For the additional conversion of the codons for cysteine into codons for serine, the 1345 Bp Eco RI fragment from pWZP dM is isolated and cloned into the dephosphorylated phage vector M13mp19am. The ssDNA of the phage MWZP dMam obtained in this way is subjected to an in vitro mutagenesis with dCl-dC6 as mutagenic primers. The ssDNA from 24 of the resulting phages is sequenced, and 4 of the phage clones, which are called MWZP dMdC, proved to be correct, with all the codons for cysteine in them having been converted into codons for serine. The RF DNA of these phages is cleaved with Eco RI, and the 1345 Bp Eco RI fragment is cloned into the dephosphorylated vector pBR 322, resulting in the plasmid pWZP dMdC.
Example 3 The piasmid pLZA into which the shortened and/or modified versions of the B-galactosidase gene can advantageously be cloned can be obtained as foLLows (Fig. 3): pg of the plasmid pBR 322 are cut with the restriction endonucleases Eco RI and Pvu II, and fractionation on a 1% agarose gel is carried out. The DNA fragment 2293 Bp in 1- ~e I -12 size is purified and partiaLLy cleaved with Hae II, and electrophoresis on a 1 agarose gel is repeated. The DNA fragment 2017 Bp in size is isolated and Ligated at 12 0
C
overnight with the DNA fragment which is 225 Bp in size and is obtained after cleavage of pUR 2 (Rither, Mol. Gen.
Genet. 178 (1980) 475-477) with Eco RI and Hae II and which contains the promoter/operator of the Lac operon, a ribosome binding site and the first 5 codons of the lac Z gene (including the start codon). Transformation of competent E. coli cells results in the plasmid pLZA.
ExamF'e 4 Plasmids which are suitable, by reason of multiple cloning sites, for the expression of polypeptides as fusion proteins with shortened B-galactosidase are obtained as follows S. F"15 (Fig. 4): 1 pg of the plasmid pLZA (Fig. 3) is opened with Eco RI, dephosphorylated and ligated at 12 0 C overnight with the modified Eco RI fragment which is 13L4 Bp in size from pWZP dMdC {Example Transformation of competent E. coli cells and selection for expression of B-galactosidase and correct distribution of the cleavage sites for various restriction endonucleases results in the plasmid pLZP RI dMdC (Fig. 4).
It proves advantageous for the cloning of a multiple cloning site into the Eco RI site at the 3' end of the B-galactosidase gene to delete the Eco RI site at the 5' end by targeted in vitro mutagenesis. This entails the Pst I- Sac I fragment which is 1767 Bp in size being cloned into M13ep19am and subjected to an in vitro mutagenesis with the mutagenic primer 5'-GCCAGTGAATCCGTAATCATGG-3' by the gapped duplex method. Sequencing and restriction analysis shows that the DNA of 2 of 4 investigated phage clones lacks, as desired, the cleavage site for Eco RI.
13 The Pst I-Sac I fragment which is 1767 Bp in size from the RF DNA of these phage clones is isolated and ligated at 160C for 4 hours with the Pst I-Sac I fragment which is 1820 Bp in size from pLZP RI dMdC. Transformation of competent E. coli cells results in the plasmid pLZP dMdC (Fig. For incorporation of the multiple cloning site, 2 pg of pLZP dMdC are opened with Eco RI, dephosphorylated and ligated with the following chemically synthesized DNA sequence (MCS) which has a multiplicity of restriction enzyme cleavage sites: f U 1* tt 4 t t6 4 4 i. t, 4 4 (Eco RI) AA TTC
G
Pst I CTG CAG GAC GTC Sac I GAG CTC CTC GAG Sma I Bar HI GCC CGG GGA TCC CGG GCC CCT AGG Xba I TCT AGA AGA TCT Sal I GTC GAC CAG CTG
CCC
GGG
Hind III Nru I Ava III Bgl II AAG CTT CGC GAT GCA TCA GAT CTA TTC GAA GCG CTA CGT AGT CTA GAT Nco I Sph I (Eco RI-) CCA TGG CAT GCC GGT ACC GTA CGG TTA A In this way, and after transformation of competent E. coli cells, are obtained the plasmids pLZPWB1 dMdC and pLZPWB2 dMdC (Fig. 5) which differ from one another in the orientation of their MCS sequence.
Example A synthetic angiogenin gene (Tab. 1) which differs from the normal human angiogenin gene by, in particular, replacement of the codon for Met-30 by a codon for leucine, isoleucine or valine is cLoned into the vector pLZPWB2 dMdC, which is described above, as follows:
~I*
-14 The synthetic gene for angiogenin is composed of 12 oLigonucleotide building blocks (see Tab. 3).
The synthesis of the gene building blocks is illustrated by the example of gene structural unit la, which embraces nucleotides 1 49 of the coding strand. For the solidphase synthesis the nucleoside Located at the 3' end, which is adenosine (nucleotide No. 49) in the present case, is covalently bonded via the 3'-hydroxyl group to a carrier. The carrier material is CPG (controlled pore glass) functionalized with long-chain aminoalkyl radicals.
In the subsequent synthetic steps, the base component is used as t;he 5-cyanoethyl ester of the dialkylamide of the 5'-0-dimethoxytrityLnuc eoside-3'-phosphorous acid, with the adenine being in the form of the N -benzoyl compound, the cytosine being in the form of the N -benzoyl compound, the guanine being in the form of the N2-isobutyryl compound, and the thymine being without a protective group.
mg of the polymeric carrier which contains 0.2 pmol of
N
6 -benzoyladenosine are treated successively with the following agents: A) acetonitrile B) 3 trichloroacetic acid in dichLoromethane C) acetonitrile D) 5 pmol of the appropriate nucleoside 3'-O-phosphite and 25 pmol of tetrazole in 0.15 ml of anhydrous acetonitrile E) acetonitri e F) 20 acetic anhydride in tetrahydrofuran containing 40 ivtidine and 10 dimethylaminopyridine G) aceionitrile H) 3 iodine in lutidine/water/tetrahydrofuran in the ratio by volume 10 1 In this context, "phosphite" is to be understood to be the monc-B-cyanoethyL ester of the 2'-deoxyribose-3 t -monophosphoroqs acir, with the third vaLency being saturated by a diisopropylamino radicaL. The yields in the individual i~c. 1 .rll-* synthetic steps can be determined after each detritylation reaction B) by measurement of the absorption of the dimethoxytrityl cation at the wavelength of 496 nm in a spectrophotometer.
After the synthesis is complete, the dimethoxytrityl group is cleaved off as described in A) to The oligonucleotide is cleaved off the carrier by treatment with ammonia and, at the same time, the B-cyanoethyl groups are eliminated. Treatment of the oligomers with concentrated ammonia at 50 0 C for 16 hours quantitatively cleaves the amino protective groups off the bases. The crude product obtained in this way is purified by polyacrylamide gel electrophoresis.
For the phosphorylation of the oligonucleotides at the terminus, 1 nmol of each of the oLigonucleotides lb to 6a is treated with 5 nmol of adenosine triphosphate and four units of T4 polynucleotie kinase in 20 pL of 50 mM tris- HCL buffer (pH 10 mM magnesium chloride and 10 mM dithiothreitol (DTT) at 37 0 C for 30 minutes. The enzyme is inactivated by heating at 950C for 5 minutes. The oligonucleotides la and 6b, which form the "protruding" singlestranded sequences in DNA sequence I, are not phosphorylated. This prevents the formation, in the subsequent Ligation, of gene fragments larger than correspond to DNA sequence I.
S The oligonucleotides la to 6b (cf. Tab. 3) are Ligated to give the complete gene as follows (Fig. 1 nmol of each of oligonucLeotides la and lb to 6a and 6b are hybridized in pairs by dissolving each of them in 20 pl of 50 mM tris- HCI buffer (pH 10 mM magnesium chloride and 10 mM DTT, heating this solution at 95 0 C for 5 minutes, and cooling it to room temperature within 2 hours. Oligonucleotides lb to 6a are used in the form of their 5'-phosphates in this.
For the subsequent Linkage of the bihelical DNA fragments which are formed, the solutions thereof are ligated together to give the complete gene as shown in Fig. 6.
I~ II_~ -16 The gene is purified by gel electrophoresis on a 6 polyacrylamide geL (without addition of urea, 40 x 20 x 0.1 cm) using as marker substance eX174 DNA (from BRL) cut with Hinf I, or pBR 322 cut with Hae III.
The Sph I-Eco RI fragment obtained in this way is Ligated at 16 C overnight with the vector pLZPWB2 dMdC which has been cut with Eco RI and Sph I. Transformation of competent E. coli cells results in ampicillin-resistant colonies which are investigated for the expression of a fus\on protein of the expected order of size. 4 positive clor~es undergo analysis of the sequence of the angiogenin part, and the sequence of 2 of the 4 clones proves to be correct.
These plasmids are called pLZPWB2 dMdC Ang.
The genes for angiogenin Ile-30 and Val-30 are obtained in the same way by Ligation with the oligonucleotides 2c and 2d, and 2e and 2f, respectively (Tab. 4).
Example 6 The bacterial strains cultivated to the desired optical density are induced with IPTG as inducer for an adequate time, for example 2 hours. The cells are then killed with 0.1 cresol and 0.1 mM phenylmethylsul'fonyl fluoiide (benzylsulfonyl f'luoride). After centrifugation or filtration, the mass of cells is disrupted in an aqueous acid solution at pH 3.0 in a suitable apparatus (French press or ('Dyno-M'hle), and the insoluble constituents are then spun down. The proteins are isolated from the supernatant by standard methods (Grau et al., Angew. Chem. 98 (1986) 530). The enrichment and the purity of the products are checked by HPLC analysis.
To liberate the angiogenin from the fusion protein, the dried residue is dissolved in 70% strength formic acid Ond incubated with 20 percent by weight of cyanogen bromide (based on the dry mass used) at RT for 4 h. Precipitation with 5 volumes of methyl t-butyl ether results in a cyanogen bromide cleavage product from which highly enriched batches 17 of reduced angiogenin can be isolated by chromatography on suitable cation exchangers (for example S-Sepharose R at pH 8 with 6-8 M urea) under reducing conditions (for example with the addition of mercaptoethanol, dithiothreitoL etc.).
Classical methods for regenerating reduced proteins using suitable mixtures of oxidized and reduced glutathione (for example Karim Ahmed et al., J. Biol. Chem. 250 (1975) 8477- 8<,82) are used to fold the ang ogenin to give the natural tertiary structure with formation of the three disulfide bridges. Highly pure angiogenin is isolated from the folded product by gel filtration followed by 'on exchange chromatography.
The r rity of the final product is checked by reversed phase HPLC and SDS gel electrophoresis. The angiogenesis ooo 15 activity is detected in the CAM assay and in the rabbit So cornea assay (Fett et al., Biochemistry 24 (1985) 5480- So 5486).
o o0 0 00 0 0 O 0 TabLe I Sp hi HIS KET G LN C CAC ATG CAG GT ACG GTG TAC GTC Ode I GLU SER ILE LED ARG GAA TCG ATT GAIGA CTT AGC TAA GAC lT XhoI ASP ASH SER ARG GAC AACTC AG CTG TTG WAOC AccI TYR THR
*LACA
ATAJ TOT 10 HIS PHE LEII CAT TTC CTG GTA AAG GAC TUiR GLN ACC CAG TGG GTC HIS TYR ASP ALA LYS PZO CAC TAT GAC GCT AAA CCG GTG ATA CTG CGA TTT GGC Sau96l 20 GLN GLY ARG ASP GAG jGGC CGG GAC GTC CG]GCC CTG Pvul ASP ARG TYR CYS GAfi CGT TAG TGG tA GCA ATG ACG Spe I ARG ARG GLY LED THR SER CGC CGT COG TTA 4ATT GCG GCA CCC AAT Y5KAT-+ 40 PRO CYS LYS CCO TGG A GGCG TTT Fok I A SP I LE NcoI ASH TIIR PilE ILE HIS GLY AAG ACT TTC ATCICAT GGT TTG TGA MAG TAG GTAJcA HindITI LYS SER SER PilE 3LN VAL AAG TGA IkGC TTC CAG GTT TTG AGT TCG AW GOTC GAA 50 ASH LYS AAG AAO TTG TTG Nde I ARO SER ILE LYS ALA I LE COT TCT ATC AMA 0CC A GGA AGA TAO 'T COG TAT BamHll CYS LYS LED HIS GLY GLY TG AAM CTT CAT 000 GL-A.
AG TTT OAA OTA CCC OCT CYS OLU TG GILA AG CTT ASH LYS AAC AMA 'TCG TTT Bst EII ASH GLY ASH PRO HIS AAC 1T ARC CGG CAT TTO CCA'FTGI 00G CTA Hhal 70 ARO OLU ASH LED ARG ILE SER CGG GAA MAC CTO C* ATC AGG OCG CTT TTG GAG [FCG TAG TCG, 80 THR THR ACA ACT TOT TGA
SER
TCC
7A4 Fnu4HI PRO TRP PRO PRO CYS GLN CCG, TOG CIG CCA TG CG GOC ACC GqC GOT ACG OTC ,RsaI NaeI 100 ALA THR ALA GIJY PilE OCT ACT GGC JGOC 'TC CGA TGA COO CCO AAG ARO ASH VAL COT AAT OTT GA TTA CAA VAL VAL, OTO OTT GAG CAA ALA CYS OLD ASH OCT TOT GAA AAC COA ACA CTT TTG 110 XbaI MboII GLY LED PRO VAL HIS LEU ASP OOT CTG CGA GTC CAT CTA OA! CCA GAG GOT GAG OTA OAT I4jP 120 Stul CLH SER ILE PilE AEG ARC PRO GAG TCT ATC TTC COA AGGICCT GTC AGA TAG AAG OCT TGC OGA EcoRI TAA TAO ATT ATG TTA A 19 TabLe 2 Mutagenic primers for converting codons for rethionine into codons for Leucine or isoLeucine and for converting ccodonls for cysteine into codlons for serine; dlM1 AG ACC GTT CAG ACA GAA CTG G dM2 AG CGC CAC CA- CCA GTG CAG G dM3 GCA AAA ATC CAG TTC GCT GGT C dM4 C GCC AAT CCA TAT CTG TGA AA C GOT AAT CCC AAT TTG ACC AC dM6 51- A CGG QGT ATA GAT GTC TGA CA dM7 G GCT GGT TTC AAT CAG TTCG CT dM8 51- C ACC AAT CCC TAT ATO GAA ACC dCl G ACC GTT CAG AGA GAA CTG GCG dC2 CAG CTC GAT GGA AAA ATC CAG TTC dC3 ATC TGC CGT CCA CTG CAA CAA dC4 CGC CAG CTG GGA GTT CAG GC 51- GCG CTC AAA ACA GGC GGC ACT dC6 GC CGT CCC GOA GCG CAG ACC
I
labLe, 3 Sphl HIS HET GLN ;GCGAG ATG CAC, GT ACG GIG TAG GTC la ASP ASH SER ARC TYR TUR HIS PilE GAG AAC TCG AGG TAT ACA CAT TTC CIG TTG AGG TCG ATA TOT GTA AAG lb LEIJ THE GLN HIS TYR ASP ALA LYS PRO GUI GLY ARG ASP ASP ARG TYR CYS CTG ACC CAG Cj! TAT GAG OCT AAA CCG CAC 0CC COG GACGCAT COT TAG TGC GAC TG GTC G G ATA CTG CGA T] i GGC OTC CCG 3CC CTG OTA GCA ATG ACG 2b LYS ASP ILE ASH 'fHR PilE ILE HIS GLY ASH LYS ARG SER ILE LYS ALA ILE AAA GAT ATC AAC ACT TTC ATC CAT GOT AAC AAG CGT TCT ATC AMA 0CC ATA TTT CTA 'TAG TTG TGA AAG TAG GTA CCA TTG TTC GA AGA TAG ITT CGG TAT 3b GLU SEE ILE LEU ARG GMA ?CG AT? CTG AGA CTT AGC TAA GAC TCT ARC ARG GLY LEO THR SEE PRO CYS CGC C GOGG *A ACT AGT CCG TGC GCG GCA CCC MAT TGA ICA GG AG G YS GLU ASH LYS
ASH
CC GAA AM, AMA AA ACO CT TIfdIT] TIC 4a GLY ASH PRO HIS ARC GLU ASH OGT MC CCG CAT COG GAA JMLC CCA TTG GGG GTA GCG CIT T
F
LEU ARG ILE SEE LYS SER SER PHE GLN GTG COG ATC ACC AAG TCA AGC TIC CG GAG CCG TAG TCC TTC ACT ?fCG AAC GTG Ab VAL THR THR CYS LYS LEU HIS GLY GLY CGfl AGA ACT TG AMA CIT CAT COG OA CAATTAAC TTr CMA GTA CCC CC? sb SEE PRO TCC CG AO GGG ?RP PRO PRO CYS GLN TFYR ARG ALA THE TGG CGG CCA TGC GAG TAG COT GCT ACT AGC GGC GOT AG GTC ATG GGA GOA TGA ALA GLY PilE CCC GC TIC COG CCG AAG ARG ASH VAL VAL VAL ALA CYS OLU COT T OTT T OTCT GCT TGT GAA GCATTA CAA GAG CAM CCA ACA CIT ASH GLY LEO AAG GOT CTO TTG CCA GAG PRa) VAL HIS LEU ASP CCA OTC CAT CTA OAT GOT CG GTA GAT CTA
I
to 6b
ECOR]
GLH SEE ILE P11K ARC CG TCT ATG TIC GOA GTC AGA TAG AAG OCT ARC PRO AGO CCT TCC GGA TMA TAG ATT ATIE TTA A- Tabie 4 TYR ASP ALA LYS PRO GLN GLY ARG ASP ASP RG TYR GYS GLU SER ILE C TAT GAC GCT AAA CCG GAG GGC CGG GAC GAT CGT TAG TGC GAA TCG ATT T GGC GTC GCG GCC CTG CTA GCA ATG ACG CTT AGC TAA 2 ARG ARG ARG GLY NHIN AGA GGC CGT GGG T NNN TCT GCG GCA CCC AAT TGA\ TCA GGC A Leu
CTG
GAC
Ie
AIC
TAG
Val1
GTT
CAA
Claims (15)
1. A process for the preparation of an angiogenin by coupling the structural gene for this angiogenin in the correct reading frame via the gene for 0-galactosidase, or a fragment of 0-galactosidase, to a regulator region, introducing this gene structure into a bacterium, expressing therein an insoluble fusion protein, isolating it after cell disruption, and cleaving off the desired angiogenin chemically or enzymatically, which comprises modifying tho p-galactcsidase gene by replacing codons for methionine and/or arginini' and/or cysteine in the gene or in a fragment of the gene, in whole or in part, by codons of other amino acids.
2. The process as claimed in claim 1, wherein the gene structure codes for a A-galactosi'jase fragment of more than 250 amino acids but significantly less than the total P-galactosidase sequence.
3. The process as claimed in claim 2, wherein the p-galactosidase fragment is composed of a fusion of an amino-terminal and/or a carboxyl-terminal part-sequence as herein defined.
4. The process as claimed in claim 1, 2 or 3, wherein the -galactosidase fragment has about 300 to about 800 amino acids. The process as claimed in claim 1, 2 or 3, wherein the A-galactosidase fragment has about 320 to about 650 amino acids.
6. The process as claimed in one or more of the preceding claims, wherein the gene for the p-galactosidase fragment corresporids to a gene fragment shown in Figure 1. III u I StI I I I I I I III= I Zi lilt 1 V I IrL V I L a i ly 1 is sequenced, with dM7 and dC5 (Tab. 2) being used as pri- mers for the sequencing in addition to the normaL 17mer -23-
7. The process as claimed in one or more of the preceding claims, wherein the structural gene for the angiogenin is coupled via an adaotor to the gene for the modified B-galactosidase fragmeint.
8. The process as claimed in claim 7, wherein the adaptor codes for a poly(amino acid) sequence.
9. The process as claimed in one or rore of the preceding claims, wherein a codon for an amno acid which allows chemical or enzymatic separation )f the angiogenin from the 1-galactosidase portion is located immediately upstream of the amin(-terminal end of the structural gene.
10. The process as claimed in one or more of the preceding claims wherein the structural gene for the angiogenin has the DNA sequence of Table 1 or Table 4.
11. A gene structure containing a regulator region, a gene for the modified p-galactosidase or a 3- galactosidase fragment, in which codons for methionine SoC, and/or arginine and/or cysteine have been replaced, in whole or in part, by codons of other amino acids, and a structural gene for an angiogenin. S12. A gene structure as claimed in claim 11, wherein the o"0 structural gene for the angiogenin is coupled via an adaptor, which ensures the correct reading frame, to the gene for the modified 1-galactosidase or the galactosidaae fragment.
13. A vector containing a gene struc~ure as claimed in claim 11 or 12.
14. A bacterium containing a vector as claimed in claim 13. i:i i 24 E. coli containing a vector as claimed in claim 13.
16. A fusion protein containing modified B-galactoeidase or a i-galactosidase fragment as claimed in one or more of claim 1 to 9, and an angiogenin.
17. A fusion protein as claimed in claim 16, wherein the angiogenin has the amino acid sequence of Table 1 or Table 4.
18. An angiogenin derivative having the amino acid sequence 1-123 of Table 1 or Table 4. DATED this 17th day of May 1988 HOECHST AKTIENGESELLSCHAFT EDWD. WATERS SONS PATENT ATTORNEYS QUEEN STREET MELBOURNE, VIC. 3000. A E IEco RI HpaI1 ~-400 EcoRI 2OOOBp EcoRI EcoRi -1i20PvuI 1200 Bp 'S ~4( 45 4 5 I I. q I S list EcoRl Avol EcoRi -1300 EcoRi Clol/loqi EcoRl -900 -6 6 B p ,X250OBp FIG.1 I I S II I I S 1 II EcoRi EcoRi -400 -3608Bp Hpa I/ Pvu 11 EcoRI BssHII Eco RI ~-1500 Bp Eco RI HpoI EcoRI ~-1050Bp E i co~ -1550 Bp v Eco R I EcoRi Sstl. Eqo RI I 1 ~-195O Bp EcoRi Sst I EcoRI Eco RI 6e o e 0 0 0 0 0000 000000 a *o 00 90 09 0 fl 00 00 .0 0 000 0 00 0 C' *0 C' o..a0 0 0 EcoRI/PvuI EcoRi 12 3 Bp (U I -J Pvu I Pvu I PvulI II I Eco RI EcoRI Eco RI Pvu I iPvuI Pvu I Eco RI 123 Bp pBR 322/EcoRl Pvu I FIG.2 Pvu I FIG.3 Ampr pBR 322 p UR 2 Psi I Pvu11/ EcoRI *00EcoRi PSI I Hoell Hoell Pvull Ampr HoeII (part.)o 0 coRl PSIlI Hoell Hoell Ampr' Eco RI Pvull1 Amnp pL? A on Hoe 11 Pstl ulI EcoRi Ec oRI FIG.L. pstK Ampr pHZ A Eco RI EcoRi PvuI Pstl EcoRi AmpK, O 07R EcoRI coRI Pst I FIG. P vul Ampf EcoRi MCS Pvu 1 a 65 0 65 65 70 7 3 4 5 L LL A 0A 0A v v 5+6v 1-2 3- R- A AR FIG.6 R U~ A A A A 382 v 390 Sph I Eco RI
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DE3716722 | 1987-05-19 | ||
DE19873716722 DE3716722A1 (en) | 1987-05-19 | 1987-05-19 | GENE TECHNOLOGICAL METHOD FOR PRODUCING ANGIOGENINES |
Publications (2)
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AU1637988A AU1637988A (en) | 1988-11-24 |
AU617999B2 true AU617999B2 (en) | 1991-12-12 |
Family
ID=6327859
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Application Number | Title | Priority Date | Filing Date |
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AU16379/88A Ceased AU617999B2 (en) | 1987-05-19 | 1988-05-18 | A genetic engineering process for the preparation of angiogenins |
Country Status (8)
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---|---|
EP (1) | EP0292763A3 (en) |
JP (1) | JPS63304987A (en) |
KR (1) | KR880014107A (en) |
AU (1) | AU617999B2 (en) |
DE (1) | DE3716722A1 (en) |
DK (1) | DK271588A (en) |
PT (1) | PT87501B (en) |
ZA (1) | ZA883517B (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
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US4966849A (en) * | 1985-09-20 | 1990-10-30 | President And Fellows Of Harvard College | CDNA and genes for human angiogenin (angiogenesis factor) and methods of expression |
US5019556A (en) * | 1987-04-14 | 1991-05-28 | President And Fellows Of Harvard College | Inhibitors of angiogenin |
DE4012818A1 (en) | 1990-04-21 | 1991-10-24 | Hoechst Ag | METHOD FOR THE PRODUCTION OF FOREIGN PROTEINS IN STREPTOMYCETES |
US5426036A (en) * | 1987-05-05 | 1995-06-20 | Hoechst Aktiengesellschaft | Processes for the preparation of foreign proteins in streptomycetes |
US5227293A (en) * | 1989-08-29 | 1993-07-13 | The General Hospital Corporation | Fusion proteins, their preparation and use |
US5358857A (en) * | 1989-08-29 | 1994-10-25 | The General Hospital Corp. | Method of preparing fusion proteins |
EP0423641A3 (en) * | 1989-10-18 | 1992-01-15 | Nippon Mining Company Limited | Expression vector, transformed microorganism, fusion protein and method for the production of platelet factor 4 or tgfalpha |
ZA909633B (en) * | 1990-02-15 | 1991-10-30 | Harvard College | Covalent angiogenin/rnase hybrids |
US5270204A (en) * | 1990-02-15 | 1993-12-14 | The President And Fellows Of Harvard College | Covalent angiogenin/RNase hybrids |
WO1996021416A2 (en) * | 1994-12-30 | 1996-07-18 | Chiron Corporation | Methods and compositions for treatment of solid tumors in vivo |
WO2001000797A1 (en) * | 1999-06-29 | 2001-01-04 | National Institute Of Advances Industrial Science And Technology | Sulfur atom-free enzyme proteins |
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AU1446988A (en) * | 1987-04-11 | 1988-10-13 | Hoechst Aktiengesellschaft | Genetic engineering process for the preparation of polypeptides |
AU1455688A (en) * | 1987-04-14 | 1988-10-20 | President And Fellows Of Harvard College | Inhibitors of angiogenin |
AU3370189A (en) * | 1987-11-19 | 1990-11-01 | Centre National De La Recherche Scientifique | 17 kd protein with angiogenic action, method of isolating it from mammalian milk, therapeutic compositions in which it is present, method of detection and/or determination and immunological reagents for detecting and determining mammalian angiogenins,homologs thereof and fragments thereof |
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NZ199391A (en) * | 1981-01-02 | 1985-12-13 | Genentech Inc | Chimeric polypeptides comprising a proinsulin sequence,and preparation by recombinant dna technique;production of human insulin |
US4987070A (en) * | 1987-03-04 | 1991-01-22 | Suntory Limited | Use of a 97 amino acid leader sequence from the E. coli B-galactosidase gene for the production of hanp and hptc as fusion proteins |
-
1987
- 1987-05-19 DE DE19873716722 patent/DE3716722A1/en not_active Withdrawn
-
1988
- 1988-05-07 EP EP88107356A patent/EP0292763A3/en not_active Withdrawn
- 1988-05-18 AU AU16379/88A patent/AU617999B2/en not_active Ceased
- 1988-05-18 DK DK271588A patent/DK271588A/en not_active Application Discontinuation
- 1988-05-18 PT PT87501A patent/PT87501B/en not_active IP Right Cessation
- 1988-05-18 ZA ZA883517A patent/ZA883517B/en unknown
- 1988-05-19 JP JP63122997A patent/JPS63304987A/en active Pending
- 1988-05-19 KR KR1019880005866A patent/KR880014107A/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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AU1446988A (en) * | 1987-04-11 | 1988-10-13 | Hoechst Aktiengesellschaft | Genetic engineering process for the preparation of polypeptides |
AU1455688A (en) * | 1987-04-14 | 1988-10-20 | President And Fellows Of Harvard College | Inhibitors of angiogenin |
AU3370189A (en) * | 1987-11-19 | 1990-11-01 | Centre National De La Recherche Scientifique | 17 kd protein with angiogenic action, method of isolating it from mammalian milk, therapeutic compositions in which it is present, method of detection and/or determination and immunological reagents for detecting and determining mammalian angiogenins,homologs thereof and fragments thereof |
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JPS63304987A (en) | 1988-12-13 |
EP0292763A3 (en) | 1990-05-23 |
DK271588A (en) | 1988-11-20 |
DE3716722A1 (en) | 1988-12-01 |
ZA883517B (en) | 1988-11-22 |
DK271588D0 (en) | 1988-05-18 |
PT87501A (en) | 1989-05-31 |
AU1637988A (en) | 1988-11-24 |
KR880014107A (en) | 1988-12-22 |
PT87501B (en) | 1992-09-30 |
EP0292763A2 (en) | 1988-11-30 |
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