IL92178A - Process for the preparation of an insulin precursor in streptomycetes - Google Patents

Abstract

A gene for a truncated proinsulin in which the insulin B chain is linked to the A chain only via Lys or Lys-Lys is coupled to the tendamistat gene, this gene construct is inserted into an expression vector, and the latter is used to transform a Streptomycetes host cell, with the result that the corresponding fusion protein is expressed and secreted. The fusion protein can easily be cleaved to give insulin precursors.

Description

A PROCESS FOR THE PREPARATION OF AN INSULIN PRECURSOR IN STREPTOMYCETES HOECHST AKTIENGESELLSCHAFT HOE 88/F 313 K Dr.KL/PP Description ^'ί)2*178/2 A process for the preparation of an insulin precursor in Streptomycetes A process for the preparation of fusion proteins has already been proposed which comprises C-terminal coupling of the structural gene for the desired protein to the optionally modified tendamistat gene, bringing about the expression of this gene structure in a Streptomycetes host cell, and isolating the secreted fusion protein from the culture supernatant (DE 37 14 866 Al or EP 0,289,936 A2 ) . The older application additionally relates to gene structures containing the optionally modified tendamistat gene to which a structural gene for another protein is C-terminally coupled, vectors which contain a gene structure of this type, Streptomycetes cells which contain a vector of this type, and fusion proteins which have an N-terminal portion of optionally modified tendamistat. The older application contains examples of fusion proteins in which the amino acid sequence of tendamistat is coupled via a bridging member to that of monkey proinsulin.

The present Patent Application is for a Patent of addition to Patent No. 86277. In a further development of the concept of this invention it has now been found that this process can be used particularly well to prepare a fusion protein in which the tendamistat portion is followed by a shortened proinsulin whose C chain comprises only one or two lysine residues. These precursors can be converted particularly straightforwardly and economically into human insulin.

Particular embodiments of the invention relate to advantageous gene structures for the amplification and expression of the gene which codes for the fusion protein. Further preferred embodiments of the invention are explained hereinafter and defined in the patent claims.

The gene structure employed according to the invention is advantageously based on a synthetic gene which codes for the shortened proinsulin derivative. It is expedient in the construction of this gene to take account of the specific codon usage of Streptomycetes . It has emerged that the yield of fusion protein is thereby increased.

It is also advantageous to incorporate a terminator sequence in the synthetic gene structure, because an increase in the synthesis rate is also thereby achieved.

A great advantage of the process according to the invention comprises the possibility of detecting the fusion proteins using the plate test which is described in EP-Al 0,161,629 in Example 3 and in German Offenlegungs-schrift 3,536,182. This considerably facilitates not only the selection of the interesting clones but also the working up because the effect of the different parameters on the yield can easily be established.

The fusion proteins obtained according to the invention are apparently present in a conformation which corres-ponds, or at least approximates, to that of mature insulin. This not only considerably facilitates the further processing to insulin but, moreover, at fermentation times long enough to provide a good yield, surprisingly there is no noticeable attack by the proteases excreted into the fermentation medium.

The modification according to the invention of the proinsulin molecule with its shortened C chain permits straightforward processing to human insulin, namely by chemical cleavage with hydroxylamine and/or by enzymatic cleavage using trypsin or, advantageously, lysyl endo-proteinases. Enzymatic cleavage is preferred. Lysyl endoproteinases carry out specific carboxy1-terminal cleavage after the amino acid lysine. The favorable arrangement of the A and the B chain in the fusion protein according to the invention means that the action of the said enzymes results in an insulin precursor in which, surprisingly, the disulfide bridges are correctly linked.

It is expedient in the construction of the gene to provide between the tendamistat portion and the start of the proinsulin molecule a bridging member which permits the proinsulin derivative to be cleaved off from the tendamistat portion with the same enzyme used to cleave the proinsulin derivative into the two insulin chains.

Cleavage of the fusion proteins according to the invention with a lysyl endopeptidase results - depending on the construction of the modified C chain - in de-B30-insulin which can be transformed into human insulin by transpeptidation, or B31-Lys-insulin or B31-Lys-B32-Lys-insulin, each of which can be transformed into human insulin, for example, by the use of carboxypeptidase B.

Particularly high yields of the desired protein are obtained when gene constructions with a shortened tendamistat gene are employed. This embodiment of the process according to the invention has the great advantage that the portion of the modified insulin in the fusion protein comprises about one half, and thus contains considerably less "ballast". The correct folding of the fusion protein is not impaired by the shortening of the tendamistat portion, so that the advantageous working up is therefore also possible with the smaller fusion protein according to the invention. Nor is this advantage achieved at the expense of an increased rate of degradation by the proteases intrinsic to the host - in fact, unexpectedly, it has emerged that the stability to these proteases is increased.

The invention thus allows a whole series of advantageous gene constructions which result in insulin precursors which can easily be separated from the "ballast portion" of the fusion protein. This straightforward working up additionally improves the yield of human insulin.

The separation of the fusion protein from the culture medium/ its further processing to the insulin precursor and the transformation thereof into human insulin can be carried out by methods known per se. Thus, the fusion protein can advantageously be isolated by adsorption or ion exchange chromatography and/or gel filtration, and the proinsulin portion can be cleaved off chemically or, advantageously, enzymatically. The construction of appropriate bridging members is generally known and described, for example, in EP-A2 0,229,998.

EP-B 0,089,007 discloses analogs of prepro- and proinsulin which carry at the C end of the prechain (or at the N-terminus of proinsulin) Lys or Arg (which is also preferred in the constructions according to the invention) , whose B chain terminates with B29-Lys and where the C peptide can be shortened to Lys or Arg so that therefore B29-Lys in the proinsulin structure is, in the simplest case, followed by only Lys or Arg, to which the A chain is attached. These compounds are used as precursors for preparing insulins with the aid of trypsin or trypsin-like endopeptidases and of an ester of a natural amino acid which, where appropriate, carries protective groups .

Insulin precursors in which the B and A chain are connected by the bridging member -X-Y-, in which X and Y are identical or different and represent Lys and Arg, are disclosed in EP-A 0,195,691. These insulin precursors are expressed from yeast and then converted into human insulin by enzymatic transformation. Insulin precursors with a shortened C chain are also disclosed in EP-A 0,163,529. EP-B 0,132,769 and 0,132,770 describe insulin derivatives and pharmaceutical agents containing them.

The invention is illustrated in more detail in the examples which follow. Unless stated otherwise, percentage data relate to weight.

The figure illustrates the gene construction according to the invention in Example 1. It is not true to scale.

Example 1 The synthetic gene ( 1 ) depicted in Table 1 is chemically synthesized in a manner known per se by the phosphoami-dite method. In the codon selection account was taken of the preference of Streptomycetes for G and C. As with the gene coding for monkey proinsulin in the earlier application (Table 2 therein), the gene (1) shown in Table 1 also has at the 5' end a protruding sequence typical for the restriction enzyme EcoRI. The structural gene is followed by two stop codons and a linker sequence with the recognition site for the enzyme Sail. The protruding sequence corresponding to the restriction enzyme HindiII is located at the 3' end.

The commercially available plasmid pUC19 is cut with the enzymes EcoRI and Hindlll, and the synthetic gene (1) shown in Table 1 is ligated in. The result is the plasmid pll (2). After amplification, the synthetic gene is cut out as fragment (3) with the enzymes EcoRI and Sail and employed for the construction described hereinafter.

The plasmid pUC19 is completely digested with Smal and ligated with the terminator sequence (4) depicted in Table 2. Plasmids which contain this sequence in the correct orientation are called pTl (5). This plasmid (5) is opened with EcoRI, and the cleavage site is filled in with DNA polymerase (Klenow fragment). The plasmid pT2 (6) is obtained by religation. This plasmid is_ opened with the enzymes Sail and SphI, and the large fragment (7) is isolated.

The plasmid pKK400 (8) (cf. earlier application, Figure 4, (20)) is cut with SphI and EcoRI, and the small fragment (9) with the tendamistat gene is isolated.

Ligation of fragments ( 3 ) , ( 7 ) and ( 9 ) results in the plasmid pKK500 (10) in which the tendamistat sequence is followed by the bridging member Phe Asn Ala Met Ala Thr Gly Asn Ser Asn Gly Lys TTC AAT GCG ATG GCC ACC GGG ATT TCG AAC GGC AAG AAG TTA CGC TAC CGG TGG CCC TAA AGC TTG CCG TTC EcoRI coding for 12 amino acids, and then by the gene for the proinsulin modified according to the invention. The correct arrangement is checked by cutting with SphI and Sstl, resulting in a fragment of 833 bp from the plasmid about 3.5 kb in size. The sequence is confirmed as correct by DNA sequencing using the dideoxy method.

Gene constructions, according to the invention, in which the Lys acting as C peptide is supplemented by another Lys are prepared analogously. For this purpose, the triplet AAG coding for Lys is doubled. The plasmid pi2, and therefrom the vector p K600, are obtained analogously.

Example 2 In analogy to the vector pGFl proposed in the earlier application, the expression plasmids pGF2 and pGF3 are prepared from the vectors pKK500 and pKK600. For this purpose, double digestion with SphI and Sstl of each of the vectors pKK500 and pKK600 is carried out to isolate the insert of 823 and 826 bp reepectively, and these DNA fragments are ligated into the expression plasmid pIJ702 cleaved with the same enzymes. The ligation mixture is transformed into S. lividans TK 24, and the plasmid DNA is isolated from thiostrepton-resistant transformants which show tendamistat activity (plate test). All the positive clones contain the insert from pKK500 or pKK600 employed.

The expression of the coded fusion protein can be carried out in a known manner. If the transformed strain S. lividans TK 24 is incubated in a shaken flask at 28°C for four days and the mycelium is separated from the culture solution by centrifugation, the fusion protein can be detected in the clear solution as follows: 20 to 200 ΐ of 15% strength trichloroacetic acid are added to 10 to 100 ΐ of solution, and the precipitated protein is concentrated by centrifugation, washed and taken up in SDS-containing sample buffer (U. Laemmli, Nature 227 (1970) 680-685). Incubation at 90°C for 2 minutes is followed by fractionation by electrophoresis on a 10-17% SDS polyacrylamide gel. A protein of molecular weight 15 kD is obtained, that is to say in the molecular weight range expected for the fusion protein composed of tendamistat and proinsulin. The fusion protein reacts both with antibodies against tendamistat and with antibodies against insulin.

Example 3 The expression vector pTF2 (DE 37 14 866 Al, Example 4) is digested with the restriction enzymes EcoRI and Sstl, and the fragment which encodes monkey proinsulin is removed. The fragment 5.65 kbp in size is used for the ligation reaction described below.

These same restriction enzymes are used to cut a DNA fragment which is 285 bp in size and which contains the shortened proinsulin gene, as well as the termination sequence, out of the plasmid pKK500 (Example 1).

Ligation of the fragment 5.65 kbp in size from pTF2 with the fragment 285 bp in size from pKK500 yields the expression plasmid pTF3.

Transformation of protoplasts of Streptoroyces lividans TK24 with the ligation mixture results in clones which are thiostrepton-resistant and secrete a fusion protein which reacts with antibodies against proinsulin. This fusion protein comprises the first 41 amino acids of tendamistat, the bridging member Pro-Ser-Leu-Asn-Ser-Asn-Gly-Lys and the shortened proinsulin.

Table 1 B1 10 ASN SER ASN GLY LYS PHE VAL ASN GLN HIS LEU CYS GLY SER HIS AAT TCG AAC GGC AAG TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC GC TTG CCG TTC AAG CAG TTG GTC GTG GAC ACG CCG AGC GTG (EcoRI) 20 30 LEU VAL GLU ALA LEU TYR LEU VAL CYS GLY GLU ARG GLY PHE PHE CTC GTG GAG GCC CTC TAC CTG GTG TGC GGG GAG CGC GGC TTC TTC GAG CAC CTC CGG GAG ATG GAC CAC ACG CCC CTC GCG CCG AAG AAG C Al 40 TYR THR PRO LYS THR LYS GLY ILE VAL GLU GLN CYS CYS THR SER TAC ACC CCC AAG ACC AAG GGC ATC GTG GAG CAG TGC TGT ACG TCC ATG TGG GGG TTC TGG TTC CCG TAG CAC CTC GTC ACG ACA TGC AGG 50 ILE CYS SER LEU TYR GLN LEU GLU ASN TYR CYS ASN STP STP ATC TGC TCC CTC TAC CAG CTC GAG AAC TAC TGC AAC TAG TAA TAG ACG AGG GAG ATG GTC GAG CTC TTG ATG ACG TTG ATC ATT GTC GAC CTG CAG CCA CAG CTG GAC GTC GGT TCG A Sail (Hindlll) Table 2 5'-CGATAAACCGATACAATTAAAGGCT CmTOGAGCLm GCTATTTGGCTATGTTAATTTC03AGGAAAACCTCGGAAAAAAAMCCTCTAA

Claims (7)

10 - 19217K 2 CLAIMS i

1. A process for the preparation of a fusion protein which comprises coupling the structural gene coding for a proinsulin derivative in which the B chain is connected to the A chain via a bridging member coding for Lys or Lys-Lys to the C terminus of the tendamistat gene or modified tendamistat gene or shortened tendamistat gene, expressing this gene structure in a Streptomycetes host cell and isolating the secreted fusion protein from the supernatant.

2. A process as claimed in claim 1, wherein the tendamistat gene is modified.

3. A process as claimed in claim 1 or 2, wherein the tendamistat gene is shortened.

4. A gene structure containing the optionally modified tendamistat gene to which the structural gene defined in claim 1 is coupled C-terminally .

5. A vector containing a gene structure as claimed in claim 4.

6. A Streptomycetes cell containing a vector as claimed in claim 5.

7. A fusion protein which has a N-terminal portion of optionally modified tendamistat to whose C terminus the proinsulin defined in claim 1 is bonded.