A PROCESS FOR PREPARING RIPE PROTEINS FROM FUSION PROTEINS, SYNTHETIZED IN PRO- OR EUKARYOTIC CELLS

Abstract: A fusion protein with the amino acid sequence: (Ym...Y2-Y1)-(Pro)p-(X1-X2...Xn) in which (Ym...Y2-Y1)-(Pro)p is the pro-sequence and the rest is the ripe protein, m is an integer greater than 2, and Y is an arbitrary amino acid, p is O ifX1 or X2 is Pro, and 1 if X1 or X2 is different from Pro, X is an arbitrary amino acid, and n is integer equal to or greater than 4, is converted into ripe proteins by enzymatic cleavage, which is carried out by stepwise cleavage off of the amino acid groups Ym...Y2 if p = 1 or X1 = Pro, or the groups Ym...Y2-Y1 if X2 = Pro, by means of an aminopeptidase, and then the two amino acids Y1-Pro if P = 1 are cleaved off enzymatically in one or two steps in a manner known per se, and similarly Y1 alone is cleaved off if X1 = Pro. The preferred aminopeptidase is leucine aminopeptidase. The process enables removal in vitro of the formyl methionine or the methionine group and optionally the pro-sequence from a protein synthetized in pro- or eukaryotic cells by genetic engineering, and thus achievement of a desired ripe protein, such as human growth hormone or human proinsulin.

Description: A process for preparing ripe proteins from fusion proteins, synthetized in pro- or eukaryotic cells The invention relates to a process for preparing ripe proteins from fusion protein, synthetized in pro- or 5 eukaryotic cells. Proteins and peptides are synthetized in all live cells in which they are essential components. Moreover, cer¬ tain proteins and. peptides, such as the peptide hor¬ mones insulin and growth hormone, play an important 10 role in intercellular communications. Other proteins and peptides, such as i munoglobulins and blood clot¬ ting factors, are of great importance to the defense mechanisms of higher organisms. Peptides and proteins consist of short and long chains 15. of amino acids, respectively, and a given protein may include one or more chains. The amino acids are selected from the about 20 normally occurring cellular amino acids. The amino acid sequence in a given pro¬ tein or peptide chain is specified in the genes of the 0 cell. The genes of the vast majority of organisms are composed of long chains of desoxyribonucleic acid (DNA) consisting of nucleotide triplets (codons) and when a protein is to be synthetized, it takes place through a ribonucleic acid (RNA) copy of the coding DNA region. 5 This so-called messenger RNA is then used by the cell as a pattern for the protein as ribonucleotide tri¬ plets (and thus indirectly the corresponding desoxyribo- nucleotide triplets in the DNA chain) specify specific amino acids in accordance uith the genetic code. OΛ'.PI The basic molecular-bio logical principl es whi ch , based on DNA , lead to the format ion o f a protein are well- known to those skilled in the ar t . When proteins are synthetized in cells, the product first formed is seldom equal to the final, ripe protein. Most often, e.g. a somewhat longer amino acid chain is synthetized which is then cut to the length of the de¬ sired ripe protein, probably enzymatically. The cut ami- no acid sequences are usually disposed in the N-terminal end of the protein, and are called pro-sequences or sig¬ nal sequences, according to their function and time of cleavage. Usually, only the ripe protein has full bio¬ logical activity. Initiation of the biosynthesis of pro teins/peptides takes place by a mechanism causing the N-termiπal amino acid in the produced protein or peptide to always be menthionine (in eukaryotic cells) or N-formyl methionine (in bacteria). This N-terminal methionine (or formylme- thionine) is often included in a pro-sequence or a signal sequence, which is cleaved later, and therefore is not present in the ripe protein. Discoveries and progress in recent years within molecu¬ lar biology have made it possible to transfer genes (DNA segments) in vitro from one organism to another, unrelated organism. Thus, e.g. human genes can be transferred to bacteria, such as E. coli. If the human DNA segment is modified so as to resemble bacterial DNA. the bacteria may in certain cases be caused to synthe- tize a protein identical or closely related to the pro- tein which is coded for by the human DNA segment. Nor¬ mally, however, great problems are associated with making bacteria synthetize a product by this genetic engineering which is structurally identical to the desired ripe eukaryotic protein. Usually, the result¬ ing bacterial product is a fusion protein between an amino acid sequence of a bacterial protein and the de- sired eukaryotic protein or it is the eukaryotic pro¬ tein provided with an N-terminal formyl methionine group. These products are only rarely further modified in bacteria. To remedy this situation, it is proposed in the DK patent application No. 1616/81 to couple a bacterial signal sequence directly to a eukaryotic protein, and the bacteria itself will hopefully then cleave off the signal sequence. However, this is not a universally use¬ ful method because the cleavage also seems to depend upon the amino acid sequence of the eukaryotic protein. Further, it is proposed in the DK patent application No. 888/81 to incorporate specific proteolytic or chemical cleavage sites at the border between the bacterial and the eukaryotic part of the protein. The usefulness of this method, however, is limited because the desired ripe eukaryotic protein must not contain the same proteolytic or chemical cleavage site. The fusion protein may be cleaved chemically at specific amino acids (e.g. after methionine using BrCN). However, this method requires that the specific amino acid in question is not also present in the ripe protein, and the method is vitiated by a certain risk due to the toxi- city of BrCN. When synthetized in bacteria, the newly synthetized protein always contains, as mentioned, N-terminal N- for yl methionine and optionally a pro-sequence which v 7 . v - " ϋ " is not present in proteins of eukaryotic cells and must therefore always be removed if the bacterially synthe¬ tized protein is to have the same primary amino acid sequence as the desired protein from e.g. humans. Similarly, proteins formed in eukaryotic cells always contain N-terminal methionine. In addition, a suitable DNA sequence may be incorporated by genetic engineering in front of the DNA segment cod¬ ing for the desired protein so that the expressed fu- sion protein is caused to contain an appropriate N- terminal amino acid sequence. This amino acid sequence may e.g. be selected so that the purification of the fu¬ sion protein will be easier, that a desired proteolytic cleavage site results or that the fusion protein is transported out of the bacteria. Such an N-terminal a- mino acid sequence must of course also be removed to provide the desired, ripe protein. Generally, it may be said that when the bacteria cannot be caused to produce the desired protein directly, then the resulting fusion protein must be treated enzymatic- ally or chemically so that the additional N-terminal a- mino acids are removed without the reactions influencing the desired protein. A process for enzymatic cleavage of a fusion protein is known from the EP Publication No. 0020290. In this pro¬ cess the enzyme collagenase is used for cleaving a bond in the signal or pro-sequence of the fusion protein, viz. the bond Xyz-Gly in which Xyz is a peptide chain incorporated in the pro-sequence. The remaining amino acid or acids in the pro-sequence are then removed en- zy atically. However, it has been found that collagenase and other endopeptidases are deficient in specificity, see Bio- chim. Biophys. Acta, 271 (1972) 133-144. Moreover, the collagenases are active only in connection with proteins of a specific steric structure, see Peptides 1980, Proceedings of the sixteenth European Peptide Symposium, Ed. K. Brunfeldt (Scriptor Copenhagen 1981). Proteolytic enzymes (proteases) are characterized in that in certain circumstances they can hydrolyze the peptide bonds between the amino acids in proteins (and peptides). The proteolytic enzymes constitute a very large and heterogenous group of proteins, both as regards mode of operation and the specificity with which the amino acid sequences are cleaved. In brief summary, the proteolytic enzymes are divided into two main groups: exo- and endoproteases. As indicated by the names, exoproteases cleave off amino acids from the end of the protein chain, whereas endoproteases can directly cleave in the interior of a protein. Amino- peptidases are exoproteases cleaving off amino acids from the N-terminal end of the protein chain. Proteases often have a high degree of specificity as they mainly cleave bonds in specific amino acid sequences. The cleavage is not only dependent upon the amino acid sequence, but also the sterical structure of the substrate may have a great influence on the cleavage rate. The ability of proteases to cleave proteins in a specific manner has been used for a number of years for characterization, analysis and modification of proteins. The present invention is based on the use of an exo- peptidase which cleaves off the amino acid groups in the pro-sequence of the fusion protein one by one until only the group Y-Pro remains in which Y is an arbitrary amino acid . The exopeptidase used is an aminopeptidase, and as such leucine aminopeptidase is preferred. The object of the present invention is to provide a process in which any protein, synthetized by genetic engineering in pro- or eukaryotic cells, can be con¬ verted in vitro by enzymatic processes to a protein having an amino acid sequence identical to the selected eukaryotic protein, and thus solve the following two problems in the preparation of ripe proteins: a) removal of the formyl methionine or the methionine group, b) release of the ripe protein from the pro-sequence. According to the invention, proteases are used, and in particular aminopeptidases, for cleaving off, if necessary, formyl methionine and optionally other N- terminal amino acids in a synthetized fusion protein. Thus, the invention relates to a process for preparing ripe proteins by enzymatic cleavage of a fusion protein with the amino acid sequence: (Y ...Y A 7 .-Yl.)-(Pro)p-(Xi 1 -X 9 ...Xn) in which (Y ... Y--Y-, )-(Pro) is the pro-sequence and the rest is the ripe protein, m is an integer greater than 2, and Y is an arbitrary amino acid, P is 0 if X, or X^ is Pro, and 1 if X, or X„ is different from Pro, X is an arbitrary amino acid, and n is an integer equal to or greater than 4, and the process is characterized by carrying out the enzymatic cleavage by stepwise cleavaqe off of the amino acid groups Y ...Y if p = 1 or X, = Pro, or the groups Y ...Y -Y. if X„ = Pro, by means of an aminopeptidase, and then the two amino acids Y,-Pro if p = 1 are cleaved off enzy atically in one or two steps in a manner known per se, and simi¬ larly, Y, alone is cleaved off if X, = Pro. According to the invention, fusion proteins as stated in step (i), which do not contain a formyl methionine group, can be obtained i.a. in one of the following ways : 1) The DNA coding sequence of a suitable prokaryotic N- terminal pro-sequence containing or constituting a signal sequence can be spliced on to the coding sequence of the desired protein at gene level. The; DNA sequence corresponding to this pro-sequence may be selected from among the large number of naturally occurring sequences or may be synthetized in vitro when their structure at nucleotide and amino acid level is known. After the synthesis of the fusion protein in bacteria, the signal sequence (containing the N-terminal f-Met.) is cleaved off by the bacteria in vivo. 2) The DNA coding sequence of the selected pro-sequence is coupled to the coding sequence of the desired protein. The fusion protein (with the N-terminal formyl methionine) synthetized in bacteria, is iso¬ lated completely or partly from the cells or the bacterial supernatant. The fusion protein is then treated with aminopeptidase I (AP I) (E.C. 3.4.11.12) from Bacillus stearothermophilus (Roncari & Zuber, Int. J. Protein Research II, 191 (1970)). This amino¬ peptidase has the same broad specificity as most of the other aminopeptidases, but is moreover able to cleave off N-formyl methionine in the above-mentioned fusion product. This cleavage reaction is to be optimized with respect to time and enzyme concentra¬ tion as, in case of prolonged incubation, amino¬ peptidase I can also hydrolyze amino acids of the desired product. This problem can be reduced by incorporating one or more of the amino acid sequences Gly-Ala, Gly-Gly or Lys-Leu in the pro- sequence because these peptide bonds are hydrolyzed only slowly by the above-mentioned aminopeptidase I. 3) The DNA coding sequence of the pro-sequence is so formed that the pro-sequence is caused to contain highly active cleavage sites for trypsin (E.G. 3.4.21.4.) or trypsin-like proteases. Such cleavage sites may e.g. consist of the amino acid sequences Arg-Arg, Lys-Lys, Lys-Arg and/or Arg-Lys. After the synthesis of the fusion protein it is isolated in vivo wholly or partly, and is treated in vitro with trypsin or the trypsin-like protease. In each of the three above-mentioned procedures it is thus possible according to the invention to provide a protein containing the amino acid sequence corresponding to the desired protein as well as an N-terminal pro- sequence without formyl methionine. In this pro-sequence the C-terminal amino acid, which is bonded directly to the N-terminal amino acid in the desired protein, must be proline, unless the desired protein itself contains proline as N-terminal or next-to-the-outermost N-terminal amino acid. The pro-sequence may then be removed speci- fically in vitro by treating the fusion protein with a suitable aminopeptidase, e.g. leucine aminopeptidase (E.C. 3.4.11.1). This commercially available amino¬ peptidase will cleave the sequence, but will stop after having hydrolyzed the bond just before the dipeptide BUR i f/" r X-Pro. After removal or inactivation of the leucine aminopeptidase, the resulting remaining fusion protein can be treated in one of the following ways if X and optionally Pro are to be cleaved off: a) X is different from Gly. First the amino acid X is cleaved off, e.g. with aminopeptidase P (3.4.11.9) or with p.rolidasε, and then, if desired, proline is cleaved off with proline iminopeptidase (3.4.11.5). b) X = Gly. Here Gly-Pro can be cleaved off in the same manner as mentioned under point a). However, when e.g. a Gly-Pro hydrolyzing dipeptidylpeptidase IV (3.4.14.-) is used, whole dipeptides Gly-Pro can be cleaved off in one'step. EXAMPLE 1 A cloned DNA sequence coding for a protein with an amino acid sequence as in human growth hormone, hGH, (191 amino acid residues of which the first four amino acids are Phe-Pro-Thr-Ile) is coupled with the following syn¬ thetically produced, dual-stranded DNA sequence so that the 3' end of the +strand is coupled to the +5' end of the above-mentioned gene, and the 5' end of the -strand of the synthetic DNA sequence is coupled to the 3 1 end of the above-mentioned gene by blunt end ligature. + 5' GATCCATGCTGGCTGTAAGC 3' - 3' TACGACCGACATTCG 5' where the 5 first nucleotides in the +strand are part of a Bam HI restriction site, and the following nucleo- tide sequences code for the amino acids Met, Leu, Ala, Val, Ser. 10 The above-mentioned gene is introduced by ordinary gene cloning techniques into an expression pla ' s ide con¬ taining a fusioned Trp-Lac promotor as well as the SD sequence AGGA; this structure should therefore express Met-Leu-Ala-Val-Ser-hGH. This plas ide structure is then introduced into an E. coli cell by prior art techniques. A suitable clone containing the above-mentioned structure is isolated and cultivated in a 30 1 scale. The cells are harvested by centrifugation and are suspended in a small volume and lyzated using a so-called "French press". The expected fusion protein could be demonstrated in the above-mentioned bacterial extract by immunological methods using hGH antibodies. The fusion product was purified from this extract by hydrophobic interaction chromatography, ammonium sulfate precipitation, gel filtration and anion exchange, Fractions containing the fusion protein were determined immunologically using hGH antibodies. The purified fusion protein was evaluated to be more than 98% pure by polyacrylic amide gel electrophoresis and ion exchange HPLC. 15 g were isolated from a 30 1 culture. Possible disulfide bridges in the purified fusion protein were reduced and S-carbamidomethylated in principle as described by L. Graf et al. : FEBS Letters ^6(2 , 233 (1976). An N-terminal sequence de¬ termination according to Edman of the purified and reduced and S-carbamidomethylated fusion protein showed the expected amino acid sequence Met-Leu-Ala-Val-. This reduced and S-carbamidomethylated product was then treated with leucine aminopeptidase TEC 3.4.11.1) as py.Pi ' 11 described by D.H. Sprek an et al., J. Biol. Chem. 212, 255 (1955) and A. Light: In: Methods in Enzymology, vol. XI, Ed.: C.H.W. Hirs (1967), Academic Press, p. 426. However, the enzymatic hydrolysis was carried out in the presence of urea and aprotinin. The the reaction mixture was fractionated by ion ex¬ change, and protein from fractions containing hGH immunoreactivity was isolated. Evaluated by polyacrylic amide gel electrophoresis and ion exchange HPLC the protein was more than 9835 pure. N-terminal determination according to Edman showed Phe as the main component. EXAMPLE 2 Production of human proinsulin A gene coding for a protein with an amino acid sequence as in human proinsulin (86 amino acid residues of which the first 4 amino acids are Phe-Val-Asn-Gln), is coupled with the following synthetically produced, dual-stranded DNA sequence so that the 3' end of the +strand is coupled to the 5' end of the +strand of the above- mentioned gene, and the 5 1 end of the -strand of the synthetic DNA sequence is coupled to the 3' end of the above-mentioned gene by blunt end ligature. Met Leu Val Ala Gly Pro + 5' ATTC ATG TTG GTT GCT GGT CCA G TAC AAC CAA CGA CCA GGT The 5 first nucleotides in the +strand are part of an Eco RI restriction site, and the subsequent nucleotide sequences code for the amino acids Met-Leu-Val-Ala-Gly- Pro. 12 The above-mentioned gene is introduced by ordinary gene cloning techniques into an expression plas ide containing the promotor for 3-phosphoglycerate kinase and the terminator for the FLP gene, both from S.cerevisiae. (Ref.: Hitzeman et al. (1983), Science 219 620-625). This plasmide structure is then introduced into an S.cerevisiae cell by prior art techniques. An S.cerevisiae clone containing the above-mentioned structure is isolated and cultivated in a suitable volume. The yeast cells are harvested and lyzated using a so-called "French press". A protein assumed to be the expected fusion protein is demonstrated in the extract by immunological methods using proinsulin antibodies. The fusion protein is purified by hydrophobic inter¬ action chromatography. The fusion protein is purified to >90J_ purity, evalu¬ ated by polyacrylic amide electrophoresis and ion- exchange HPLC. An N-terminal sequence determination according to Edman is carried out to confirm the expected amino acid sequence (corresponding to Met- Leu-Val-Ala-Gly-Pro-Phe ). This purified product is treated with leucine amino- peptidase (EC 3.4.11.1.) as described by D.H. Sprekman et al., 3 . Biol. Chem. 212, 225 (1955) and A. Light: In: Methods in Enzymology, vol. VI, Ed.: C.H.W. Hirs (1967), Academic Press, p. 426. Then the reaction mixture is fractionated by ion exchange, and protein from fractions containing proinsulin immunoreactivity is isolated. The purification is better than 90% 13 evaluated by polyacrylic amide electrophoresis and ion-exchange HPLC. Gly-Pro-Phe is found in the isolated product by three consecutive N-terminal degradations according to Ed an. Gly-Pro is removed enzymatically from this product in a manner known per se in a 1-step reaction using post- proline dipeptidyl aminopeptidase (EC 3.4.14.-), cf. R. Walter, W.H. Simmons & T. Yoshimoto: Molecular & Cellular Biochemistry 3_TJ(2) (1980), 111-127. Or, alter¬ natively, in a 2-step process, cf. R.I. Delange & E.L. Smith, in: The Enzymes. Ed.: P.D. Boyer, Academic Press (1971), p. 115, to produce human proinsulin. EXAMPLE 3 An 0.1 M Tris buffer is used in which pH is adjusted to 8.5 with 1 M hydrochloric acid. This buffer is ad¬ 2+ mixed with MnCl . 2 ,, 4 -H. , r „0 so that the concentration of Mn is 0.5 mM. Leucine aminopeptidase (Sigma) is incubated at 37°C for 45 minutes in the above-mentioned buffer (1:9). 950 nmoles~*l mg Gly-Phe-Phe-Tyr-Thr-Pro-Lys- (Ala) is dissolved in 2 ml of the above-mentioned buffer. This mixture is admixed with 1 ml of the incubated enzyme solution, 22 units leucine aminopeptidase. The mixture is left to stand at 37°C. After 30, 60, 90 and 120 minutes 300 μl (~9.5 n oles) are removed, and the reaction is stopped by the addition of 50 jul EDTA (12 mg/ml) and 10 ul concentrated hydrochloric acid. Amino acid analysis is carried out on 100 j ul (corre¬ sponding to 26.4 moles) without preceding acid hydro¬ lysis. The result of the analysis is in nmole: Gly: 28.7, Tyr: 22.6 and Thr : 43.9. Thr could not be demon¬ strated by the amino acid analysis. 14 EXAMPLE 4 0.8 mg (1.0 umole) Ile-Ser-Pro-Ser-Arg-Se ' r-Gln is dis¬ solved in 2 ml of the buffer described in example 1. 1 ml (22 units) of incubated leucine aminopeptidase is added, like in example 1. The mixture is left to stand at 37°C. After 30, 60, 90 and 120 minutes, 300 jul nmoles) are removed. The reaction is stopped as described in example 1. Amino acid analysis is carried out on 100 μl (corresponding to 27,78 nmoles) without preceding analysis. 18 nmoles lie are seen in all the samples. EXAMPLE 5 A synthetic peptide 0.9 mg (93 nmoles) Tyr-Pro-Ala-Gln- Ile-Lys-Val is treated with leucine aminopeptidase as described in example 1. 300 μl are removed after 30, 60 and 240 minutes. Amino acid analysis is carried out on 100 /J1 (corresponding to 25.8 nmoles) without preceding hydrolysis. Between 0 and 11 nmoles of Thr or Ser, Glu, Ala, Val, He and Tyr could be detected. This is probably present because the synthetic peptide was not quite pure and a proline is presumably missing, iU LΛ -y vvι. " o "

Claims: 15 P a t e n t c l a i m s: 1. A process for preparing ripe proteins by enzymatic cleavage of a fusion protein with the amino acid se¬ quence : (Ym Y 2 - ~(Pro) -(X 1 -X 2 ...X n ) in which (Y Y--Y. )-(Pro) is the pro-sequence and the rest is the ripe protein, is an integer greater than 2, and Y is an arbitrary amino acid, P is 0 if X, or X 2 is Pro, and 1 if X, or X„ is different from Pro, X is an arbitrary amino acid, and n is an integer equal to or greater than 4, c h a r a c t e r i z e d by carrying out the enzymatic cleavage by stepwise cleavage off of the amino acid groups Y ...Y if p = 1 or X. = Pro, or the groups m Z r 1 Ym...Y_.-Y1. if XZ„ = Pro, by means of an aminopeptidase, and then the two amino acids Y,-Pro if p = 1 are cleaved off enzymatically in one or two steps in a manner known per se, and similarly Y, alone is cleaved off if X. = Pro. 2. A process according to claim 1, c h a r a c t e r i z e d in that the aminopeptidase is leucine aminopeptidase. 3. A process according to claim 1 or 2, c h a r a c t e r i z e d in that the fusion protein includes the amino acid sequence occurring in a human growth hormone. G5MPE 16 4. A process according to claim 1 or 2,. c h a r a c t e r i z e d in that the fusion protein contains proinsulin.

Inventors: BALSCHMIDT PER, CHRISTENSEN THORKILD, DAHL HANS-HENRIK MARSTRAND, HEJNAES KIM RY, BALSCHMIDT, PER, CHRISTENSEN, THORKILD, DAHL, HANS-HENRIK, MARSTRAND, HEJNAES, KIM, RY
Applicants: NORDISK INSULINLAB, NORDISK INSULINLABORATORIUM
Application year: 1983
Main classification: C12P21
IPC codes: C07K14, C12N9, C12N15, C12P21