PH26508A - A method for the selective cleavage of fusion proteins - Google Patents

Abstract

Polypeptides or proteins such as, for example, insulins or de-B<3><0>-insulins - especially human insulin or de-B<3><0>-human insulin - are prepared by enzymatic cleavage, using a trypsin-like endopeptidase, of specific fusion proteins which can be obtained by genetic manipulation and which contain the residue of a peptide-like alpha -amylase inhibitor in the molecule. The use of the specific fusion proteins results in a reaction product which can be worked up advantageously; the working-up can take place, where appropriate, by crystallisation.

Description

Deseription. » The invention relates to a methed fer the prepara- tion of polypeptides er proteins by emsymatio cleavage of a fusion protein using as trypsin-like s endopeptidase, Lo

The increasing importance of recombinant DNA tech- - nology for obtaining polypeptides and proteins re- quires the development of new gethods fer enriching and purifying the products, which are appropriate for the (new) starting materiale.

In the senisynthetic preparation of human insulim, which is employed for the treatment of diabetes mellitus, by the so-called two-stage process, the feplacement of the amino acid ester in the ° po- “sition (porcine insulin = Ala « human insulin = : Thr) takes place in two separate, consecutive re- : | action stages. In the firat reaction stage the 2? : ~Ala residue of porcine insulin is eifninated ensy- matically using trypsin-like proteases, and them in | Y : 20 the second reaction stage a threonine derivative - e.g. threonine tert.- butyl ester (ThroBu®)- is coupled on, which likewise takes place in the pre- \ : sence of trypsin-like enzymes. An example of an en- } - syne for the two reaction stages is the Achromobaocter 5 protease I described by K. Morihara et al,, Biochemi-

) | Y 7s eal end Biophysical Research Bomvunications Vel. 92, No. 2, 1980, pages 396-403. This is a so- ¢alled endepeptidase vhich cleaves the peptide chains specifically on the carboxyl side of the enmine scid Lysine and, where appropriate, also reconstricts then.

After the alanine elémination and coupling on the threonine derivative have been completed, the pre-

B tective groups introduced with the threonine deri- vative are eliminated again for the. purpose of ob- taining the humam insulin without protective groups; " warious methods are ¥mown for this, of which elimi- sation of the protective groups using CF COOK is pre- ferred where appropriate.

Elimination of the B>C-Ala residue from porcine in- : eulin and coupling on of a threonine derivative can also take place in a single reaction step (“ene- : stage process"). Ia this case too it is nedessary to have an ¢nsyme present, suitable ensymes being virtually exclusively trypsin and trypsin-like ‘ endopeptidases; cf., for example, EP-B %6 951, After . . the "¢ranspaptidation" is compléte, the protective groups introduced with the threonine derivative have te be eliminated again,

For obvious reasons, attempts have algo beeson made in

Cae ‘

: | od qe the preparation of human insulin to get away from . the natural starting material porcine insulis, "which has also succeeded - at least in part-since the development of genatic engineering. Thue, for ~~ S example, a process for the preparation ef insulins oo

So especially of human insulin-from prepreoinsulin ana- legs ohtainable by genetic enginesring is desorived . in EP-B 89 007, in which preproinsulis analogs are : roactsd at a pH below the isoelectric point of the relevant insuline, using trypsin of a trypsin-1ike ) endopeptidase, with an exter of natural amine acids : ‘or derivatives therepf containing protective groups ‘(transpeptidation), and then the ester group and the . protective groups which Bre present where appropriate oo 15 are eliminated. The amine acid derivative preferably suployed for the preparation of human insulin in this “reaction is Thr (Bu®)-omu®.

Tne starting preproinsulin analogs specified im the said EP-B are these which carry on the N terminus eof : the proinsulim Lysine or arginine or the acylamine- ’ agyl redical thereof ass carboxyl, end of a PEON the formula of these prepreinsulin analegs according } to the I'-B is: | } gi ot “ww . - h - :

& 2

C chain ;

Lo om 2 eel . [ 88) bo Mo 1 cechain i —_— Of # p , - 8 ; ty CT I SA b

B chain oo in which Y denotes Lys or Arg and R' denotes hydro- f gen, an L-amino acid or she peptide residue X described hb herein after, X can be any peptide acting as signal sequence. A suitable proinsulin part is every preduct ~ encoded by a proinsulin gene which is natural or has i been synthetically modified by known nucleotide prepa- N ration processes. Analogous preproinsulins with a - shortened C peptide segment, such as is the case, for = example, in bovine C peptide by comparison with por- ot oine C peptide, are likewise suitable for the process. ' 4 ~ Buman preproinsulin shalogs are just as suitable, It | 7 ] ' is essential for the structure of the C peptide that : jt is linked via a basic L amino acid Y = L-Arg or L- ¥

Lys te the glycine of the {insulin A chaing i.e that a . structure Y-(AA) -T 1s present, where AA denotes all : encoded amino acids snd n is 0-35. Amine acid esters 3 and/or derivative thereof with a frees amino group cam ; ~ + “also be selected for the process in such a way that, ’ : if desired, an insulin analog with an unnatural se- : quence i3 produced. {

In the easymatic reaction, the pre part rl.y® and } the C chain y*0_p» are eliminated, and the appro- priate amino acid derivatives is coupled en at posi- tion 3°, | oo

It is conceivable that the d0-33%01nsulins can alse be odtained without addition of the appropriate amino acid derivative in this case,

Although this process has considerable advantages, es- pecially because it does aot depend on animal insulins as starting materials, neverthelsss the necessity for purification of the reaction products by column chroma- tography, and the associated less of substahce -which is often not negligible- is & certain disadventage. The : : necessity for purificatiom by column chromatography arises because peptide cleavage products are often ge- aerated in the enzymatic reaction and can be separated from the desired insulin (derivatives) only with diffi- culty, especially because of a similar mokecular chain ’ length,

The object now was to modify the process of the above- mentioned EP-B 89 007 in such a way that polypeptides or proteins - preferably de-3>°Zinsulins or humen in- sulins-are obtained and that the desired preducts re- sults in a from which can be easily purified with few losses during purification. :

Ts object has baen achieved according to the invent-

gE } i fon Wy ensymatic cleavage of specific fusiom pre- . teins using a trypsin-like endopeptidase. The specific fusion proteins are compounds of the fel- } lowing I : R-Lys -P - (Lys = r%), - (Lye) (1) in which

R denotes -(AA) o-(Peptide-like « -amylase inhibi- ter), vith AA = genetically econdable anine acid apart frem Lys, o = integers frem 0.200, preferably 5-100, in particular 10-12, and peptide- like eg -amylase inhibitor = residue of the ec amylase inhibiters knewa frem the literature,

P denotes the desired polypeptide er protein, : denotes =(AA) =o with AA = same meaning as under . | . 15. R (genetically encedable amine mcid apart frem

Lys), , p = integers from 1-35, preferably 1-10, ia par- ticular 1-3, and . n donetes O or 1 > na,

Hence the invention relates to a precess for a prepa- ration of pelypeptides ar preteinas by ensymatic cleavage oo oY 9%» " of a fusien pretein using a trypsim-like ende- peptidase, which cemprises using as specific fu- - sion proteins compeunds ef the fermula I abeve. ' The pelypeptidase er proteins generated in this enzymatic reaction-insulins such as, for example, do-BC-insulins or human insulin are preferred- are particularly easy to purify because of the « ~ amylase inhibiter residue in the fusion protein,

The compeunds are readily soluble in squeous me- | dium, are easily crystallized and have an affini- ty fer oc -amylase, which property in-tura oan be used te separate them off. For example, the de-p° ~insulins can be purified with less effort and fewer

Jesses than can the reaction products of the process of EP-B 89 077. In favorable cases, the working up of the reaction mixture can even be carried out merely vy crystallization - that is to say without : ' the necessity for purification by celumn chronatog- : - raphy. It is surprising that the residue of the of ~ : amylase inhibiters Ynewa frem the literature is met . cleaved by the trypsin-like endopeptidase in the re- action according to the invention, because these o - tnhibitors have the ir peptide chain a Lysyl radical . at which no cleavage takes place-~ in contract to, for example, the lysyl radicals in positions A, »®

CC - 8 -

: and 32? of the starting compounds ef the ae-3° insulins (see page 3); a cleavage of this type would generate fragments which can be separated frem the de-3>°_ insulins only with considerable : effort in some instances.

Suitable genetically encodable amino acids (apart from Lys) for AA are Bly, Ala, Ser, Thr, Val ,Leu, Ile,

Asp, Asn, Glu, Gln, Cys, Met, Arg, His, Iyr, Phe, Trp,

Pro, (neutral amino acids underlined). . 10 The radical P preferably represents the A and B peptide chains of human, porcine or devine insulia, in parti- , lar of human or porcine insulin, : } The radical R2 preferably comprises only the amine acids Ala or Thr, in particular only Thr. ‘A particularly preferred part - (An) - of the radical

R is the peptide chains '-Giy-Asn-Ser-Asn-Gly-Thr-A1a-Met-Ala-Asn-Phe-.

Buitable as peptide -like q -amylame inhibitor radi- cal in R are virtually all radicals of the og ~amylase - 20 inhibitors known from the literature, as are described,

Tor example, in: L. Vertesy ot al., Eure. J. Biochem. ih, 505-512 (198%); L. Vertesy, D. Iripier. TEBS~

Letters ¥ol. 18% , 187-190 (1985); O., Hoffmann et al.,

Biol. Chem Hoppe-Seylers Vol. 366, 1161-1168 (1985);

} of

H. Mural et al.., J. Biochem. 97, 1129-33 (1988), 8. Murao et al,, Agric. Biel Chem. 49, 107-110 (1985) and 49, 793-797 (1983); Japan Kekai 73/77594 (20. Nev. 1973) H. Gote et al.

;

A , - 7 -

S70 A preferred peptide-like a-amylase inhibitor radical is: -Leu-Cys-S-S-Cys-Leu-Gly-Glu-Thr-Asp-Asp-Glu-Tyr-Val-Val-Lys—

Arg

Ala Tyr

Leu Ala

Tyr val }

Arg Ala . Ala Pro

His Gly

Co Gly Gln i

A His lle g

Ser Thr

Gly Th

Ile Val - .

Tyr Gly : .. Gly-— Asp

LVal-Thr-Val-Thr-Glu-Ala-Cys-5-5-Cys-Ser-Pro-Ala-Pro-Glu-Ser-Val-Thr-Thr-Asp 6ly NH,

Co Asn val :

Asp Thr

Co Ala Leu gin Tyr ,

Ser Gin

Tyr Ser .

Arg — Trp . » : # - un - 2 Cm

: of

Particularly preferred products are the compounds : of the formula I with P= peptide sequence of hu- man er porcine insulin, RZ = Thr and R = the above- mehtioned preferred peptide sequences.

The fusion proteins of the formula I are prepared in the microorganism by generally known metheda by inserting a sequence of a foreign protein (of amylase inhibitor) in front of the amino acid sequence of the ~ desired polypeptide or protein (see, for example, © F.A.0. Harsten, Biechem. J. 240 (1986) 1-12). The preferred preproinsulin ahalogs, fusion proteins ; of the formula I in which P are the A and B peptide oC by genetic engineering, preferably by the process of the patent application "A" process for the prepara- 18 tion of an insulin precursor in Streptomycetes" (HOE Co 88/7313) filed at the same time. The insulih pre- cursors, which are usually excreted into the culture ’ medium, are preferably isolated from the filtrates of the fermemtation liquids. It has been found that many properties of the compounds defined by the formula I resemble the behavior of the said ec ~amylase inhibi- tors. Thus, many steps in the isclation and puri fi- ‘ . cation of the micrebial —— inhibitors mentioned can be employed to obtaim the fusion protein precur- : a5 | sors.

Cee Stn r———— of yo

Such steps in the purification are percipitations with salts, such as sedium chloride, ammonium sujl- fate inter alia, with acids such as, for example, metaphosphorie acid, tannin, trichlereacetic acid, s sufifuric acid and the like, with heavy metal salts and other precipitants such as, for example, pely- ethyleneimines, bentonite or others,

Whereas proteins obtained by genetic engineering often display undesired solubility behavior, and oo 10 thus are not amenable to easy precessing, the com- pounds of the formula I, which give clear solutions, - such as the pPreproinsulina analogs can, for example, ‘ be very straightforwarily concentrated by ultrafiltration and simultaneously have salts removed. Commercially available cellulose membranes, for example, are suit Co able for the ultrafiltration, :

It iw likewise possible to use adsorption resins ‘tor enriching and purifying precursors of the come . pounds of the formula I, such as, for example, in- sulin precursors. Such adsorption resins based en ~ polystyrene or styrene/-divinylbepsene copolymer i are commercially available and cen be purchased un- der the names X Amberlite XAD (Rohm & Haas, USA), ® Diaton BP-20 (Miteubishi Chemical Corp.) inter alia, ~ 13 a of ob

The isolation ef a protein using adsorption resins such as, for example Diaion HP-20 has already been described in German Offenlegungsschrift 3038130. . However, enrichment of proteins obtained by genetic engineering, such as, for example, insulin precur- sors, using adeorption resins has been impossible or possible only with great losses. This was owing, inter alia, to the excessively strong adhesion eof the desired substance to the support. It has nov deen found, surprisingly, that adsorption reains cam in fact be enployed advantageously 1f it is ensured ! that there is more mild, leas binding of the mate-~ rial. This inactivation can be effected, for exan- ple, by treating the support and/or by suitable ad- ditives in the material to be separated. An exsmple of the treatment of the support which may be mention- . ed ie: washing with aqueous organic solvents (con~ taining 2-70%, preferably 5.30%, solvents). Suit- - able organic solvents are these which are miscible oo with watery preferably lower alcohols and acetone.

The pretreatment of the support can additionally be effected by a washing with aqueous deteArgent solu~ tions, for example with 0.02-3% strength ‘Iriten x- | oo 100 solution, but preferably with 0.1-1% solution, however, a laArge number of other deteArgents are, oo - 1h -

oo ng f CL of qourse, also suitable. Likewise suitable is the oo addition of suitable additives to the molution ef the material to be separated and/er te the eluent. : Additives of this type are lower water-miscible i 5 erganio solvents and “‘atergents, in each case in the abovementioned concentrations, or so-called chaotropic avbstances such as, for example, peta- 3 : ssium perchlorate (0.1-3%) urea (1-8 molar) or } guanidinium hydrechloride, inter alia,

The fusion protein precursors (for example insulin prescursors) which have been, where appropriate, enriched and from which salts have been removed can : : be further purified by ion exchanger chromatography on cation exchangers or else on anion exchangers.

Examples of suitable cation exchangers are BP =~

Rsephadex, (Pharmacia, Sweden), Cm-cellulese, S~

R Sepharose (Pharmacia, Swedsn), R Fractegel TSK CM : (E. Merck, Darmtadt), R Fractegel TSK SP (E. Merok,

Darms@adt ) and other. Suitable anion exchangers ‘are “Fractogel TSK DEAE (E. Merck, Darmtadt), DEAE- cellulose, DEAE~ R Sephadex (Pharmacia, Sweden), Q-

Sepharose, QAE-Sephadex, QAE-cellulose and others.

The separations are carried out in a manner known : per se in agueous solution. It has proven advanta- 2% geous, however, in some cases in the separations te = 15 =-

BAD ORIGINAL AN add solubilizers and/or chaotropic substances to the water. Additives of this type are 2-8 molar urea, vetaine, lower alcohols, such as methanol, isepre- panel, ethanol, ethylene glycol and others. Suit- able purification operations are,in,addition, hydre- phobic chromatography, fer example en phenyl-Sepha-

Co rose, molecular sieve chromatography, for example using Biegel, preparative high-pressure liquid chro~ matography or chromatography en a column loaded with antibodies sgaint of -amylase inhibitor. ‘The properties of the ipsulin precursor which resemés- ble those of o<-amylase inhibitor also make pmmifica- tion by crystallization possible. It is possible to crystallize both in the neighborhood of the particu- lar iescelectric peint or in acid medium, where appre- priate using additives such as, for example, sodium chleride or other, Crpstallisation from aqueous selu- tien is also poseible, by addition of agents promoting orystallization, such as for example, of heavy metal ions such as 22*, Cu? and others, or of picric acid and the like, :

It im, An addition, very advantageous to utilize the eg -amylase- inhibiting property ef the proetins eof the formula I or of the cleavage products thereof for the purificatic:. Thie is bec- “se it has been found

Co -a6

N | we . | 4 that the proteins which have been obtained by gene~ ‘tic engimerring snd which carry the amino acid me- quence of the inhibitors also inhibit the <-srylase by complex formation. The inhibiter/q -amylase complex are less soluble than the starting cemponments ’ and can therefore be employed ror the separation. It is also possible selctively to separate the cempounds of the fermula I out of the culture filtrate using amylase : immobilized on suppotte and subsequently to separate, using suitable agents such as, for example, pH or salt ’ gradient, the complex found. A procedure of this type is also advantageous in the workigg up of the protecly-~ tic hydrolysate of the compound of the formula I. In this case, the inhibitor cleavege product which is no longer required is removed from the reaction mixture Co using immobilized q -amylase. The remaining product : (compound of the formula I, in particular de-3"-hu- . man insulin) is then already sufficiently pure te bp subjected immediately to crystallization. The orys- tailization is carried out by methods known per se. ’ Employd for the onzymatic cleavage of: the compounds gorresponding to the formula I re purified precur- sors, but it is also possible to use only partially purified starting materials.

The concentration of the starting material of the for-

Co - 17 -

of r formula I in the reaction mixture can vary withim relatively wide limits. A preferred concentration is of about 0.02 -15% by weight, in particular of about 0,05-5% by weight, based on the total rwact- : 5 ~ ion mixture. ’

Suitable trypsin-like endopeptidases are the endo- peptidases which are known from the literature as trypein-like, i.e, these which specifically cleave ) oo peptide linkages at the carboxyl end of basic amine + 10 acids§ some sppropriate citations are indicated in . oo the abovementioned It -B 89 007, or those which se- : lectively cleave behind Lyeine, see, for example,

EP-A 092 829 and US-A 4,514,332. The preferred trypsin- like endopeptidase is Lysyl endopeptidase from Lysobac- ter enzymogeries, which cleaves peptide linkages at the carboxyl end specifically of the basic amino scid ly- sine. Relevant literature relating to 1ysyl sndopepti- ’ . ' dase is, for exemple, P.A. Jeckel et al., Analytical

Biochemistry, Volume 13B (1983), pages 3497-354 and T.

Masaki et al,, Biochem. Biophys. Akad,, Volume 660, pages b4,f and S1.f (1981).

The concentration ef the trypsin-like endopeptidase in the reaction mixture is expediently adjusted to between ‘ : about one fiftieth and one ten-thousandth, preferably

CC | | x | : .

. ; : CL | - oo wt between 0.5 and 1.5% (w/w), in particular of about one thousandth, of the weight of the fusion protein of the formulas I.

The enzymatic cleavage of the compounds of the for- mula I i8 carried out in aqueous solution, vhere appropriate in the presence of a pay bALLTeE {such : - a, for example, ureds isopropanol etOey)y Bt 2 oH petween about 5 and 114 preferably petween about 7 and 9.5. It is expedient to use one of the customary ’ buffer pystems guch as, for examples phosphate puffer,

Trig/HCYL » NaC) /NeHCO3 for adjusting and maintaining constant the pHa ~The temperatureof the enzymatic cleavage CAD be between about 1 and ¢0%cg it is preferably between about 15 and 40°C.

The cleavage te generally complete in about 0.1 to 2 - hours, put shorter of jonger times may result depend~ ing on the specific reaction conditions.

After the enzymatic cleavage 1a completes which can »e established, for example by chromatography any further, undesired cleavage is stopped by changing the reaction medium in 8 kbown manner, for example BY changing the ’ 2K, cooling, adding inhibitors etoes and the 40-8 insulin formed is jsolated bY methods known per 86. _ 19 -

ob } : .

Although the isolation can be carried out by chro- matography, as in the known relevant methods, alse possible in the present case im, in principle, is0~ lation by crystallization, vecause the de-B°-insu- - s | 1in differs considerably in respect of chain length, and thus of molecular weight, from the by-products. :

A"transpeptidation" is carried out in aqueous/erganic solution (for example water; DMF, waters DMSO) in all mixing ratios, preferably mixtures of 1th to 1:1 (sol- | [, vents water). The pH ought to be between 4 and 8, . preferably between 5 and 6 the temperature between 1 and 50°C, preferably between 15 and 30°¢C.

After the enzymatic "transpeptidukdon" is complete, which can be established, for example by Le, a . possible reverse reaction, such as, for example to de- . 3°. insulin, is prevented by addition of inhibitors such ss, for example, aprotinin, butylamime or tosyl-

L-Lysine chioromethyl ketone. It is then possible , without difficulty to dilute and puréfy by prepmmative \ column cgromatography, and thua alse to separate eff \ from unreacted starting material, Crystallisation is ~ 1ikevise possidle in this case. The elimination of the protective groups is carried out by methods known the literature. :

Rr CoRR

: ¢ we

The process according to the invention is dew to be expalined in more detail dy the examples which follows The examples (of the invention) are, more~ ever, followed by a comparison example which shows that the ensymatic cleavage of a preproinsulia ana- logs of the general formula in EP-B $9 007 using

Lysyl endopeptidase yield a reaction predust which ean be worked up with difficulty and much less.

Examplel 30 1 of culture broth which has been fermented to eobd- taiz the construct pGF2 is centrifuged and gubse- quently sterile filtered omce again. The solutiom is adjusted te pH 7.2 and, after addition of 3 1 »f ispropancl, loaded on a 4 1 Fpisien HP-20 column which has previously beem washed with 10% strength _ isepropanel. The newly constructed protein pra2 is eluted from the celumn by applying a 10-50% strength , 1sopropanol gradient. The fractions containing the desired product are subseqpently dirsctly loaded en : a prepared DEAE-" Spharesté (Pharmacia, Sweden) fast flew column, pH 7.2 300 ml (3 x 15 on’), washed with phosphate buffer and finally eluated with a 0 to . 0.5 molar sodium chloride gradient , pH 7.2. The protein pFN2 is to be found ia the fractions im which 2s the conductivity of the solution is 20 to 30 mS. The ot 9 latter are combined and precipitated with a ammonium sulfate (35% saturation). The precipitate which forms is removed By centrifugation after 16 hours, dissolved in water, reprecipitated »y adjusting the pH to k.6 and, in this instance, collected by eon- trifugation after only 2 hours. It contains the pro- tein pGE2 which is already greatly enriched. The fi- nal purification is carried out on R Macrobe Nucleo- si1 (Macherey and Nagal), Durem 12--10 Ch, with the dimensions of the column being chosen such that 100 mg of pGH 2 is separated on a column volume of 100 ml. : The preparative HPLC column is likewise develope using the 0.1 trifluoroacetic acid/acetonitrile system. The . fusion protein is eluted with 33% strength acetonitrile.

The solid substance is obtained by drying in vacuo.

Example 2 ( 10 mg of fusion protein obtained as in Example 1 i= dissolved in O.4 ml sodium acetate buffer, PH 5.3, and 0.1 ml of DMF. To this are added 140 mg of Thr (But)oBu® and BO jul 1ysyl endopeptidase (2.7 mg/ml} . dissolved in water). The mixture is left to s:ant : at room temperature. The reaction is followed by HPLC a analysis (system as in Example 3).

Samples are taken at various times and precipitated using twice the volume of methanol and 10 times the volume of methyl t-dutyl ether. The precipitate is taken up in 10 times the volume of 0.1% strength s triflueroacetic acid and analysed by HPLC. About 89% of human insulin ester has been produced after 40 he. The remainder is essentially 4e-3"°-1nsulia.

Example 3

The precipitate obtained as in Example 1 is taken up in the minimum amount of 0.1% strength triflnore- acetic acid and subjected to preparative HPLG. The stationary phsse employed is a Bakerbond vide-Pore™™

Cy/3 Jom with the dimensions 4.6 x 250 mm. The mo~- bile phase has the follewing composition: 13 “Ar 0. & TFA

Bs A/CH CN 10/90

The flow rate is 1.3 ml/yin. The gradient has the fel- lowing composition: ’ % %3- 0 2% 20 hs 2s bs 28 25 23 :

of wd

De-3"C-insulin elutes at 15.1% — insulin eater at 20.92 min. The solvent is removed from the fractions im vacuo, the selid is then washed with acetonitrile and the selvent is remevedd ia - 5 . Yacue,

Example & 100 mg of the protein pGH2 are dissolved in 10 ml of Tris/Hel buffer pE 8, and incubated with 50 =¥ of Lysyl endepeptidase. After 2.5 hours, the mix- ture is acidified to pB 3 with trifluoreacetioc acid to stop, and the reaction mixture is subjected to a reverse~phsse fractionatiom. A sharp peak eluted . with 30% strength acetonitrile uarks the appearance of 3>%.dethreonine human insulin in the column ef- ’ fluent. The preduct from which solvent has been re- moved weighs 35 mg, correppending to 95% of theory. . 90 mg of X ~anylase from porcine pancreas are dis- solved in 35 ml of 0.5 M potassium phosphate buffer, ce | pH 7.8, and dialysed against the same buffer over- Te night. ) . 50 g * Atfigel 10 {bdoRad 1535-ED46) are washed im socordance with the manufacturer's instructioas and equilibrated with 0.5 M potassium phosphate buffer

Sah -

§ wd (see above). The moist gel obtained in this way is left to react with the enzyme solution,shaking gent- 1y, at room temperature for 2 hr. It is then fil- tered off with suction and briefly washed binding suffer. The support is subsequently {nactivated with © 0.1 M ethanolamine, pH 8.0, for 1/2 h and finally giltered off with suction again and washed several times with 1 M NaCl snd binding buffer alternately.

About i44 g ef moist gel are obtained and are stored under 0.02% azide in 50 mM potassium phosphate buf- gor at 4°C. The resulting binding balance is as fellows 3

Initial 1004 units = 100%

Combined washing water 705 units = 70%

Support: 181 units = 10%

Agaay method: Behring nTestomar" asday kity

Procedure in accordance with EN [ manufacturer's instructions

Example 6 . | i hi g of amylase immobilised as in Example kb are stir- red with 10 mg of pGH2 degraded as in Example 4, with- ' -out purification, with the addition of = protasse in- hibitor in 200 ml ef water. “fter 1/4 hour the mix- ture is filtered with suction, and the clear liquid : phase is freese-dried. The result is $ mg of a coler-

less product which, taken up in a little water, results in crystalline do-3° -fnsulin by addi- tion of 202. to vo i oo : .

N\ - 26 = Co

Claims (1)

1. PATENT CLAIMS:

   1. In a process for the preparation of a polypep- tide er protein by ensymatic cleavage of a fu- sion protein using a trypsin-like endopepti- dase, the improvement comprising using as fu- sion protein a compound of the formula I . } - \ R-Lys -P - (Lys - R ) a - (Lys) , (1) in whieh L R denotesii(AA) -(peptide-like « -amylass inhibiter), with AA = genetically encedable amino acid aprt from Lys, e = integers from 0-200, and peptide- like o -amylase inhibitor = residue of the « ~ amylase inhibitors known from the literature, 'P denotes the desired polypeptide or protein, 2 denotes =(AR) =o with AA = same meaning as under - R (genetically encedable amino acid apart frem Lys), p = integers from 1-35, snd u denotes O or 1 > Re . ' ’ 2e A process as claimed in claim 1, wherein a compound of the formula I where R> denotes ~(AR) oy with Ake CL <i qt & same meaning as vunder:R and peintégers from i " 1-3.

   3. A process as classed 1, wherein a cempeund of the formula I where R denotes -(AR) - _ peptide-like ac -amylase inhibitor), with AA= genetically encodable amine acid apart frem Lys, O= integeras from 5-100. 4, A process a claimed in claim 1, wherein a com- pound of the formula I where R denotes-(AA) - peptide-1ike of -amylase inhibitor), with AAs genetically bmcodable amine acid apass from : Lys, O = integers from-1-10.

   5. The process as claimed in claim 1, wherein a compound of the formula I where rl is composed only of Ala or Thr, and m and n each denote 1, is used as fusion protein.

   : 6. The process as claimed in claim 1, wherein a compound of the formula I where rR is composed only of Thr and m and n each denote 1, is used fusion proteins. -

   7. The process as claimed in claim'l, whereina cem- pound of the formula I with .

   ee mm ns = re Ea Le Sa we sR een ed! id eden ces LE Ely Bri ib 4 Lim nel ante From 03) yim Sey ae 3 we Thr ea ba-Hat-ala Aerie I eee eee ee eee toro omer ee £4 08 4. i RAR 11 4A A Ra 1 m7 a en ree Lg pet em Ge Ure Lat (3 Dy = GB hue Thr fig pr-eesp SA ue Tyra lam] Lye i i } Arg to i 1 1 1 1 ' 1 Ala Tvr t i i ) 1 . 1 1 1 Loss fla ! . 1 ¥ + 1 ' i Tyr Val ! ’ F 1 1 1 t C - Arg Ala ! ; - : Ala Fro ! i : } ! } ¢ His 3 1 kd }

   1. 1 3 I. 1 ) 1 { — “ Ely Gin } 5 t 1 + { . ' ! J : . ‘ 4 His Iles \ 1 + 1 1 1 1 Ser The ! ! { Bly Thr ! : I 1 1 : , i 1 1 oo lle Val ! Lr ) ! ! : 1 1 ' Tor 31 - \ | 1 1 1 1 Gly r————Asn : i Mal-The=al-Thr-Glu-Al a-Lye-G-S-Dve-Ser-Fro-Al a-Fro-Glu-ter-Val ~Thr—Ttr sp ! Gl | Pb ‘ ' * ! ; ‘ Amn Val 1 1 1 1 NE [3 hr ’ t 1 . ! ! yk a [= F . | . C : ; ui Lin Tor 1 1 1 + . Sent Gin r ’ ! ! Tor Ser . 1 ¥ v . ar gem ING fo ! im used as fusion protein. - 29 = ” La Sve sid AR =~ : air t mees aspen: AHHGEEARY } Cr Le anmERY si SRE TE a To

   - Co x, : oo ef NY

   8. The process as claimed in claim 1, wherein : the fusion protein of the formula I is em- pleyed in a concentration of about 0.02-15% by weight, based on the total resection mix- ture. oo Ce

   9. The process as claimed in claim 8, wherein the . fusion proteim of the formula I is empleyed in Co A a concentration of about 0.5-95% by weight, based on the total reaction mixture. 10, The process as claimed in claim 1, wherein 1ysyl endopeptidase is used as trypsin-like endopepti- dase,

   11. The process as claimed in claim 1, wherein the I trypsin-like endopeptidase is empleyed in a | : : concentration between about one fiftieth and one . tentfousandth of the weight of the fusion pro- ; tein of the formula I, oo

   > . . . ’ '

   12. The process as claimed in claim 1 wherein the on-~ synatic oleavage is carried out in aqueous solu~

   : . tion, where appropriate in the presence of a solu- sulizer, of pH between 5 and 11.

   13. The process as claimed im claim 12, wherein the enzymatic cleavage is carrisd out in aqueous solu- - a

   . Se | Ce TETAS eg 7 = J ’ or I | 08. . 7 - * tiom, where appropriate in the presence of a solubiliser, of pM between about 7 and 9.5%, : “1he The process as claimed in claim 1, wherein the reaction mixture is worked up by erystallization | ; after the ensymatic cleavage is complete. nn TT : 3 LABSLO VERTESY ee KALUS SAUBER Le EBERHARD EELERS : -- Inventors — . 7 } | . . : ! } ~~ rN - : oo TT v . :

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