PROCESS FOR PREPARING A DESIRED PROTEIN

Abstract

A desired protein having the formula:

Description

[0001] The present invention concerns a process for preparing a desired protein having the formula stated in the introductory portion of claim 1.

[0002] It is known from the U.S. Pat. No. 4,342,832 to produce biosynthetic hGH by fermentation of a recombinant host cell, in particular E. coli, which codes for hGH with associated methionine. However, this known process results in hGH whose N terminus has attached to it the amino acid methionine which is not present in ripe hGH.

[0003] Owing to the risk of antigenic reactions and other side effects in the use of a growth hormone which is not quite identical with hGH, it is inexpedient to use biosynthetic Met-hGH.

[0004] Accordingly, there is a great need for a process which enables production of biosynthetic hGH with a correct amino acid sequence. A solution to this problem has been proposed by U.S. Ser. No. 488,232 (DK Patent Application 2046/84), which concerns a process for producing hGH from pre-hGH in a recombinant prokaryotic microorganism, such as Pseudomonas aeruginosa or E. coli.

[0005] The use of Ps. aeruginosa for the production of hGH without methionine for therapeutic use, however, is vitiated by the risk that this bacterium and many other Pseodomonas bacteria, which are potentially pathogenic, synthetize toxic toxines which are problematic.

[0006] The expression of pre-hGH followed by proteolytic cleavage to obtain the ripe hGH in an E. coli (which is not pathogenic) is indicated in the DK Patent Application 2046/84, but it is not documented in that specification that the proteolytic cleavage unambiguously leads to the formation of ripe hGH, i.e. with a correct amino acid sequence.

[0007] As mentioned above, risks may be involved in using Met-hGH. Though methods have been proposed for enzymatic cleavage of the methionine group by means of aminopeptidases, the problem would not be solved by this because the known enzymatic processes of this type do not lead to a 100% conversion. A mixture of hGH and Met-hGH would occur, which cannot be separated completely by conventional preparative purification processes.

[0008] The present invention is based on the finding that the enzyme dipeptidyl aminopeptidase I (DAP I) or cathepsin C (EC(3,4,14,1)) is suitable for cleaving an N-terminal amino acid sequence with an even number of amino acids to form a desired protein having the formula:

A-B-C-P

[0009] wherein

[0010] a) A is Lys or Arg, and B and C are arbitrary amino acids, or

[0011] b) A is an arbitrary amino acid different from Pro, Lys and Arg, and B and/or C is Pro,

[0012] and P are in both cases the residual amino acid sequence in the desired protein.

[0013] Thus, DAP I has been found suitable not only for production of hGH in which the three first amino acids are Phe-Pro-Thr, but proteins in general which satisfy the conditions of the sequence A-B-C-P.

[0014] Thus, the process of the invention is characterized in that a biosynthetically formed amino terminal extended protein having the formula:

X-A-B-C-P,

[0015] wherein A, B, C and P are as defined above, and X is an amino acid sequence having an even number of amino acids, of which the first one, seen from the N-terminal end, is different from Lys and Arg, all other uneven amino acids are different from Pro, Lys and Arg, and all even amino acids are different from Pro, is reacted with the enzyme dipeptidyl aminopeptidase I (DAP I).

[0016] Examples of proteins which may be produced by the process of the invention are the following:

Proteins with lysin on 1st site
	Name	Origin	N-terminal sequence
	Cholecystokinin	Porcine	Lys-Ala-Pro-
	Neurotoxin I	Scorpion	Lys-Asp-Gly-
	Penicillinase	Staphylococcus	Lys-Glu-Leu-
		Aureus
	Ribonuclease	Bovine	Lys-Glu-Ser-
	Proparathyrin	Human	Lys-Ser-Val-
	Lactalbumin	Human	Lys-Glu-Phe-
	Kallidin II	Human	Lys-Arg-Pro-
	Purothionine A-I	Wheat	Lys-Ser-Cys-
	Viscotoxin A3	Eru. Mistelten	Lys-Ser-Cys-
	Lysozyme	Human	Lys-Val-Phe

[0017]

Proteins with arginine on 1st site
	Name	Origin	N-terminal sequence
	Beta Casein	Bovine	Arg-Glu-Leu-
	Posterior Pituitary	Bovine	Arg-Gly-Glu-
	Peptide
	Serum Albumin	Bovine	Arg-Gly-Val
	Precursor
	Long Neurotoxin I	Black Mamba	Arg-Thr-Cys-
	Tuberculin-Active	Mycobacterium	Arg-Leu-Leu
	Protein	Tuberculosis
	Bradykinin (Kalli-	Bovine	Arg-Pro-Pro
	din I)
	Amyloid Protein AA	Human	Arg-Ser-Phe

[0018]

Proteins with proline on 2nd site
	Name	Origin	N-terminal sequence
	Choriogonadotropin	Human	Ala-Pro-Asx-
	Follitropin (α-chain)	Human	Ala-Pro-Asp-
	Pancreatic Hormone	Bovine	Ala-Pro-Lys-
	Aspartate Aminotrans-	Porcine	Ala-Pro-Pro-
	ferase
	Plasminogen	Human	Glu-Pro-Leu-
	Insulin-like	Human	Gly-Pro-Glu-
	Growth Hormone
	Prealbumin	Human	Gly-Pro-Thr-
	Prolactin	Porcine	Leu-Pro-Ile-
	Lipid-binding Pro-	Human	Thr-Pro-Asp-
	tein C-I
	Cholera Enterotoxin	Vibria	Thr-Pro-Glu-
	(β-chain)	Cholerae
	Prolactin	Bovine	Thr-Pro-Val-
	Lymphotoxin	Human	Lys-Pro-Gly-
	Interleukin-2	Human	Ala-Pro-Thr-
	Erythropoietin	Human	Ala-Pro-Pro-

[0019]

Proteins with proline on 3rd site
	Name	Origin	N-terminal sequence
	Neurocarzinostatin	Streptomyces	Ala-Ala-Pro-
		Carzinostaticus
	Somatotropin	Bovine	Ala-Phe-Pro-
	Carbonic Anhydrase B	Human	Ala-Ser-Pro-
	Toxin II	Sea Anemone	Gly-Val-Pro-
	Allergin RA5	Wormwood	Leu-Val-Pro-
	Lac Repressor	E. coil	Met-Lys-Pro-
	Alcohol Dehydrogenase	Yeast	Ser-Ile-Pro-
	Orosomukoid	Human	Glx-Ile-Pro-
	Interleukin-1	Murin	Ser-Ala-Pro

[0020] Examples of starting materials which may be cleaved with DAP I are the following:

	Met-Glu-Ala-Glu	hGH	to obtain hGH
	Met-Phe-Glu-Glu	hGH	(proline on 2nd site)
	Met-Thr-Glu-Glu	hGH
	Met-Glu-Glu-Glu	hGH
	Ala-Ala-Glu-Glu	hGH
	Met-Phe-	Glu-hGH	to obtain Glu-hGH
	Met-Leu-	Glu-hGH	(proline on 3rd site)
	Ala-Glu	Glu-hGH
	Met-Ala-	Glu-hGH

[0021] The present process is thus suitable for production of biosynthetic proteins, such as hGH having attached to it a pre-sequence which can be cleaved enzymatically in a high yield, and which gives products by the enzymatic cleavage which may be separated satisfactorily by known purification methods, such as ion exchange.

[0022] Examples of suitable amino terminal extensions which may be cleaved by means of DAP I are those in which the last amino acid in the amino acid sequence X, before A, is an amino acid with a charged side chain, such as Glu or Asp.

[0023] These amino terminal extensions may be obtained by fermentation in a suitable substrate of a microorganism which is transformed with a plasmid coding for the desired extended protein.

[0024] After expression, the methionine residue is optionally cleaved enzymatically in the microorganism so that the recovered protein is attached to the desired amino terminal extension with an even number of amino acids which may be cleaved selectively and in a high yield. Isolation of the resulting protein takes place in a manner known per se, e.g. by chromatographic methods.

[0025] By selecting an amino extension which contains at least one amino acid with a charged side chain, such as a carboxyl group, it it possible to perform the separation and the purification of amino terminal extended protein from the ripe protein.

[0026] At least one of the charged amino acids may be attached directly to the N-terminal end of the protein because it may then be observed whether the entire amino terminal extension has been cleaved. This is particularly important when the microorganism in vivo only partly cleaves the N-terminal methionine residue.

[0027] It is most expedient that an amino acid with charged side chains in the amino terminal extension to the protein is either exclusively positively or negatively charged. This prevents amino terminal extended protein, partly enzymatically converted amino terminal extended protein and authentic protein from having the same net charge at any time.

[0028] In hGH, slight deamidation of certain Gln and Asn residues takes place, i.e. Gln and Asn are converted to Glu and Asp, respectively—i.e. amino acids with negatively charged side chains. For this reason it will therefore be most expedient that the charged amino acid in the amino terminal extension are the negatively charged Glu and/or Asp, because this avoids the situation of one or more deamidations in hGH neutralizing the positive charge/charges present in the extension. Such neutralization of charges will make it impossible to separate possibly unreacted deamided amino terminal extended hGH by ion exchange from the enzymatically formed hGH.

[0029] Examples of particularly suitable amino terminal extensions which may be cleaved with DAP I are

[0030] 1. Met-Glu-Ala-Glu

[0031] 2. (Ala-Glu)r, wherein r is an integer from 1 to 12

[0032] 3. Met-Phe-Glu-Glu

[0033] 4. Thr-Glu-Ala-Glu

[0034] 5. Met-Asp-Ala-Asp

[0035] 6. Met-Glu-Ala-Asp

[0036] These and other suitable amino terminal extensions may be obtained by fermenting in a suitable substrate a micro-organism transformed with a plasmid, which codes for the desired protein with these attached amino terminal extensions.

[0037] In some specific pre-sequences, methionine, which is the N-terminal amino acid in all proteins formed in E. coli, is cleaved enzymatically in the microorganism after expression of the protein. This results e.g. in the above-mentioned amino terminal extended proteins.

[0038] These proteins are purified by conventional purification methods. The amino terminal extension is cleaved selectively and in a high yield. The formed protein may then easily be separated from any residues of partly converted amino terminal extended protein by known chromatographic methods.

[0039] The process of the invention will be illustrated more fully below by means of some working examples.

EXAMPLE 1

[0040] Preparation of hGH by Means of DAP I

[0041] A cloned DNA sequence which codes for a protein having an amino acid sequence like human growth hormone, hGH (191 amino acid residues, the first four amino acids of which are Phe-Pro-Thr-Ile) 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 above-mentioned gene, and the 5′ end of the synthetic DNA sequence strand is coupled to the 3′ end of the above-mentioned gene by blunt end ligature

[0042] +5′ CGATG GCT GAA

[0043] −3′ TAC CGA CTT

[0044] where the 2 first nucleotides in the +strand are a ClaI restriction site overhang, and the following nucleotide sequences code for the amino acids Met-Ala-Glu-.

[0045] The above-mentioned gene is introduced by ordinary gene cloning techniques into an expression plasmid containing a fusioned Trp-Lac promotor as well as the SD sequence AGGA. This structure expresses Met-Ala-Glu-hGH.

[0046] This plasmid 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 5 l scale. The cells were harvested by centrifugation and are suspended in a small volume and lyzated using a so-called “French press”.

[0047] The expected fusion protein could be demonstrated in the above-mentioned bacterial extract by immunological methods using hGH antibodies, corresponding to a concentration of 200 mg/l in the culture medium.

[0048] The fusion protein is purified conventionally by anion exhange, ammonium sulfate precipitation and hydrophobic chromatography.

[0049] The purified Met-Ala-Glu-hGH was evaluated to be more than 99% pure, evaluated by SDS electrophoresis.

[0050] An amino terminal sequence determination showed that the purified hGh material had the sequence Ala-Glu-hGH, which means that Met has been cleaved by an E. coli enzyme.

[0051] 100 mg of AE-hGH in 10 mM Tris-Cl. pH 4.2 (1.5 mg/ml) were admixed with 5 mg of DAP I (3,4,14,1).

[0052] The reaction mixture was then incubated at 40° C. After 4½ hours the mixture was cooled to 4° C. The cooled reaction mixture was then fractionated by anion exchange, and following this the main peak (hGH product) was isolated. The yield was 90%.

[0053] The hGH product was shown to be more than 99% pure, evaluated by SDS electrophoresis. An amino terminal determination (Edman degradation) showed that the amino terminal sequence of the hGH product was Phe-Pro-Thr-Ile-Pro-, i.e. as for authentic hGH.

[0054] The biological activity of the hGH product was determined by a tibia test and was found to be 2.5 IU/mg, which is also the case with authentic hGH.

EXAMPLE 2

[0055] Preparation of hGH from Met-Glu-Ala-Glu-hGH with Dipeptidyl Aminopeptidase I, (DAP I)

[0056] Met-Glu-Ala-Glu-hGh is produced by gene techniques in principle as described in example 1. Met-Glu-Ala-Glu-hGH is purified from the fermentation product by anion exchange and hydrophobic interaction chromatography.

[0057] The purified Met-Glu-Ala-Glu-hGh was evaluated to be more than 99% pure by ion exchange and SDS electrophoresis.

[0058] An amino terminal sequence determination showed that the purified hGH had the sequence Met-Glu-Ala-Glu-Phe-Pro-Thr-Ile-Pro-Leu, where the last six amino acids correspond to the N-terminus in hGH. 200 ml of Met-Glu-Ala-Glu-hGH 2.0 mg/ml) in 20 mM Tris, 10 mM citric acid, 25 mM Nacl, pH 5.2 were admixed with 10,000 mU (corresponding to 3.3 mg) dipeptidyl aminopeptidase I (E.C. 3,4,14,1) from Boehringer Mannheim. Other makes may be used as well. The pH value is optionally readjusted to 4.2.

[0059] The reaction mixture was then incubated at 40° C. for 60 minutes, resulting in a more than 98% conversion of Met-Glu-Ala-Glu-hGh to hGH. The reaction mixture was cooled to 4° C. after completed reaction. The further purification comprises isoprecipitation, gel filtration and an anion exchange.

[0060] The hGH product was shown to be more than 99% pure evaluated by IE-HPLC and SDS electrophoresis. An amino terminal sequence determination by Edman degradation showed that the amino terminal sequence of the HGH product was Phe-Pro-Thr-Ile-Pro-Leu, i.e. as for authentic hGH.

[0061] The biological activity of the hGH product was determined by a tibia test and was found to be equipotent with pituitary hGH.

EXAMPLE 3

[0062] Preparation of hGH from Met-Phe-Glu-Glu-hGH with Dipeptidyl Aminopeptidase I

[0063] Met-Phe-Glu-Glu-hGh is produced by gene techniques in principle as described in example 1. Met-Phe-Glu-Glu-hGH is purified from the fermentation product by anion exchange and hydrophobic interaction chromatography.

[0064] The purified Met-Phe-Glu-Glu-hGH was evaluated to be more than 99% pure by IE-HPLC and SDS electrophoresis.

[0065] An amino terminal sequence determination showed that the purified hGH product had the sequence Met-Phe-Glu-Glu-Phr-Thr-Ile-Pro-Leu, where the last six amino acids correspond to the N-terminus in hGH.

[0066] 100 ml of Met-Phe-Glu-Glu-hGH (1.5 mg/ml) in 20 mM Tris, 10 mM citric acid, 25 mM NaCl, 1 mM L-Cysteine pH 4.2 were admixed with 15,000 mU (corresponding to 5.0 mg) aminopeptidase I (E.C. 3,4,14,1) from Boehringer Mannheim. Other makes may be used as well. The pH value is optionally readjusted to 4.2.

[0067] The reaction mixture was then incubated at 40° C. for 60 minutes, resulting in a more than 98% conversion of Met-Phe-Glu-Glu-hGH to hGH. The reaction mixture was cooled to 4° C. after completed reaction. The further purification comprises isoprecipitation, gel filtration and an anion exchange.

[0068] The hGH product was shown to be more than 99% pure evaluated by IE-HPLC and SDS electrophoresis. An amino terminal sequence determination by Edman degradation showed that the amino terminal sequence of the hGH product was Phe-Pro-Thr-Ile-Pro-Leu, i.e. as for authentic hGH.

[0069] The biological activity of the hGH product was determined by a tibia test and was found to be equipotent with pituitary hGH.

EXAMPLE 4

[0070] Preparation of hGH from Ala-Glu-Ala-Glu-hGH with Dipeptidyl Aminopeptidase I

[0071] Met-Ala-Glu-Ala-Glu-hGH is produced by gene techniques in principle as described in example 1. Met is cleaved in vivo so that the protein formed by fermentation is Ala-Glu-Ala-Glu-hGH. This is purified conventionally by anion exchange and hydrophobic interaction chromatography.

[0072] The purified Ala-Glu-Ala-Glu-hGH was evaluated to be more than 99% pure by IE-HPLC and SDS electrophoresis.

[0073] An amino terminal sequence determination showed that the purified hGH product had the sequence Ala-Glu-Ala- Glu-Phe-Pro-The-Ile-Leu-Pro-Leu, where the last six amino acids correspond to the N-terminus in hGH.

[0074] 100 ml of Ala-Glu-Ala-Glu-hGH (2.0 mg/ml) in 20 mM Tris, 10 mM citric acid, 25 mM NaCl, pH 4.2 were admixed with 20,000 mU (corresponding to 6.7 mg) Dipeptidyl Aminopeptidase I (E.C. 3,4,14,1) from Boehringer Mannheim. Other makes may be used as well. The pH value is optionally readjusted to 4.2.

[0075] The reaction mixture was then incubated at 40° C. for 60 minutes, resulting in a more than 98% conversion of Ala-Glu-Ala-Glu-hGH to hGH. The reaction mixture was cooled to 4° C. after completed reaction. The further purification comprises isoprecipitation, gel filtration and an anion exchange.

[0076] The hGH product was shown to be more than 99% pure evaluated by IE-HPLC and SDS electrophoresis. An amino terminal sequence determination by Edman degradation showed that the amino terminal sequence of the hGH product was Phe-Pro-Thr-Ile-Pro-Leu, i.e. as for authentic hGH.

EXAMPLE 5

[0077] Preparation of ILIβ from Met-Glu-Ala-Glu-ILIβ

[0078] Biosynthetically produced Met-Glu-Ala-Glu-ILIβ was purified and isolated by chromatography, and the eluate was admixed with 0.38 unit of DAP I (from Boehringer Mannheim, called cathepsin C, 21.9 IU/ml) per mg of protein, calculated on the basis of E (280, 0.1%)=0.6. The reaction mixture was left to stand for 45 min. at 37° C. The solution was dialyzed against 20 mM Na-citrate, 2 mM EDTA, pH=4.0 at 4° C. for 18 hours.

[0079] The dialysate was applied to an FF-Q Sepharose CL6B column in Tris-Cl pH =8.0 with an NaCl gradient to 0.2 M.

[0080] The ILIβ fraction was concentrated by ultrafiltration with a 10 ml Nova cell to a volume of 2.0 ml (c=7.0 mg per ml). The pooled concentrate was applied to a Sephacryl column in 0.5 M Na-acetate, pH=3.5.

[0081] The product was characterized by amino acid analysis and N-terminal sequence analysis. The sequence was shown to be identical with the first 42 N-terminal amino acids in authentic ILIβ.

EXAMPLE 6

[0082] Preparation of Human Lysozyme (hLZ)

[0083] Usual biotechnological methods are used for preparation of the gene for the protein MFEE-hLZ, where hLZ has the amino acid sequence:

[0084] 1 K V F E R C E L A R T L K R L G M D G Y R G I S L A N W M C

[0085] 31 L A K W E S G Y N T R A T N Y N A G D R S T D Y G I F Q I N

[0086] 61 S R Y W C N D G K T P G A V N A C H L S C S A L L Q D N I A

[0087] 91 D A V A C A K R V V R D P Q G I R A W V A W R N R C Q N R D

[0088] 121 V R Q Y V Q G C G V *

[0089] The gene is introduced into a suitable expression system and cultivated to form MFEE-hLZ. This protein was purified and treated with DAP I under the conditions stated in example 1. Thereby, authentic pure human lysozyme is isolated.

EXAMPLE 7

[0090] Preparation of IGF-1

[0091] Usual biotechnological methods are used for the preparation of a plasmid which codes for an extended human insulin-like growth factor 1 having the formula Met-Phe-Glu-Glu-IGF-1, where the sequence IGF has the following structure:

1                                  10
Gly-Pro-Glu-Thr-Leu-Cys-Gly-Ala-Glu-Leu-Val-Asp
20
Ala-Leu-Gln-Phe-Val-Cys-Gly-Asp-Arg-Gly-Phe-Tyr-
30
Phe-Asn-Lys-Pro-Thr-Gly-Tyr-Gly-Ser-Ser-Ser-Arg-
40
Arg-Ala-Pro-Gln-Thr-Gly-Ile-Val-Asp-Glu-Cys-Cys-
50                                      60
Phr-Arg-Ser-Cys-Asp-Leu-Arg-Arg-Leu-Glu-Met-Tyr-
70
Cys-Ala-Pro-Leu-Lys-Pro-Ala-Lys-Ser-Ala

[0092] The plasmid is introduced into E. coli, which is cultivated under usual conditions. The formed fusion product is isolated and purified in a known manner and treated with the enzyme DAP I to form authentic human IGF-1.

EXAMPLE 8

[0093] Preparation of Bovine Growth Factor, bGH

[0094] Usual biotechnological methods are used for the preparation of plasmid which codes for an extended bovine growth hormone having the formula MFEE-bGH, where the sequence bGH has the following structure:

[0095] 1 A F P A M S L S G L F A N A V L R A Q H L H Q L A A D T F K

[0096] 31 E F E R T Y I P E G Q R Y S I Q N T Q V A F C F S E T I P A

[0097] 61 P T G K N E A Q Q K S D L E L L R I S L L L I Q S W L G P L

[0098] 91 Q F L S R V F T N S L V F G T S D R V Y E K L K D L E E G I

[0099] 121 L A L M R E L E D G T P R A G Q I L K Q T Y D K F D T N M R

[0100] 151 S D D A L L K N Y G L L S C F R K D L H K T E T Y L R V M K

[0101] 181 C R R F G E A S C A F *

[0102] The plasmid is introduced into E. coli, which is cultivated under usual conditions. The formed fusion product is isolated and purified by chromatographic methods, followed by a treatment with the enzyme DAP I. The reaction mixture was processed to develop pure bGH.

EXAMPLE 9

[0103] Preparation of Pickwale Ribonuclease, pwR

[0104] Usual biotechnological methods are used for the preparation of a plasmid which codes for an extended protein having the formula MFEE-pwR, where the sequence pwR has the following structure:

[0105] 1 R E S P A M K T Q R Q H M D S G N S P G N N P N Y C N Q M M

[0106] 31 M R R K M T Q G R C K P V N T F V H E S L E D V K A V C S Q

[0107] 61 K N V L C K N G R T N C Y E S N S T M H I T D C R Q T G S S

[0108] 91 K Y P N C A Y K T S Q K E K H I I V A C E G N P Y V P V H F

[0109] 121 D N S V *

[0110] The plasmid is introduced into E. coli, which is cultivated under usual conditions. The formed fusion product is isolated and purified chromatographically, and it is treated with the enzyme DAP I. The reaction mixture is processed to isolate pure pwR.

Claims

1. A process for preparing a desired protein having the formula:

2. A process according to claim 1, characterized in that the last amino acid in the amino acid sequence X, before A, is an amino acid with a charged side chain.

3. A process according to claim 2, characterized in that the last amino acid in the amino acid sequence X is Glu or Asp.