US20050186564A1

US20050186564A1 - Production of proteins

Info

Publication number: US20050186564A1
Application number: US11/099,302
Authority: US
Inventors: Gunter Stempfer; Jorg Windisch; Franz Knauseder
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-08-09
Filing date: 2005-04-05
Publication date: 2005-08-25
Also published as: ES2269173T3; JP2003506093A; HUP0202519A2; HU230231B1; NO20020633D0; CZ2002481A3; IL147456A0; CZ302366B6; NO328339B1; MXPA02001424A; DE60029968T2; NZ516869A; AU775662B2; TR200200253T2; CA2379571A1; AU6993300A; CN1369014A; PL200225B1; HK1047128A1; WO2001011057A1

Abstract

The present invention relates to a process for the recombinant production of a desired heterologous polypeptide with a clearly defined homogenous N-terminus in a bacterial host cell. A fusion protein comprising a peptide with the autoproteolytic activity of an autoprotease N^proof a pestivirus and the heterologous polypeptide is initially expressed in the form of cytoplasmic inclusion bodies in the host cell, the inclusion bodies are isolated and subsequently treated so that the desired heterologous polypeptide is cleaved autoproteolytically by the N^proactivity of the fusion protein.

Description

The present invention relates to a process for the recombinant production of a desired heterologous polypeptide with a clearly defined homogeneous N-terminus in a bacterial host cell, wherein initially a fusion protein which comprises a peptide having the autoproteolytic activity of an autoprotease N^proof a pestivirus and the heterologous polypeptide is produced in the form of cytoplasmic inclusion bodies in the host cell, and then the inclusion bodies are isolated and treated in such a way that the desired heterologous polypeptide is cleaved from the fusion protein by the N^proautoproteolytic activity.
In the production of recombinant proteins in heterologous organisms such as the expression of human or other eukaryotic proteins in bacterial cells it is often difficult to obtain a clearly defined N-terminus which is as nearly 100% homogeneous as possible. This applies in particular to recombinant pharmaceutical proteins whose amino acid sequence ought in many cases to be identical to the amino acid sequence naturally occurring in humans/animals.
On natural expression, for example in humans, many pharmaceutical proteins which are in use are transported into the extracellular space, and cleavage of the signal sequence present in the precursor protein for this purpose results in a clearly defined N-terminus.
Such a homogeneous N-terminus is not always easy to produce for example in bacterial cells for several reasons.
For production on an industrial scale, many pharmaceutical proteins are produced in the cytoplasm of bacterial cells (for example Escherichia coli) because accumulation thereof in adequate quantities is possible, and moreover insoluble inclusion bodies (IBs) are often formed which have great advantages in the working up and purification. In addition, the protein expressed in the form of IBs is protected from protease degradation by intracellular proteases (R. Rudolph, in: Protein Engineering: Principles and Practices. Eds. Jeffrey L. Cleland and Charles S. Craik John Wiley & Sons Inc., (1995), ISBN 0471-103543).
However, production of IB material requires in vim refolding of the expressed protein. This can in many cases be effected by methods known per so (R. Rudolph et al. (1996), in: Protein Function: A Practical Approach, Ed.: Creighton, T. E., 1-39). To do this, the proteins in the form of lBs are solubilized by adding strong denaturing agents under reducing conditions and are then renatured.
Only in rare cases is export into the bacterial periplasm with the aid of a pro- or eukaryotic signal sequence suitable, because it is usually possible to accumulate only very small quantities of product here because of the low transport capacity of the bacterial export machinery.
However, the bacterial cytoplasm differs considerably from the extracellular space of eukaryotes. On the one hand, reducing conditions are present therein and, on the other hand, there is no mechanism for cleaving N-terminal leader sequences to form mature proteins. The synthesis of all cytoplasmic proteins starts with a methionine which is specified by the appropriate start codon (ATG=initiation of translation). This N-terminal methionine is retained in many proteins, while in others it is cleaved by the methionine aminopeptidase (MAP) present in the cytoplasm and Intrinsic to the host. The efficiency of the cleavage depends essentially on two parameters: 1. the nature of the following amino acid, and 2. the location of the N-terminus in the three-dimensional structure of the protein. The N-terminal methionine is preferentially deleted when the following amino acid is serine, alanine, glycine, methionine or valine and when the N-terminus is exposed, i.e. not “hidden” inside the protein. On the other hand, if the following amino acid is a different one, in particular a charged one (glutamic acid, aspartic acid, lysine, arginine), or if the N-terminus is located inside the protein, in most cases cleavage of the N-terminal methionine does not occur (Knippers, Roif (1995) Molekulare Genetic, 6th edition, Georg Thieme Veriag. Stuttgart, New York. ISBN 3-13-103916-7).
And even if an amino acid promoting the cleavage is present at position 2, the cleavage is rarely complete. It is usual for a not inconsiderable proportion (1-50%) to remain unaffected by the MAP.
In the early days of the production of recombinant pharmaceutical proteins in bacterial cells the procedure was simply to put a methionine-encoding ATG start codon in front of the open reading frame (ORF) for the mature i.e. without signal sequence or other N-terminal extension) protein. The expressed protein then had the sequence H₂N-Met-target protein.
Only in a few cases was it possible to achieve complete cleavage of the N-terminal methionine by the MAP intrinsic to the host. Most of the proteins produced in this way therefore either are inhomogeneous in relation to their N-terminus (mixture of Met form and Met-free form) or they all have an additional foreign amino acid (Met) at the N-terminus (only Met form).
This inhomogeneity or deviation from the natural sequence is, however, unacceptable in many cases because these products frequently show different immunological (for example induction of antibody formation) and pharmacological (half-life, pharmacokinetics) properties. For these reasons, it is now necessary in most cases to produce a nature-identical product (homogeneous and without foreign amino acids at the N-terminus). In the case of cytoplasmic expression, the remedy here in most cases is to fuse a cleavage sequence (leader) for a specific endopeptidase (for example factor Xa, enterokinase, KEX endopeptidases, IgA protease) or aminopeptidase (for example dipeptidyl aminopeptidase) to the N-terminus of the target protein. However, this makes an additional step, with expenditure of costs and materials, necessary during further working up, the so-called downstream processing, of the product. In addition, in the presence of lBs there is in many cases interference with or even complete prevention of the refolding by the leader sequence.
There is thus a need for a process for producing a desired heterologous target protein which is expressed in the form of inclusion bodies in bacterial cells, from which the target protein can then be prepared with a uniform, desired N-terminus. Such a process for producing a desired target protein from inclusion bodies using the viral autoprotease N^profrom pestiviruses has been developed within the scope of the present invention.
Pestiviruses form a group of pathogens which cause serious economic losses in pigs and ruminants around the world. As the pathogen of a notifiable transmissible disease, the classical swine fever virus (CSFV) is particularly important. The losses caused by bovine viral diarrhoea virus (BVDV) are also considerable, especially through the regular occurrence of intrauterine infections of fetuses.
Pestiviruses are small enveloped viruses with a genome which acts directly as mRNA and is 12.3 kb in size and from which the viral gene products are transcribed in the cytoplasm.
This takes place in the form of a single polyprotein which comprises about 4000 amino acids and which is broken down both by viral and by cellular proteases into about 12 mature proteins.
To date, two virus-encoded proteases have been identified in pestiviruses, the autoprotease N^proand the serine protease NS3. The N-terminal protease N^prois located at the N-terminus of the polyprotein and has an apparent molecular mass of 23 kd. It catalyses a cleavage which takes place between its own C-terminus (Cys168) and the N-terminus (Ser169) of nucleocapsid protein C (R. Stark et al., J. Virol. 67 (1993), 7088-7095). In addition, duplications of the N^progene have been described in cytopathogenic BVDV viruses. In these there is a second copy of N^proat the N-terminus of the likewise duplicated NS3 protease. An autoproteolytic cleavage of the N^pro-NS3 protein is observed in this case too (R. Stark et al., see above).
N^prois an autoprotease with a length of 168 amino acids and an apparent M of about 20,000 d (in vivo). It is the first protein in the polyprotein of pestiviruses (CSFV, BDV (border disease virus) or BVDV) and undergoes autoproteolytic cleavage from the following nucleocapsid protein C (M. Wiskerchen et al., J. Virol. 65 (1991), 4508-4514; Stark et al., J. Virol. 67 (1993). 7088-7095). This cleavage takes place after the last amino acid in the sequence of N^pro, Cys168.
It has now surprisingly been found within the scope of the present invention that the autoproteolytic function of the autoprotease N^procan be utilized for cleaving a heterologous polypeptide from a fusion protein which is expressed in the form of inclusion bodies in a bacterial expression system and which comprises an autoprotease N^proof a pestivirus and the heterologous polypeptide. This entails uncleaved N^profusion protein being isolated from inclusion bodies, solubilized and cleaved during refolding.
In one aspect, the present invention thus relates to a process for the recombinant production of a heterologous polypeptide, comprising

(i) cultivation of a bacterial host cell which is transformed with an expression vector which comprises a nucleic acid molecule which codes for a fusion protein, the fusion protein comprising a first polypeptide which exhibits the autoproteolytic function of an autoprotease N^proof a pestivirus, and a second polypeptide which is connected to the first polypeptide at the C-terminus of the first polypeptide in a manner such that the second polypeptide is capable of being cleaved from the fusion protein by the autoproteolytic activity of the first polypeptide, and the second polypeptide being a heterologous polypeptide, wherein cultivation occurs under conditions which cause expression of the fusion protein and formation of corresponding cytoplasmic inclusion bodies,
(ii) isolation of the inclusion bodies from the host cell,
(iii) solubilization of the isolated inclusion bodies,
(iv) dilution of the solubilizate to give a reaction solution in which the autoproteolytic cleavage of the heterologous polypeptide from the fusion protein is performed, and
(v) isolation of the cleaved heterologous polypeptide.

A polypeptide with the autoproteolytic function of an autoprotease N^proof a pestivirus is, in particular, an autoprotease N^proof a pestivirus or a derivative thereof with autoproteolytic activity.
For the purpose of the present invention, the term “heterologous polypeptide” means a polypeptide which is not naturally cleaved by an autoprotease N^proof a pestivirus from a naturally occurring fusion protein or polyprotein. Examples of heterologous polypeptides are industrial enzymes (process enzymes) or polypeptides with pharmaceutical, in particular human pharmaceutical, activity.
Examples of preferred polypeptides with human pharmaceutical activity are cytokines such as interleukins, for example IL-6, interferons such as leukocyte interferons, for example interferon α2B, growth factors, in particular haemopoietic or wound-healing growth factors, such as G-CSF, erythropoietin, or IGF, hormones such as human growth hormone (hGH), antibodies or vaccines.
In the process according to the invention the pestivirus is preferably selected from the group of CSFV, BDV and BVDV, with CSFV being particularly preferred.

Further preference is given to a process according to the present invention in which the first polypeptide of the fusion protein comprises the following amino acid sequence of the autoprotease N^proof CSFV (see also EMBL database accession number X87939) (amino acids 1 to 168, reading from N-terminal to the C-terminal direction)


(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTTLRDL

PRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDEAQFCEVTKRIGRVTGSDGKLYH

IYVCVDGCILLKLAKRGTPRTLKWIRNFTNCPLWVTSC-(168),

or the amino acid sequence of a derivative thereof with autoproteolytic activity.

A polypeptide exhibiting the autoproteolytic activity of an autoprotease N^promay also be a derivative of an autoprotease N^proof a pestivirus which is derived from this autoprotease N^proof a pestivirus by mutagenesis, in particular amino acid substitution, deletion, addition and/or amino acid insertion, as long as the required autoproteolytic activity, in particular for generating a desired heterologous polypeptide with homogeneous N-terminus, is retained. Methods for generating such derivatives by mutagenesis are familiar to the skilled person. It is possible by such mutations to optimize the activity of the autoprotease N^proin relation to different heterologous proteins to be cleaved from the fusion protein. After production of a nucleic acid which codes for a fusion protein which, besides the desired heterologous protein, comprises an autoprotease N^proderivative which exhibits one or more mutations by comparison with a naturally occurring autoprotease N^pro, it is established whether the required function is present by determining the autoproteolytic activity.
The autoproteolytic activity can be detected, for example, by initially solubilizing the isolated/purified IBs in 7 M guanidine/HCl solution and then diluting 1:100 in reaction solution. After incubation for about 24 h, the reaction solution is examined by SDS-PAGE for cleavage having taken place. A Western blot is carried out to identify the proportions processed and unprocessed. The proportion of cleaved material is determined by densitometric analysis of the Coomassie-stained SDS-PAGE gel.
A preferred process according to the present invention is, for example, one in which the expression vector comprises a nucleic acid molecule which codes for a fusion protein which has an N-terminal region in which one or more amino acids have been deleted or substituted in the region of amino acids 2 to 21 as long as the resulting derivative continues to exhibit the autocatalytic function of the autoprotease N^proto the desired extent. For the purpose of the present invention, autoprotease N^proderivatives which are preferred in the fusion protein comprise, for example, the amino acid sequence of the autoprotease N^proof CSFV with a deletion of amino acids 2 to 16 or 2 to 21. It is also possible by amino acid substitution or addition to exchange or introduce amino acid sequences, for example in order to introduce an amino acid sequence which assists purification.
A nucleic acid molecule which is particularly preferred in the process according to the present invention codes for a fusion protein in which the first polypeptide comprises the amino acid sequence Glu22 to Cys168 of the autoprotease N^proof CSFV or a derivative thereof with autoproteolytic activity, the first polypeptide furthermore having a Met as N-terminus, and the heterologous polypeptide being directly connected to the amino acid Cys168 of the autoprotease N^proof CSFV.
A nucleic acid molecule which is likewise preferred in the process according to the present invention codes for a fusion protein in which the first polypeptide comprises the amino acid sequence Pro17 to Cys168 of the autoprotease N^proof CSFV or a derivative thereof with autoproteolytic activity, the first polypeptide furthermore having a Met as N-terminus, and the heterologous polypeptide being directly connected to the amino acid Cys168 of the autoprotease N^proof CSFV.
The said nucleic acid molecule is, in particular, in the form of a DNA molecule in the process according to the present invention.
An expression vector to be employed in the process according to the present invention preferably comprises at least one expression control sequence. Expression control sequences are, in particular, promoters (such as lac, tac, T3, T7, trp, gac, vhb, lambda pL or phoA), ribosome binding sites (for example natural ribosome binding sites which belong to the abovementioned promoters, cro or synthetic ribosome binding sites), or transcription terminators (for example rmB T1T2 or bla). The above-mentioned host cell is preferably a bacterial cell of the genus Escherichia, in particular E. coli. However, it is also possible to use other bacterial cells (see below). In a preferred embodiment, the expression vector is a plasmid.
A bacterial host cell to be employed in the process according to the present invention can be selected, for example, from the group of the following microorganisms: Gram-negative bacteria such as Escherichia species, for example E. coli, or other Gram-negative bacteria, for example Pseudomonas sp., such as Pseudomonas aeruginosa, or Caulobacter sp., such as Caulobacter crescentus, or Gram-positive bacteria such as Bacillus sp., in particular Bacillus subtilis. E. coli is particularly preferred as host cell.
The bacterial host cell, i.e. the expression strain, is cultivated in accordance with microbiological practice known per se. The strain is generally brought up starting from a single colony on a nutrient medium, but it is also possible to employ cryopreserved cell suspensions (cell banks). The strain is generally cultivated in a multistage process in order to obtain sufficient biomass for further use.
On a small scale, this can take place in shaken flasks, it being possible in most cases to employ a complex medium (for example LB broth). However, it is also possible to use defined media (for example citrate medium). For the cultivation, a small-volume preculture of the host strain (inoculated with a single colony or with cell suspension from a cryoculture) is grown, the temperature for this cultivation not generally being critical for the later expression result, so that it is possible routinely to operate at relatively high temperatures (for example 30° C. or 37° C.). The main culture is set up in a larger volume (for example 500 ml), where it is in particular necessary to ensure good aeration (large volume of flask compared with the volume of contents, high speed of rotation). Since it is intended that expression take place in the form of insoluble inclusion bodies, the main culture will in most cases also be carried out at relatively high temperature (for example 30 or 37° C.). Inducible systems are particularly suitable for producing inclusion bodies (for example with trp, lac, tac or phoA promoter). After the late logarithmic phase has been reached (usually at an optical density of 0.5 to 1.0 in shaken flasks), in these cases the inducer substance (for example indoleacrylic acid, isopropyl β-D-thiogalactopyranoside ═IPTG) is added and incubation is continued for 1 to 5 hours. During this time, most of the N^profusion protein is deposited as inclusion bodies in the bacterial cytoplasm. The resulting cells can be harvested and processed further.
On a larger scale, the multistage system consists of a plurality of bioreactors (fermenters), it being preferred to employ defined nutrient media in this case in order to be able to improve the process engineering control of the process. In addition, it is possible greatly to vase biomass and product formation by metering in particular nutrients (fed batch). Otherwise, the process is analogous to the shaken flask. For example, a preliminary stage fermenter and a main stage fermenter are used, the cultivation temperature being chosen similar to that in the shaken flask. The preliminary stage fermenter is inoculated with a so-called inoculum which is generally grown from a single colony or a cryoculture in a shaken flask.
Good aeration and a sufficient inducer concentration must also be ensured in the fermenter—and especially in the main stage thereof. The induction phase must, however, in some cases be made distinctly longer compared with the shaken flask. The resulting cells are once again delivered for further processing.
In the process according to the present invention, the inclusion bodies are isolated from the host cell in a known manner, as described, for example, in R. Rudolph et al., in: Creighton, T. E. (Ed.), Protein Function, A Practical Approach, IRL Press, (1996), 1-39; R. Rudolph, in: H. Tschesche (Ed.), Modem Methods in Protein and Nucleic Acid Research, De Gruyter, Berlin, (1990), 149-171; R. Rudolph, in: J. L. Cleland & C. S. Craik (Eds.), Protein Engineering: Principles and Practices, Wiley-Liss Inc., (1995), 283-298.
For example, after the fermentation has taken place, the cells are harvested by centrifugation. The inclusion bodies (IBs) present therein can be obtained, after disruption of the cells, for example by means of high-pressure dispersion, by a simple centrifugation at low rotor speed. The purity in relation to the desired target protein can then be improved by multiple resuspension of the IBs in various buffers, for example in the presence of NaCl (for example 0.5-1.0 M) and/or detergent (for example Triton X-100). This usually leads to most of the foreign proteins in the IBs being removed. Any residual foreign proteins usually do not interfere with the autoproteolytic cleavage.
The solubilization routinely takes place by dissolving the IBs for example in a guanidine-containing buffer (for example 0.1 M tris/HCl, 6.0 M guanidine/HCl, 5 mM EDTA, 50 mM DTT) (see, for example, R. Rudolph et al., (1996); R. Rudolph (1990), R. Rudolph (1995), supra). After removal of
Insoluble material, for example by centrifugation, a protein determination can be carried out on the supernatant.
The autoproteolytic cleavage of the heterologous polypeptide from the fusion protein is achieved by diluting the solubilizate with a cleavage buffer (for example 1 ml of solubilizate+99 ml of cleavage buffer), for example with a tris-containing cleavage buffer, preferred concentration 0.8-1.0 M, or with an arginine-containing cleavage buffer, preferably with 0.4-0.6 M arginine, for example at a neutral pH, preferably pH 7.0-7.5, and at a temperature of, for example, less than 30° C., preferably 10-20° C., thus forming the reaction solution (see also R. Rudolph (1996), supra). The cleavage solution is subsequently incubated for a particular period, for example 12 h, and the extent of the N^procleavage can be analysed by SDS-PAGE.
In a preferred embodiment, the solubilizate is diluted with an arginine-containing buffer so that the final concentration of arginine is up to 1.0 M, preferably 0.4-0.6 M. Alternatively, dilution is also possible by dialysing the solubilized inclusion bodies against an appropriate arginine-containing cleavage buffer.
The temperature of the reaction solution for the cleavage is, for example, between 0° C. and 30° C. The temperature can preferably be 10° C.-20° C.
The pH of the reaction solution is, for example, 5.0-9.0. The pH is preferably 7.0-8.0, in particular 7.0-7.5.
Where appropriate, the reaction solution contains DTT in a concentration of 0.5-100 mM. The DDT concentration is preferably about 5.5 mM.
The protein concentration in the reaction solution during the cleavage can be, for example, in the region of 20-150 μg/ml. The protein concentration is preferably less than 40 μg/ml.
The reaction solution can contain tris/HCl in a concentration of, for example, up to 1.0 M during the cleavage. The tris/HCl concentration is preferably between 0.8 M and 1.0 M.
Other buffer systems are also possible in place of arginine-containing and/or tris/HCl-containing buffers.
In a particularly preferred embodiment, the pH in the cleavage buffer is 7.0-7.5, the temperature during the cleavage is 10° C.-20° C., the protein concentration does not exceed 40-50 μg/ml, and the cleavage buffer contains about 5 mM DTT as reducing agent.
Finally, the heterologous polypeptide which has been cleaved from the fusion protein is isolated in a manner known per se (see, for example, M. P. Deutscher, in: Methods in Enzymology: Guide to Protein Purification, Academic Press Inc., (1990), 309-392).
The following examples illustrate the present invention, without in any way limiting the scope thereof.

EXAMPLES

Example 1

Expression and In Vitro Cleavage of N^pro-hGH (Human Growth Hormone)

The fusion proteins are produced by heterologous expression in Escherichia coli in an expression system (vector p160TP1 with host W3110). All the elements in this system for constructing the vector originate from the E. coli genome.
p160TP1 is a pBR322-derived vector in which the expression is under the control of a modified E. coli tip promoter with deleted attenuator. The Shine-Dalgamo sequence of the attenuator peptide from the same operon is used as ribosome binding site (RBS). The double terminator T1T2 from the E. coli rmB gene ensures, together with the bla gene terminator which remains in the construct, efficient termination of the transcription. The expression cassette is inserted starting from the pBR322 EcoRI cleavage site in the counter-clockwise direction (i.e. contrary to the orientation of the tetracycline resistance gene). The β-lactamase gene (bla) is deleted together with the promoter. The structural genes for the heterologous proteins to be expressed are introduced (generally as PCR fragment cut to size) via the XbaI cleavage site at position 3506.
An expression plasmid (NPH-pET) serves as source of the N^pro-hGH structural gene. The plasmid comprises the known expression vector pET11a (F. W. Studier et al., Methods. Enzymol. 185 (1990), 6089). Firstly a fusion protein composed of N^proand the CSFV nucleocapsid protein is cloned into the expression vector. This entails the first 16 amino acids of the natural N^prosequence (MELNHFELLYKTSKOK) being replaced by a 10 amino acids-long oligo-histidine purification aid (MASHHHHHHH). A SpeI cleavage site is introduced at the junction between N^proand nucleocapsid protein into the resulting expression plasmid by a targeted mutagenesis. This makes it possible to delete the structural gene for the nucleocapsid protein from the vector by restrictions with SpeI (at the 5′ end) and XhoI (at the 3′ end). The corresponding linearized N^pro-pET11a vector is removed by preparative gel electrophoresis from the nucleocapsid gene fragment. It is then possible to introduce the hGH structural gene via the “sticky” SpeI and XhoI ends.

For this purpose, the following preparatory work on the hGH gene is necessary. The structural gene is amplified from a cDNA bank from human brain, which can be processed (Clontech), using a high precision PCR (for example Pwo polymerase from Roche Biochemicals, procedure as stated by the manufacturer). The following oligonucleotides are employed for this purpose:


Oligonucleotide 1 (“N-terminal”):
5′- ATAATTACTA GTTGTTTCCC AACCATTCCC TTATCCAGGC
C -3′

Oligonucleotide 2 (“C-terminal”):
5′- ATAATTGGAT CCTCGAGTTA TTAGAAGCCA CAGCTGCCCT
CCAC -3′

A SpeI cleavage site is introduced at the 5′ end, and an XhoI cleavage site is introduced at the 3′ end via the oligonucleotide used. In addition, the double ochre stop codon (TAATAA) is introduced at the end of the structural gene for efficient termination of translation. The SpeI cleavage site at the front end permits ligation in reading frame with the N^pro-pET11a vector described above. The XhoI cleavage site at the rear end makes directed cloning-in possible. The plasmid produced in this way is named in NPH-PET.
This plasmid NPH-pET is that used, as already mentioned, as source of the structural gene of the N^pro-hGH fusion protein. For this purpose, once again a high-precision PCR with Pwo polymerase is employed using the plasmid NPH-pET linearized with XhoI as template. The following primers are used to produce a clonable fragment:
Front end (corresponding to the N-terminus of the protein) of the structural gene:

5′-

AGGGTATCTA GAATTCTATG CCAGTGGGAG TGGAGGAACC G-3′
Rear end (corresponding to the C-terminus of the protein) of the structural gene:

5′-

AGAATTAAGC TTCTAGATTA TTAGAAGCCA CAGCTGCCCT CCAC-

3′
The XbaI cleavage sites used subsequently for the cloning (front and rear) are shown underlined, and the start (ATG) and stop (MTAA) codons are written in larger characters.
These two oligonucleotides result in amplification of a structural gene with an open reading frame (ORF) for a fusion protein (FP) in which the His, purification aid and the preceding amino acids (with the exception of the methionine necessary for the start of translation) have been completely deleted.

The sequence of the PCR fragment (1072 bp) which contains the open reading frame for the N^pro-hGH fusion protein is depicted below. The start codon and the two stop codons are printed in bold, and the XbaI cleavage sites used for the cloning are shown underlined.


5′-AGGGTATCTAGAATTCTATGCCAGTGGGAGTGGAGGAACCGGTGTATGACACCGCGGGGAGACC

ACTATTTGGGAACCCAAGTGAGGTACACCCACAATCAACGCTGAAGCTGCCACACGACAGGGGGAGA

GGAGATATCAGAACAACACTGAGGGACCTACCCAGGAAAGGTGACTGTAGGAGTGGCAACCATCTAG

GCCCGGTTAGTGGGATATACATAAAGCCCGGCCCTGTCTACTATCAGGACTACACGGGCCCAGTCTA

TCACAGAGCTCCTTTAGAGTTCTTTGATGAGGCCCAGTTCTGCGAGGTGACTAAGAGAATAGGCAGG

GTCACGGGTAGTGATGGTAAGCTTTACCACATATATGTGTGCGTCGATGGTTGCATACTGCTGAAAT

TAGCCAAAAGGGGCACACCCAGAACCCTAAAGTGGATTAGGAACTTCACCAACTGTCCATTATGGGT

AACTAGTTGTTTCCCAACCATTCCCTTATCCAGGCCTTTTGACAACGCTATGCTCCGCGCCCATCGT

CTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGT

ATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAG

GGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGG

CTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGAGAGCA

ACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGG

CAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGAT

GACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACAT

TCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAATAA TCTAGAAGCTTAAT

TCT-3′

This ORF thus codes for the fusion protein shown below, the proline at position two corresponding to the proline at position 17 of the natural N^proprotein. The sequence of the N^prohGH fusion protein (344 amino acids, of which 153 N^proand 191 hGH) is thus as follows (read from the N-terminal to the C-terminal direction), with proline 17 (position 2 of the fusion protein) from the natural N^prosequence being shown in italics, and the hGH


MPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTTLRDLPRKGDCRSGNHLGPVSGIY

IKPGPVYYQDYTGPVYHRAPLEFFDEAQFCEVTKRIGRVTGSDGKLYHIYVCVDGCILLKLAKRGTP

RTLKWIRNFTNCPLWVTSCFPTIPLSRPFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNP

QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLK

DLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQC

RSVEGSCQF

The FP in the reduced state has an M_rof 39 316.92 d, and the M, after a possible cleavage would be 17 220.02 d for the N^propart (reduced) and 22 114.88 d for the hGH part (reduced). N^prohas six cysteines, and hGH four. In the bacterial cytoplasm these cysteines are likely to be in reduced form for the most part. During the subsequent processing there is presumably at least partial formation of disulfide bridges. It must be expected that the N-terminal methionine in the fusion protein (or the N^propart) will be mostly cleaved by the methionine aminopeptidase (MAP) intrinsic to the host, which would reduce the M_rby, in each case, 131 d to 39 186 d (FP) and 17 089 d (N^pro) respectively.
The PCR fragment with the structural gene for the FP described is purified and cut with XbaI, and the two sought cleavage products are removed from the target fragment. This fragment is ligated to the expression vector p160TP1 as described.
For this purpose, the vector is initially linearized with XbaI and 5′-dephosphorylated with calf intestine phosphatase (CIP). T4 DNA ligase is used for ligation. The ligated DNA is introduced by electroporation into Escherichia coli K-12 DH10B and plated out on Luria broth (LB) agar plates with 15 mg/L tetracycline. Numerous clonal colonies result from this transformation. Plasmid DNA is isolated from several clones and examined by restriction analysis for the presence of an insert with the correct size and orientation. One clone is selected and subjected to detailed characterization by restriction analysis and DNA sequencing. The plasmid behaves in accordance with the calculations in all the investigations. This plasmid is called pNPH1 and is used for the subsequent work.

The sequence of the N^pro-hGH expression plasmid pNPH1 (4840 bp) is shown below. The start codon and the stop codon for the N^pro-hGH fusion protein are underlined. The open reading frame runs in the reverse direction when shown in this form.


5′-GAATTCTCATGTTTGACAGCTTATCATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAAT

TGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCA

CCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCA

TTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGC

GCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTG

GAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCAT

CGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGG

GAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCG

TGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGG

CCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCC

TTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTA

TGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGA

GGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCC

CTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCG

GCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCC

CATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAG

GTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCA

CTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGAT

TGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACC

TCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCA

ATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATC

TCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGC

TCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGAT

ACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTC

TTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGA

TCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCA

TTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGT

TCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTA

TCATTACCCCCATGAACAGAAATTCCCCCTTACACGGAGGCATCAAGTGACCAAACAGGAAAAAACC

GCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGG

ACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTG

CCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCT

TGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTC

GGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCA

GAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAAT

ACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG

AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAG

AACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC

ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC

AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTG

CCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCT

GTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCA

GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG

CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCT

TGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCC

AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT

TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT

CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA

AAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG

TAAACTTGGTCTGACAGTTACCTCGAGGCCATCCGTCAGGATGGCCTTCTGCTTAATTTGATGCCTG

GCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCGCAACGTTCAAATCCGCTC

CCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTC

TTTCGACTGAGCCTTTCGTTTTATTCTAGATTATTAGAAGCCACAGCTGCCCTCCACAGAGCGGCAC

TGCACGATGCGCAGGAATGTCTCGACCTTGTCCATGTCCTTCCTGAAGCAGTAGAGCAGCCCGTAGT

TCTTGAGTAGTGCGTCATCGTTGTGTGAGTTTGTGTCGAACTTGCTGTAGGTCTGCTTGAAGATCTG

CCCAGTCCGGGGGCTGCCATCTTCCAGCCTCCCCATCAGCGTTTGGATGCCTTCCTCTAGGTCCTTT

AGGAGGTCATAGACGTTGCTGTCAGAGGCGCCGTACACCAGGCTGTTGGCGAAGACACTCCTGAGGA

ACTGCACGGGCTCCAGCCACGACTGGATGAGCAGCAGGGAGATGCGGAGCAGCTCTAGGTTGGATTT

CTGTTGTGTTTCCTCCCTGTTGGAGGGTGTCGGAATAGACTCTGAGAAACAGAGGGAGGTCTGGGGG

TTCTGCAGGAATGAATACTTCTGTTCCTTTGGGATATAGGCTTCTTCAAACTCCTGGTAGGTGTCAA

AGGCCAGCTGGTGCAGACGATGGGCGCGGAGCATAGCGTTGTCAAAAGGCCTGGATAAGGGAATGGT

TGGGAAACAACTAGTTACCCATAATGGACAGTTGGTGAAGTTCCTAATCCACTTTAGGGTTCTGGGT

GTGCCCCTTTTGGCTAATTTCAGCAGTATGCAACCATCGACGCACACATATATGTGGTAAAGCTTAC

CATCACTACCCGTGACCCTGCCTATTCTCTTAGTCACCTCGCAGAACTGGGCCTCATCAAAGAACTC

TAAAGGAGCTCTGTGATAGACTGGGCCCGTGTAGTCCTGATAGTAGACAGGGCCGGGCTTTATGTAT

ATCCCACTAACCGGGCCTAGATGGTTGCCACTCCTACAGTCACCTTTCCTGGGTAGGTCCCTCAGTG

TTGTTCTGATATCTCCTCTCCCCCTGTCGTGTGGCAGCTTCAGCGTTGATTGTGGGTGTACCTCACT

TGGGTTCCCAAATAGTGGTCTCCCCGCGGTGTCATACACCGGTTCCTCCACTCCCACTGGCATAGAA

TTCTAGATACCCTTTTTACGTGAACTTGCGTACTAGTTAACTAGTTCGATGATTAATTGTCAACAGC

TCATTTCAGAATATETGCCAGAACCGTTATGATGTCGGCGCAAAAAACATPATCCAGAACGGGAGTG

CGCCTTGAGCGACACGAAITATGCAGTGATTTACGACCTGCACAGCCATACCACAGCTTCCGATTGG

CTGCCTGACGCCAGAAGCATTGGTGCACCGTGCAGTCGAGATGCGCGTCGGCACCCTGGCGATCACC

GACCATGACACCACAGCAT-3′

The Escherichia Coal host strain for expressing the N^pro-hGH fusion protein is selected on the basis of considerations of productivity and biological safety. Escherichia coli K-12 is the best-characterized line of the species E. coli (B. J. Bachmann; in: J. L. Ingraham et al., (Ed.) Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington D.C. (1 987a), pp. 1191-1219; B. J. Bachmann 1987b; Barbara J. (1987b) Linkage map of Escherichia coli K-1 2, edition 7. In: Escherichia coli and Salmonella typhimurium, Vol. 2. Ed.: F. C. Neidhard. Am. Soc. Microbiol., Washington D.C. (1987b), 807-876; K. F. Jensen, J. Bacterol. 175 (1993), 3401-3407) and its representatives are generally regarded as safe.
Escherichia coli K-12 W31 10 (ATCC 27325) is a prototropic derivative, which is very close to the wild type, of Escherichia coli K-12 and is frequently employed as host strain for expressing heterologous proteins. It has been deposited at the American Type Culture Collection (ATCC) and has the following genotype:
[F⁻ mcrA mcrB IN(rrnD-rrnE)1 Lambda⁻]
The strain can be purchased from the ATCC. Its growth is excellent on fully synthetic media without complex nitrogen sources and process engineering control thereof it possible very effectively.
The expression strain W3110[pNPH1] is produced by transformation of the expression plasmid pNPH1 described above into W31 10. The transformation takes place by electroporation, using 10% glycerol as suspending medium. 50 ng (1 μL) of a pNPH1 solution in water were mixed with 30 μL of a W31 10 cell suspension and exposed to an 1800 V pulse in a 1 mm cuvette (Eppendorf Electroporator 2510). The cells are resuspended in SOC medium, shaken at 37° C. for 1 h and then plated out on Luria broth agar plates with 15 mg/L tetracycline. Numerous clones result from this transformation after incubation at 37° C. (over night).
A medium-sized colony with distinct margins is picked and forms the basis for the expression strain W3110[pNPH1]. The clone is cultivated and preserved in cryoampoules at −80° C. (master cell bank MCB). For daily use, the strain is streaked on LB-tetracycline agar plates.
The strain W3110[PNPH1] is subcultured from a single colony on an agar plate and this is used to inoculate a preculture in Luria broth+15 mg/L tetracycline (200 mL in a 1 L baffle flask). The preculture is shaken at 250 rpm at 30° C. for 14 h and, during this, reaches an OD₆₀₀of about 1.0. 10 mL portions of preculture are then used to inoculate the main cultures (in each case 330 mL of citrate medium in 1 L baffle flasks) (3% inoculum). The main cultures are grown at 37° C. (250 rpm) until the OD₆₀₀is 0.8 and then production of the fusion protein is induced with 50 μg of indoleacrylic acid per mL of culture (final concentration; stock solution 5 mg/mL in 100% analytical grade EtOH). The cultures are cultivated further at 37° C. and 250 rpm for 4 h, during which the OD₆₀₀reaches about 1.5 to 3.0.
The cultures are transferred into sterile 500 mL centrifuge bottles and centrifuged at 10,000 g for 30 min. The centrifugation supernatant is completely discarded, and the pellets are frozen at −80° C. until processed further.
About 12 g (wet weight) of BL12 I(DE3) cell pellet are then suspended in 30 mL of 50 mM tris/Cl, 5 mM benzamidine, pH 8.0 and homogenized using an Ultraturrax. After addition of 0.5 mM EDTA and 0.1% (wh) lysozyme, the cell suspension is incubated in an ice bath for 30 min. Subsequent ultrasound treatment (7×20 sec with 20 sec pause in each case) leads to a complete disruption of the cells. Then 10 mM MgCl₂and 0.006% (w/v) DNase are added to the disrupted suspension and incubated at 4° C. for 30 min. Insoluble and soluble constituents are subsequently separated by centrifugation (JA 20; 7700 g). The pellet is suspended in 40 mL of 50 mM tris/HCl, 5 mM benzamidine, 5 mM EDTA, pH 8.0 and again centrifuged (JA 20; 7700 g). This step is repeated twice more, and the resulting pellets are suspended in 40 mL of 20 mM tris/HCl, pH 7.0.20 mL of 1.5 M NaCl, 60 mM EDTA, pH 7.0 are added to the suspension and incubated in an ice bath for 30 min. Finally, a further centrifugation (JA 20; 7700 g) and an H₂O washing step are carried out. The resulting pellet represents the N^pro-hGH IB (inclusion body) material.
This is routinely followed by suspending about 100 mg of IB material in 2 mL of 6.0 M guanidine hydrochloride, 0.1 M tris, 5 mM EDTA, 50 mM DTT, pH 9.0 at RT for 30 min. Insoluble constituents are subsequently removed by centrifugation (10 min/30000 g). A protein determination is carried out on this supernatant (protein concentration about 20 mg/mL). The solubilizate produced in this way is either immediately employed in cleavage tests or stored at −20° C. (max. 7 days).

Example 2

Cleavage of the Inclusion Body Fusion Proteins under Various Conditions

Cleavage of the N^proportion is made possible in principle by a 1:100 (or larger) dilution of the solubilizate (see above) in cleavage buffer (residual concentration of guanidine hydrochloride ≦0.06 M). The protein concentration during this is routinely 20 μg/mL (in cases of exception, this is pointed out hereinafter).
It is furthermore possible to achieve cleavge of the N^proportion by dialysis (1:100) of the solubilized material against cleavage buffer.
The extent of the cleavage of the N^proportion can be measured by detecting the cleavage products after SDS-PAGE. This entails the polyacrylamide gels being photographed after SDS-PAGE has taken place, and the intensity of the appropriate bands being measured. The appropriate bands are identified by Western blot in preliminary tests.

Example 2.1

In Vitro Cleavage of N^pro-hGH by Arginine

The cleavage takes place by dilution (1:100) of the solubilizate in cleavage buffer (20 mM tris/HCl, 2 mM EDTA, 5 mM DTT, pH 7.0 and the particular arginine concentrations) at RT. The protein concentration in the cleavage mixtures is 20 μg/mL. The extent of the cleavage is measured after 24 h by detecting the cleavage products after SDS-PAGE.
It emerges that the N^procleavage efficiency can be distinctly increased by adding arginine in the cleavage buffer. Maximum cleavage takes place at a concentration of 0.40-0.6 M arginine.

Example 2.2

Cleavage of N^profrom N^pro-hGH by Arginine at Various Temperatures

The cleavage takes place by dilution (1:100) of the solubilizate in cleavage buffer (20 mM tris/HCl, 2 mM EDTA, 5 mM DTT, pH 7.0, 0-1.0 M arginine) at the particular temperatures. The protein concentration in the cleavage mixture is 20 μg/mL. The extent of the cleavage is measured after 24 h by detection of the cleavage products after SDS-PAGE.
It emerges that a maximum cleavage takes place at temperatures between 10 and 20° C.

Example 2.3

Extent of the Cleavage of N^profrom N^pro-hGH as a Function of the pH at Various Temperatures

The cleavage takes place by dilution (1:100) of the solubilizate in cleavage buffer (20 mM tris/HCl, 2 mM EDTA, 5 mM DTT, 0.5 M arginine) at the particular pH. The protein concentration in the cleavage mixture is 20 μg/mL. The extent of the cleavage was measured after 24 h by detecting the cleavage products after SDS-PAGE.
A systematic-variation of the pH in the cleavage buffer form pH 5.0 to 9.0 is carried out. With the arginine concentration employed, a pH of 7.0-7.5 is optimal for cleavage of the N^profusion protein.

Example 2.4

Cleavage of N^profrom N^pro-hGH at Various DTT Concentrations

The dependence of the cleavage efficiency on the DTT concentration is investigated.
The cleavage takes place by dilution (1:100) of the solubilizate in cleavage buffer (20 mM tris/HCl, 2 mM EDTA, pH 7.0, 0.5 M arginine, 0-100 mM DTT at 15° C.). Because of the DTT concentration in the solubilizate, the mixture with 0.0 mM DTT still contains 0.5 mM DTT. The protein concentration in the cleavage mixture is 20 μg/mL. The extent of the cleavage is measured after 24 h by detecting the cleavage products after SDS-PAGE.
A maximum cleavage of the N^proportion is reached at about 5.5 mM DTT.

Example 2.5

Variation of the pH in Solubilizate for Cleavage of N^pro-hGH

The cleavage takes place by dilution (1:100) of the solubilizate at various pH values (pH 6.0, pH 7.0, pH 8.0, pH 9.0) in the cleavage buffer (20 mM tris/HCl, 2 mM EDTA, 5 mM DTT, pH 7.0, 0-0.8 M arginine at 15° C.). The pH values are adjusted by titrating the solubilization buffer to the appropriate pH values before mixing with the IBs. The protein concentration in the cleavage mixture is 20 μg/mL. The extent of the cleavage is measured after 24 h by detecting the cleavage products after SDS-PAGE.
It emerges that a pH of 7.0-8.0 in the solubilizate is optimal.

Example 2.6

Kinetics of N^proCleavage from N^pro-hGH

Reactivation of denatured N^pro-hGH is started by dilution (1:100) in 20 mM tris/HCl, 0.5 M arginine, 2 mM EDTA, 10 mM DTT, pH 7.0 at 15° C.
It emerges that the end point of the cleavage is reached after about 24 h.

Example 2.7

Cleavage of N^profrom N^pro-hGH by Dilution at Various Protein Concentrations

The cleavage takes place by dilution (1:100) of the solubilizate in cleavage buffer (20 mM tris/HCl, 0.5 M arginine, 2 mM EDTA, 10 mM DTT, pH 7.0 at 15° C.). A check is carried out to determine whether the cleavage has taken place after about 24 h by detection of the cleavage products after SDS-PAGE.
It emerges that protein concentrations ≧40 μg/mL lead to a distinct decline in the cleavage efficiency. Further tests reveal that the optimal protein concentration is 20-40 μg/ml.

Example 2.8

N^proCleavage from N′-hGH by Dialysis as a Function of the Protein Concentration

Guanidine hydrochloride is removed from solubilized N^pro-hGH IB material by dialysis (1:100) against 20 mM tris/HCl, 0.5 M arginine, 2 mM EDTA, 10 mM DTT, pH 7.0 at 4° C., and thus the cleavage is initiated. After about 24 h, SDS-PAGE is used to analyse for N^procleavage having taken place. It emerges that cleavage of the N^proportion can be achieved not only by dilution but also by dialysis of the solubilized material.

Example 2.9

Efficiency of Cleavage of N^profrom N^pro-hGH as a Function of the Tris/HCl Concentration

The cleavage takes place by dilution (1:100) of the solubilizate in cleavage buffer (2 mM EDTA, 20 mM DTT, pH 7.0, 0.1-1.0 M tris/HCl at 15° C.). The cleavage was checked after about 24 h by detecting the cleavage products after SDS-PAGE.
It emerges that an increasing tris/HCl concentration up to 0.8 M in the buffer improves the N^profusion protein cleavage yield. An increase above this causes no measurable enhancement of the N^procleavage.
The reaction ought thus preferably to take place at tris/HCl concentrations of 0.8-1.0 M.

Example 3

Expression and In Vitro Cleavage of NP-PGH (Porcine Growth Hormone)

Production of the N^pro-pGH expression vector starts from pNPH1 (see Example 1). The plasmid is linearized with BglII and amplified by means of PCR. This entails the entire plasmid apart from the region of the hGH structural gene (from the codon for Phe1 to the codon for Phe 191) being amplified with Pwo polymerase and primers without 5′-phosphate. The purified PCR fragment is employed as vector in the ligation with the pGH structural gene. Removal of 5-phosphates (for example using calf intestine phosphatase, Arctic shrimp alkaline phosphatase) prevents circularization by self-ligation.
The pGH structural gene is likewise produced by PCR amplification. The template used is a porcine cDNA bank (for example porcine liver 5′-stretch cDNA library from Clontech or porcine brain or pituitary cDNA bank). The following primers are used for the amplification, and are in each case provided with a 5′-phosphate during the synthesis:
Front end (corresponding to the N-terminus of the protein) of the structural gene (Phe1 to Ser7):

5′- TTC CCA GCC ATG CCC TTG TCC -3′
Rear end (corresponding to the C-terminus of the protein) of the structural gene (Phe190 to Val184):

5′- GAA GGC ACA GCT GCT CTC CAC -3′
The PCR fragment contains exclusively the ORF (structural gene) for mature pGH. Ligation (T4 DNA ligase) results in an ORF for N^pro-pGH which is analogous to that of pNPH1. The fusion protein described hereinafter is encoded by this ORF. The ligated DNA is introduced by electroporation in Escherichia coli K-12 DH10B (electrocompetent cells supplied by Life Technologies, genotype Escherichia coli K-12 F⁻ mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔ15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara, leu)7697 gal/U gal/K λ-rpsL nupG) and plated out on Luria broth (LB) agar plates with 15 mg/L tetracycline. This transformation results in numerous clonal colonies. The plasmid DNA is isolated from several clones and examined by a restriction analysis for the presence of an insert with the correct size and orientation. One clone is selected and subjected to detailed characterization by restriction analysis and DNA sequencing. The plasmid behaves in accordance with calculations in all the investigations. This plasmid is called pNPP1 and used for the subsequent work.

In the following amino acid sequence (read from the N-terminal to the G-terminal direction) of the N^pro-pGH fusion protein (342 amino acids, of which 153 are N^proand 189 are pGH), proline 17 (position 2 of the fusion protein) from the natural N^prosequence is put in italics, and the pGH sequence is shown in bold print.


MPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTTLRDLPRKGDCRSGNHLGPVSGIY

IKPGPVYYQDYTGPVYHRAPLEFFDEAQFCEVTKRIGRVTGSDGKLYHIYVCVDGCILLKLAKRGTP

RTLKWIRNFTNCPLWVTSCFPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIGNAQ

AAFCFSETIPAPTGDKEAQQRSVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLE

EGIQALMRELEDGSPRAGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRVMKCRRF

VESSCAF

The FP in the reduced state has an M_rof 38 819.82 d, and the M_rafter a possible cleavage would be 17 220.02 d for the N^propart (reduced) and 21 617.79 d for the pGH part (reduced). N^prohas six cysteines, and pGH four. In the bacterial cytoplasm these cysteines are likely to be in reduced form for the most part. During the subsequent processing there is presumably at least partial formation of disulfide bridges. It must be expected that the N-terminal methionine in the fusion protein (or the N^propart) will be mostly cleaved by the methionine aminopeptidase (MAP) intrinsic to the host, which would reduce the M_rby, in each case, 131 d to 38 689 d (FP) and 17 089 d (N^pro) respectively.
The expression strain W3110[pNPP] is produced as described in Example 1 by transformation (electroporation) of the expression plasmid pNPP1 in Escherichia coli K-1 2 W3110. In this case too, even detailed characterization revealed no deviations from the expected restriction pattern.
A medium-sized colony with distinct margins is picked from the transformation plate and forms the basis for the expression strain W3110[pNPP1]. The clone is cultivated and preserved in cryoampoules at −80° C. (master cell bank MCB). The strain is streaked on LB-tetracycline agar plates for daily use.
Expression of the N^pro-pGH fusion protein takes place as described in Example 1 for the N^pro-hGH fusion protein.
Preparation of the N^pro-pGH lBs takes place as described in Example 1 for a N^pro-hGH fusion protein.

Example 4

In-Vitro Cleavage of N^profrom N^pro-RGH

Solubilization and renaturation tests corresponding to those described above for N^pro-hGH are carried out for the N^pro-pGH fusion protein. After variation of the parameters already described (see above) it emerges that in-vitro cleavage of the N^proportion is possible after solubilization and renaturation also in the case of the N^pro-pGH fusion protein.

Claims

1. A process for the recombinant production of a heterologous polypeptide, comprising, (i) cultivation of a bacterial host cell which is transformed with an expression vector which comprises a nucleic acid molecule which codes for a fusion protein, the fusion protein comprising a first polypeptide which exhibits the autoproteolytic function of an autoprotease N^proof a pestivirus, and a second polypeptide which is connected to the first polypeptide at the C-terminus of the first polypeptide in a manner such that the second polypeptide is capable of being cleaved from the fusion protein by the autoproteolytic activity of the first polypeptide, and the second polypeptde being a heterologous polypeptide, wherein cultivation occurs under conditions which cause expression of the fusion protein and formation of corresponding cytoplasmic inclusion bodies,

(ii) isolation of the inclusion bodies from the host cell,

(iii) solubilization of the isolated inclusion bodies,

(iv) dilution of the solubilizate to give a reaction solution in which the autoproteolytic cleavage of the heterologous polypeptide from the fusion protein is performed, and

(v) isolation of the cleaved heterologous polypeptide.

2. A process according to claim 1, wherein the pestivirus is selected from the group of CSFV, BDV and BVDV.

3. A process according to claim 2, wherein the pestivirus is CSFV.

4. A process according to claim 3, wherein the first polypeptde comprises the following amino acid sequence:

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTTLRDLPRKG DCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDEAQFCEVTKRIGRVTGSDGKLYHIYVCVDGC ILLKLAKRGTPRTLKWIRNFTNCPLWVTSC-(168),

5. A process according to claim 3, wherein the first polypeptde comprises the amino acid sequence Glu22 to Cys168 of the autoprotease N^proof CSFV or a derivative thereof with autoproteolytic activity, wherein the first polypeptide has a Met as N-terminus, and wherein the heterologous polypeptide is directly connected to the amino acid Cys168 of the autoprotease N^proof CSFV.

6. A process according to claim 3, wherein the first polypeptide comprises the amino acid sequence Pro17 to Cys168 of the autoprotease N^proof CSFV or a derivative thereof with autoproteolytic activity, wherein the first polypeptide has a Met as N-terminus, and wherein the heterologous polypeptide is directly connected to the amino acid Cys168 of the autoprotease N^proof CSFV.

7. A process according to claim 1, wherein the nucleic acid molecule is a DNA molecule.

8. A process according to claim 1, wherein the expression vector is a plasmid.

9. A process according to claim 1, wherein the bacterial host cell is an E. coli cell.