NZ627054B2 - Novel prongf mutants and uses thereof in the production of beta-ngf - Google Patents
Novel prongf mutants and uses thereof in the production of beta-ngf Download PDFInfo
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- NZ627054B2 NZ627054B2 NZ627054A NZ62705412A NZ627054B2 NZ 627054 B2 NZ627054 B2 NZ 627054B2 NZ 627054 A NZ627054 A NZ 627054A NZ 62705412 A NZ62705412 A NZ 62705412A NZ 627054 B2 NZ627054 B2 NZ 627054B2
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- prongf
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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- C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
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- C07K—PEPTIDES
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Abstract
Discloses a proNGF mutant wherein the protease cleavage site R1SK3R4 is substituted at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wildtype proNGF sequence (SEQ ID NO: 1) by any amino acid selected from a non-basic amino acid and Histidine, wherein the sequence is defined as in the complete specification, and related methods of preparing a biologically active human beta-NGF. s defined as in the complete specification, and related methods of preparing a biologically active human beta-NGF.
Description
Novel proNGF mutants and uses thereof in the production of beta-NGF
FIELD OF THE INVENTION
The present invention relates to novel proNGF mutants having substitutions at the native
protease cleavage site. The present invention further discloses a method of producing a
biologically active human beta-NGF from an inactive insoluble proNGF mutant and the use of
a proNGF mutant for producing human beta-NGF.
BACKGROUND OF THE INVENTION
Nerve growth factor (beta-NGF) is a neurotrophic factor playing a crucial role in the growth
and survival of neurons (sensory and sympathetic) (Levi-Montalcini, R., Science 237 (1987)
1154; Thoenen, H., et al., Physiol. Rev. 60 (1980) 1284; Yankner, B. A., et al., Annu. Rev.
Biochem. 51 (1982) 845). Beta-NGF belongs to a cysteine-knot superfamily of growth factors
assuming stable dimeric protein structure. Furthermore, beta-NGF promotes the growth,
differentiation and vitality of cholinergic neurons of the central nervous system (Hefti, F. J., J.
Neurobiol. 25 (1994) 1418). Possible therapeutic indications for recombinant human nerve
growth factor include peripheral sensory neuropathies, e.g. associated with diabetes or as a
possible side effect in AIDS therapy. Other indications for beta-NGF are central neuropathies,
e.g. Alzheimer's disease. In this case, the loss of memory is the result of a loss of cholinergic
neurons. to treatment cutaneous
lHU'H.l.Cl"•-' et
to retinal cells from degeneration and apoptosis in an
experimental animal model of glaucoma and to improve visual function in a few patients
affected by glaucoma (Lambiase A, et al. PNAS 2009).
Mature human beta-NGF is a 118 amino acid protein which is translated as a preproprotein
consisting of 241 amino acids. The signal peptide (prepeptide) of 18 amino acids is cleaved
during translocation into the endoplasmic reticulum (ER). The resulting proprotein (proNGF)
is processed at its N-terminus by removing the pro-sequence by protease cleavage. Mature
human NGF shows a high degree of identity (about 90%) to rodent (murine and rat) beta
NGF. For clinical studies or therapeutic uses, beta-NGF has to be provided in high
concentrations. Submaxillary glands of mice are a natural source of beta-NGF. However,
these beta-NGF preparations are heterogeneous mixtures of different dimers and thus not
suitable for therapeutic uses. Furthermore, it is desirable to administer the human form of the
protein to patients. In human tissue, however, neurotrophic factors are present only in low
concentrations.
The prosequence is a domain separate from the mature protein (see the sequence data in
Figure 1, wherein the prosequence is indicated in bold). These two domains are separated by
an exposed protease cleavage site with a basic amino acid target sequence of the type Arg
Ser-Lys-Arg located at positions 101 to 104 of the human proNGF sequence (SEQ ID NO: 1).
This motif is naturally a cleavage site for the serine endoprotease Furin. Additionally, the
cleavage site may be specifically processed by other suitable proteases, preferably by
proteases with substrate specificity of cleavage after the amino acid Arginine (Arg, R). For
example, the protease trypsin cleaves after basic amino acids such as Lysine (Lys, K) or
Arginine (Arg, R).
Methods for the preparation of biologically active beta-NGF from its inactive proform are
well-known in the field of the art. For example, EP 0 994 188 Bl describes a method for the
preparation of biologically active beta-NGF from its inactive pro-form having a poor
solubility. According to this method, beta-NGF is obtainable from recombinant insoluble
inactive proNGF which solubilized in a denaturing solution. Afterwards, the solubilized
proNGF is transferred into a non- or weakly denaturing solution. The denatured proNGF
assumes a biologically active conformation as determined by the disulfide bonds present in
native beta-NGF. Subsequently, the prosequence of proNGF is cleaved off whereby active
beta-NGF is obtained.
Human proNGF contains a native protease (Furin) cleavage site Arg-Ser-Lys-Arg, thus
1 3 4
having the following sequence motif: R SK R . For specific production processes such as
those requiring "Good Manufacturing Practice" (GMP) quality levels, materials such as
enzymes have to be provided in high quality. The protease Furin is currently not available as
GMP-grade protease.
Therefore, an alternative protease, Trypsin (EC 3.4.21.4), was chosen to cleave proNGF to
result in a mature beta-NGF protein. The serine protease Trypsin cleaves peptide chains at the
carboxyl side of basic amino acids Arginine or Lysine. In human proNGF, the naturally
occuring cleavage site in human proNGF contains three positions with basic amino acids
1 3 4
(positions 101, 103, and 104 of SEQ ID NO: 1; alternatively referred to as R , K and R
herein). Thus, cleavage of proNGF by Trypsin may lead to numerous different cleaved
products depending on where exactly cleavage occurs. Typical cleavage products are SK R -
beta-NGF and R -beta-NGF and mature beta-NGF. This problem is exacerbated since
dimerization of the beta-NGF protein will lead to an even higher number (up to six) of
inhomogenous products which have to be purified in following steps (see Figure 2a).
TECHNICAL PROBLEMS UNDERLYING THE PRESENT INVENTION AND
THEIR SOLUTION
Methods for producing betaNGF have been described in the prior art. However, the currently
available production processes have several drawbacks, such as inhomogenous beta-NGF
products and low yields of beta-NGF.
Cleavage of the wild-type pro-NGF with Trypsin to produce beta-NGF has shown low
efficiency that obliges to use very high amounts of the enzyme in order to obtain a sufficient
yield of cleaved beta-NGF. This has several drawbacks that impact on the subsequent process
of purification. First of all, it further decreases the selectivity of the cleavage which leads to
several products of digestion. Secondly, the purification of beta-NGF from the enzyme is
necessary since the enzyme has to be absent in the final sample of the protein. This implies
several purification procedures to remove the abundant Trypsin. Thus, the use of Trypsin as
cleavage enzyme in the procedure of the prior art leads either to very low yields of beta-NGF
or to problems of purification of the protein.
Needless to say that there remains a need in the art for a method of producing beta-NGF
without the drawbacks as described above. It is thus a problem underlying the present
invention to provide a novel method of producing beta-NGF to be obtained in high quality,
high efficiency and in high yields. Further, it is a problem underlying the invention to provide
a production process for beta-NGF which results in high yields of beta-NGF, is efficient,
robust, scalable and reproducible.
An advantage of the invention is the production of a beta-NGF from a novel proNGF mutant.
The novel mutant results in homogenous beta-NGF products in good yield because the novel
proNGF mutant prevents inhomogeneous digestion by proteases and thus inhomogenous beta
NGF products. The problem of the invention is solved by providing the proNGF mutant of the
invention and the method of producing beta-NGF from the proNGF mutant as described by
the present invention.
The novel mutant results in an unexpected and striking increase in the efficiency of the
cleavage of trypsin at the relevant site in the mutated proNGF of the invention compared to
the wild type. This allows to use extremely low amounts of the protease trypsin as compared
to the amount to be used on the wild type and, as a consequence, results in reduced problems
of purification of beta NGF from the enzyme itself and from by products of the cleavage.
The above-described problems are solved and the advantages are achieved by the subject
matter of the independent claims. Preferred embodiments of the invention are included in the
dependent claims as well as in the following description, examples and figures.
The above overview does not necessarily describe all problems solved by the present
invention. Further problems and how there are solved may be apparent for the skilled person
after having studied the present application.
SUMMARY OF THE INVENTION
In a first aspect the present invention relates to a proNGF mutant, wherein the protease
1 3 4 1 3
cleavage site R SK R is substituted at least at positions R and K corresponding to positions
101 and 103 of the human wildtype proNGF sequence (SEQ ID NO: 1) by an amino acid
selected from non-basic amino acids and Histidine.
In a second aspect the present invention relates to a method of preparing a biologically active
human beta-NGF from an inactive insoluble proNGF mutant substituted at the native protease
1 3 4 1 3
cleavage site R SK R at least at positions R and K corresponding to positions 101 and 103
of the human wildtype proNGF sequence (SEQ ID NO: 1), comprising (i) providing a
proNGF mutant according to this invention, and (ii) cleaving the proNGF mutant in order to
obtain active human beta-NGF.
In particular, the invention relates to the following process:
a. dissolving the proNGF mutant in a denaturating solution;
b. transferring the proNGF mutant into a refolding solution where the denatured proNGF
assumes a biologically active conformation;
c. purifying the proNGF mutant from the refolding solution;
d. cleaving the pro-sequence of the proNGF mutant to obtain the active beta-NGF.
A third aspect of the invention relates to the use of a proNGF mutant wherein at least
Arginine at position 101 and the Lysine at position 103 of the native protease cleavage site
1 3 4
R SK R at positions 101 to 104 of the human wildtype proNGF (SEQ ID NO: 1) is
substituted by non-basic amino acids for the preparation of human beta-NGF.
A further aspect of the present invention relates to pharmaceutical compositions comprising
beta-NGF produced from the proNGF mutant wherein at least Arginine at position 101 and
1 3 4
Lysine at position 103 of the native protease cleavage site R SK R at positions 101 to 104 of
the human wildtype proNGF (SEQ ID NO: 1) are substituted by an amino acid selected from
non-basic amino acids and Histidine and a pharmaceutically acceptable carrier or diluent.
This summary of the invention does not necessarily describe all features of the present
invention. Other embodiments will become apparent from a review of the ensuing detailed
description.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Before the present invention is described in detail below, it is to be understood that this
invention is not limited to the particular methodology, protocols and reagents described herein
as these may vary. It is also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not intended to limit the scope of
the present invention which will be limited only by the appended claims. Unless defined
otherwise, all technical and scientific terms used herein have the same meanings as commonly
understood by one of ordinary skill in the art to which this invention belongs.
Throughout this specification and the claims which follow, unless the context reqmres
otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be
understood to imply the inclusion of a stated integer or step or group of integers or steps but
not the exclusion of any other integer or step or group of integer or step.
Several documents (for example: patents, patent applications, scientific publications,
instructions etc.) are cited throughout the text of this specification. Nothing herein is to be
construed as an admission that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
Sequences: All sequences referred to herein are disclosed in the attached sequence listing that,
with its whole content and disclosure, is a part of this specification.
The term "about" when used in connection with a numerical value is meant to encompass
numerical values within a range having a lower limit that is 5% smaller than the indicated
numerical value and having an upper limit that is 5% larger than the indicated numerical
value.
The term "proNGF" or "pro-NGF" refers to the pro-form of human beta-NGF. The full
human proNGF sequence is defined in SEQ ID NO: 1 (Figure la). In order to obtain mature
beta-NGF, the propeptide proNGF has to be cleaved by proteases. The prosequence of beta
NGF is a domain separate from the mature beta-NGF. Between these two domains, there is a
1 3 4
native protease cleavage site Arg-Ser-Lys-Arg (referred herein to R SK R , SEQ ID NO: 9) at
positions 101 to 104 of SEQ ID NO: 1. The cleavage site may be specifically processed by
suitable proteases, in particular furin protease.
The term "proNGF mutant" or "proNGF mutein" refers to modifications of the pro-form of
human beta-NGF by substitutions of amino acids. The proNGF mutein of the present
1 3 4
invention is substituted at the native protease cleavage site R SK R (SEQ ID NO: 9) at least
at both positions K and R corresponding to positions 101 and 103 of the human wild type
proNGF sequence (SEQ ID NO: 1) by an amino acid selected from non-basic amino acids and
Histidine.
In a preferred embodiment of the invention, amino acid Lysine in Position K (corresponding
to position 103) is substituted with Alanine (see Figure ld, SEQ ID NO: 4, Figure le, SEQ ID
NO: 5, Figure lg, SEQ ID NO: 8).
In another preferred embodiment of the invention, ammo acid Arginine in position R
(corresponding to position 101) is substituted with Valine (see Figure lb, SEQ ID NO: 2,
Figure le, SEQ ID NO: 5, Figure lg, SEQ ID NO: 8).
In another embodiment of the invention, the amino acid arginine R corresponding to position
104 of the wildtype proNGF sequence (SEQ ID NO: 1) may also be substituted by any amino
acid which allows processing of the proNGF by proteolytic cleavage to obtain beta NGF,
preferably a basic amino acid such as Arginine or Lysine. For example, the presence of
Alanine in Position R avoids processing of proNGF to beta NGF. Therefore, the mutant of
invention cannot contain Alanine in position 104.
Table 1. Protease cleavage sites of pro NGF and proNGF muteins
(X refers to any amino acid but not Arg or Lys)
SEQ ID NO: Protease cleavage site (pos. 101-104 of SEQ ID NO: 1)
1 RSKR (wild-type) (SEQ ID NO: 9)
2 VSXR (SEQ ID NO: 10)
XSXR (SEQ ID NO: 11)
4 XSAR (SEQ ID NO: 12)
VSAR (SEQ ID NO: 13)
6 RXKR
7 XXXR (SEQ ID NO: 14)
8 VXAR (SEQ ID NO 15)
The term "non-basic amino acid" refers to any amino acid which is not positively charged.
The term refers to an amino acid residue other than a basic amino acid. The term excludes
amino acids Lysine or Arginine which are amino acids with positive side chains. Non-basic
amino acids are negatively charged amino acids Glutamic Acid and Aspartic Acid, amino
acids with polar uncharged side chains (Serine, Threonine, Asparagine, Glutamine ), amino
acids with hydrophobic side chains (Alanine, Valine, Isoleucine, Leucine, Methionine,
Phenylalanine, Tyrosine, Tryptophane) and amino acids Cysteine, Glycine and Praline.
The term "biologically active pro-NGF" or "proNGF with biologically active conformation"
as such refers to the biological activity of pro-NGF. A biologically active conformation of
proNGF is determined by the presence of disulfide bridges occurring in natural beta-NGF.
The activity may be, for example, determined according to an assay as described by Chevalier
et al. 1994, Blood 83: 14 79-1485, 1994, which is incorporated herein by reference. Example
11 describes an assay for the biological activity of proNGF via stimulation of the proliferation
of TFl cells.
The term "beta-NGF" refers to a mature beta-nerve growth factor, preferably from human.
The sequence for the mature beta-nerve growth factor is shown in Figure 1 (SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 7, and SEQ
ID NO: 8), starting at position 105.
The term "activity of beta-NGF" or "biologically active beta-NGF" as such means the
biological activity of beta-NGF. Biologically active beta-NGF exists in the form of a dimer.
Beta-NGF must be present in a dimeric form to have a biologically active conformation. The
prerequisite of a biologically active conformation of beta-NGF is the correct formation of the
disulfide bridges to a cystine knot. The activity may be, for example, determined according to
the DRG assay (dorsal root ganglion assay), see for example Levi-Montalcini, R. et al.,
Cancer Res. 14 (1954) 49, and Varon, S. et al., Meth. in Neurochemistry 3 (1972) 203. In this
assay the stimulation and survival of sensory neurons from dissociated dorsal root ganglia of
chick embryos is monitored by means of neurite formation.
The term "substitution" or "substitutions" refers to modifications of the pro-form of human
beta-NGF by replacement of amino acids. The term comprises the chemical modification of
amino acids by e.g. substituting or adding chemical groups or residues to the original amino
acid. The step of modification of the selected amino acids is performed preferably by
mutagenesis on the genetic level. Preferably, the modification of proNGF is carried out by
means of methods of genetic engineering for the alteration of a DNA belonging to proNGF.
The modifications are mutations that cause the replacement of a single base nucleotide with
another nucleotide of the genetic material. Point mutations results in encoding different amino
acids compared to the wild-type sequence. Preferably, expression of the modified proNGF
protein is then carried out in prokaryotic or eukaryotic organisms, most preferably in
prokaryotic organisms.
The term "denaturating" or "denaturation" refers to a process in which the folding structure of
a protein is altered. The term refers to unfold the tertiary structure of proNGF or proNGF
mutein. The alteration of the folding structure is due to exposure to certain chemical or
physical factors. As a result, some of the original properties of the protein, especially its
biological activity, are diminished or eliminated. Due to the denaturing process, proteins
become biologically inactive. Further, denatured proteins can exhibit a wide range of
characteristics, including loss of biological function, loss of solubility and/or aggregation.
The term "refolding" or "renaturating" or "renaturation" refers to a process by which the
protein structure assumes its native functional fold or conformation. Due to renaturation or
refolding processes, the protein becomes biologically active.
The term "recombinant" refers to the cloning of DNA into vectors for the expression of the
protein encoded by the DNA in a suitable host. The host is preferably a prokaryote, most
preferably a bacterium. A "recombinant expression" as used herein refers to expression of
proNGF or the proNGF mutein in in prokaryotic host cells, for example E. coli strains suitable
for expression of recombinant proteins could be used.
The term "soluble" refers to a protein which is susceptible of being dissolved in some solvent.
The term "insoluble" refers to a protein which is not susceptible of being dissolved in some
solvent.
Description of the invention
ProNGF mutants of the invention
In a first embodiment of the invention, the present invention provides a proNGF mutant
1 3 4 1 3
wherein the protease cleavage site R SK R is substituted at least at positions R and K
corresponding to positions 101 and 103 of the human wildtype proNGF sequence (SEQ ID
NO: 1) by an amino acid selected from non-basic amino acids and Histidine. In other words,
at least Arginine R at position 101 and the Lysine K at position 103 of the native protease
1 3 4
cleavage site R SK R at positions 101 to 104 of the human wild type proNGF sequence (SEQ
ID NO: 1 ) are substituted by any amino acid but not Arginine or Lysine.
In the human wildtype proNGF (SEQ ID NO: 1), the native protease cleavage side refers to
amino acids positions 101 to 104 (ArgSerLysArg, RSKR, SEQ ID NO: 9). Amino acid Lysine
K in position 103 of the wild-type proNGF sequence and amino acid Arginine R in position
101 are replaced with any amino acid but not Arg or Lys to result in a proNGF with improved
properties in particular for producing beta-NGF. In order to achieve the above-identified
object, i.e. to generate a mutein with improved features for producing beta-NGF, Arginine
(R ) or Lysine (K ) may be substituted by all naturally occurring amino acids, or artificial
amino acids as well, provided they do not constitute a cleavage site for trypsin.
According to the invention, the ammo acid modifications to one or more positions
corresponding to residues 101-103 may be substitutions that replace basic amino acids with
non-basic amino acids. Theses substitutions can be used to create proNGF mutants according
to the invention, in particular, for the production of beta-NGF. Mutants comprise substitutions
at least at positions ArglOl and Lys103. The amino acid residues are replaced by a non-basic
amino acid or Histidine. Particularly, the mutants of the invention have the substitutions
Arg101Val and Lys103Ala. For example, said natural non-basic amino acids may be selected
from the group consisting of the naturally occurring amino acid residues Alanine, Asparagine,
Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Isoleucine, Leucine,
Methionine, Phenylalanine, Praline, Serine, Threonine, Tryptophan, Tyrosine, Valine.
Amino acids for substitutions at positions 101 and 103 are not selected from basic (positively
charged) amino acids Arginine (Arg) and Lysine (Lys). Also less preferred are amino acids
Isoleucine (Ile), Leucine (Leu), or Phenylalanine (Phe), Cysteine (Cys), Praline (Pro) or
Tryptophan (Trp). Serine (Ser) is naturally occurring in position 102 of the human proNGF
wild-type sequence. X in position 102 (SEQ ID NO: 7 and SEQ ID NO: 8) is preferably
selected from Serine (Ser) which is naturally occurring in position 102 of the human proNGF
sequence, but may also be selected from any other amino acid wherein the amino acid must be
a non-basic amino acid (i.e. not Arginine or Lysine). It is important that the amino acid in
position 102 is a non-basic amino acid (i.e. not Arg or Lys).
The ammo acid in position 104 of the wild-type human proNGF sequence is preferably
Arginine (Arg) which is naturally occurring in position 104 of the human proNGF sequence,
but may also be substituted by any other amino acid, preferably a basic amino acid, more
preferably Lysine, which allows processing of the proNGF by proteolytic cleavage to obtain
betaNGF.
For example, the presence of Alanine in position 104 of wild-type human proNGF avoids
processing of proNGF to beta NGF. Thus, Ala in position 104 is excluded.
Table 2 summarizes the preferred substitutions for the protease cleavage site of proNGF.
Table 2A. Preferred amino acids in positions 101-104 of wild-type proNGF
The star(*) shows the naturally occurring amino acid in the protease cleavage site (wild-type
proNGF, SEQ ID NO: 1).
101 Val, Ala, Asn, Asp, Glu, Gln, Gly, Ser, Thr, Tyr, Met, His, Cys, Pro, Phe, Trp, Ile, Leu
102 Ser*, Gly, Asp, Tyr, Thr, Asn, Glu, Ala, Val, Gln, His, Met, Cys, Pro, Phe, Trp, Ile,
103 Ala, Val, Asp, Asn, Glu, Gln, Gly, Ser, Thr, Tyr, Met, His, Cys, Pro, Phe, Trp, Ile, Leu
104 Arg*, Lys
Amino acids Cys, Pro, Phe, Trp, Ile and Leu are less preferred substitutions at Positions 101,
102, and 103.
Table 2B. Most preferred amino acids in positions 101-104 of wild-type proNGF
101 Val, Ala, Gly, Ser, Thr, Asn, Asp, Glu, Gln, Tyr, Met, His
102 Ser*, Val, Ala, Gly, Thr, Asn, Asp, Glu, Gln, Tyr, Met, His
103 Ala, Val, Gly, Ser, Thr, Asn, Asp, Glu, Gln, Tyr, Met, His
104 Arg*, Lys
It is essential that there are any amino acid but not Arg or Lys at positions 101, 102, and 103
of the proNGF-mutein and that there is a basic amino acid (Arg or Lys) at position 104 of the
proNGF-mutein. It was surprisingly shown that specifically two amino acid replacements in
positions 101 and 103 result in high efficiencies of beta-NGF production.
In the sequence of the most preferred proNGF mutant of the invention (SEQ ID NO: 5), the
preferred substituted amino acid in position 101 of human wild-type proNGF is Valine, in
position 103 of human wild-type proNGF is Alanine, in position 102 of human wild-type
proNGF Serine, and in position 104 of human wild-type proNGF Arginine. In particularly
preferred embodiment of the invention, the proNGF mutant of the invention presents has a
sequence corresponding to that of SEQ ID NO: 5.
The present invention is also directed to nucleic acids coding for the proNGF mutants
described herein as well.
Method of preparing a human beta-NGF from a proNGF mutant of the invention
In a second aspect, the present invention is directed to a method of preparing a biologically
active human beta-NGF from an inactive insoluble proNGF mutant substituted at the native
1 3 4 1 3
protease cleavage site R SK R (SEQ ID NO: 9) at positions 101 and 103 (R and K ) of the
human wildtype proNGF sequence (SEQ ID NO: 1), comprising providing a proNGF mutant
1 3 4 1 3
substituted at the native protease cleavage site R SK R at positions 101 and 103 (R and K )
of the human wildtype proNGF sequence, and cleaving the proNGF mutant in order to obtain
active human beta-NGF.
Preferably, the proNGF mutant is obtained by recombinant expression in prokaryotic cells.
Suitable bacterial strains are well known in the art, e.g., E. coli, Bacillus sp., and Salmonella,
and kits for such expression systems are commercially available. The preferred host cells for
recombinant expression are E. coli, for example E. coli BL21, JM 108/109 (Kl2), JM106,
JM83 und TB 1 or derivatives thereof. Any other E. coli strain suitable for expression of
recombinant proteins could be used.
Polynucleotides are operatively linked to expression control sequences allowing expression of
the fusion proteins of the invention in prokaryotic host cells. Such expression control
sequences include but are not limited to inducible and non-inducible promoters, operators,
repressors and other elements that are known to those skilled in the art and that drive or
otherwise regulate gene expression. Such regulatory elements include as for example T7,
TAC, PBAD, LAC promoters, Laci, LacIQ repressors.
The sequence of the proNGF mutant is introduced into the prokaryotic host cell by a suitable
vector. Suitable Vectors could be for example but not limited to: pBR322, pMAL, pUC19
and all derivatives. The prokaryotic host cell includes but is not limited to prokaryotic cells
such as bacteria (for example, E. coli or B. subtilis), which can be transformed with, for
example, plasmid DNA, recombinant bacteriophage DNA, or cosmid DNA expression vectors
containing the polynucleotide molecules of the invention. In one embodiment of the
invention, plasmid vectors are use. For example, but by no way limited to, plasmid vectors
described in EP1697523Bl may be used (which is incorporated by reference herein).
In order to express proNGF muteins, an expression vector is used that contains
a. a strong promoter to direct transcription (e.g. a tac or T7 promoter),
b. a coding sequence for proNGF or proNGF mutein
c. a transcription/translation terminator (e.g. tO-terminator of the bacteriophage
lambda)
d. a first selectable marker gene, e.g. a gene coding for antibiotic resistance (e.g.
Kanamycin resistence, kan),
e. a second selectable marker gene, e.g. a gene coding for proB and I or proA.
f. a repressor gene (e.g. a lacI gene)
g. a high copy number origin of replication
In one embodiment of the invention, proprietary expression vectors (Scil Proteins GmbH, see
EP1697523B1 for the structure of a suitable expression vector) or commercially available
vectors may be used for cloning. Regarding general information on the vectors which might be
used in the method of the present invention, it is referred to the above mentioned details.
However, any suitable vectors might be used as known in the art.
The structure of the proprietary expression vector pSCIL101 as one example for a suitable
vector for the transformation of prokaryotic host cells is depicted in Figure 7.
The method of the preparation of a proNGF mutant is comprising the following initial steps:
i. preparing a nucleic acid encoding a proNGF mutein
ii. introducing said nucleic acid into a procaryotic expression vector;
iii. introducing said expression vector into a host cell;
iv. cultivating the host cell;
v. subjecting the host cell to suitable culturing conditions.
Due to its expression in prokaryotic host cells, the proNGF mutein is in the form of its inactive,
insoluble form.
In a preferred embodiment, the method of production of beta-NGF from a proNGF mutant
according to the present invention comprises the steps of:
1 3 4
a. dissolving the proNGF mutant substituted at the native protease cleavage site R SK R
at positions R and K corresponding to positons 101 and 103 of the human wildtype
proNGF sequence (SEQ ID NO: 1) by solubilisation of inclusion bodies in a
denaturating solution;
b. transferring the proNGF mutant into a refolding solution where the denatured proNGF
assumes a biologically active conformation;
c. purifying the proNGF mutant from the refolding solution;
d. cleaving the pro-sequence of the proNGF mutant to obtain the active beta-NGF.
In the following, the preferred steps of a method for producing beta-NGF from a proNGF
mutant according to the present invention are discussed.
Step a: Solubilisation of proNGF mutant
Step a) corresponds to dissolving the proNGF mutant substituted at the native protease
1 3 4 1 3
cleavage site R SK R at positions R and K corresponding to positons 101 and 103 of the
human wildtype proNGF sequence (SEQ ID NO: 1) by solubilisation of inclusion bodies in a
denaturating solution. It is noted that the proNGF mutant of the invention in step a) usually is
in the form of its inactive, insoluble form due to its expression in prokaryotic host cells.
Inactive proNGF showing a poor solubility is formed during overexpression of the protein in
the cytosol of prokaryotes. In this case, proNGF prepared by recombination remains in the
cytoplasm in an insoluble and aggregated form. These protein aggregates, the isolation thereof
as well as their purification are described for example in Marston, F. A., Biochem. J. 240
(1986).
To isolate these inactive protein aggregates (inclusion bodies), the prokaryotic cells are
disrupted following fermentation. Cell disruption may be performed by conventional
methods, e.g. by means of high pressure homogenization, sonification or lysozyme (Rudolph,
R., et al. (1997); Folding proteins. In: Creighton, T. E. (ed.): Protein Function: A Practical
Approach. Oxford University Press, pp. 57-99).
Further, the inclusion bodies are solubilized. Inclusion bodies (IB) are accumulations of
usually defective or incompletely folded proteins. They form inside cells, for example
bacteria cells, such as E. coli, in the event of excessive expression of recombinant proteins.
The inclusion bodies employed according to the invention preferably comprise the proNGF
mutein. This means that they contain at least 60, at least 70, at least 80 or at least 90 wt.% of
pro-NGF (based on the total amount of protein).
The invention provides a method for the production of proNGF mutein thereof, wherein
inclusion bodies which non-folded, inactive, insoluble proNGF mutein or a derivative thereof
are solubilized in a denaturing buffer (solution).
The denaturating solution of step a) preferably compnses a solution containing (i) a
chaotropic agent, (ii) a chelator, (iii) a buffer, and (iv) a reducing agent.
The denaturation buffer compnses at least one chaotropic substance (agent). Chemical
substances which dissolve ordered hydrogen bridge bonds in water are called chaotropic.
Since the hydrogen bridge bonds are broken open, the chaotropic substances interfere with the
water structure and ensure disorder (increase in entropy). The reason for this is that the
formation of the H 0 cage structures necessary for the salvation is prevented. In the case of
amino acids, they reduce the hydrophobic effects and have a denaturing action on proteins,
since a driving force of protein folding is the assembling together of hydrophobic amino acids
in water. Generally, any substance which exerts the hydrophobic effect in the solubilization
buffer and therefore has a denaturing action on the proteins can be employed as a chaotropic
substance. Chaotropic substances are in general salts or low molecular weight compounds,
such as urea. Chaotropic substances are clearly distinguished from detergents, since they
contain no hydrophobic radical, such as an alkyl radical, in the molecule. Generally, the
chaotropic action is accompanied by an improvement in the solubility of the protein, in this
case the prethrombin.
In a preferred embodiment of the invention, the chaotropic compound is chosen from
guanidinium salts, in particular guanidinium hydrochloride and guanidinium thiocyanate,
iodides, barium salts, thiocyanates, urea and perchlorates.
The chaotropic compounds are employed in conventional amounts. For example, 4 - 8 M
guanidinium hydrochloride or 4 - 9 M urea can be employed.
The denaturation buffer comprises a reducing agent compound, for example a disulphide
compound such as Glutathione (GSH). The disulphide compound is capable of forming mixed
disulphides with thiol groups (-SH) of cysteines of the polypeptides in the inclusion bodies.
The disulphide is added to the solution. The disulphide does not designate proteins which the
inclusion bodies comprise and which possibly comprise disulphide bridges. Preferably, the
disulphide is not a true peptide. Preferably, the disulphide is a low molecular weight
compound. The molecular weight is, for example, lower than 2,000 g/mol or than 1,000
g/mol. The disulphide is employed, for example, in a concentration of from 5 mM to 1 M, in
particular 10 mM to 0.5 M.
In a preferred embodiment of the invention, the disulphide compound is glutathione
disulphide. Glutathione (GSH), also y-L-glutamyl-L-cysteinylglycine, is a pseudo-tripeptide
which is formed from the three amino acids glutamic acid, cysteine and glycine. GSH is
present in the cytoplasm of both prokaryotes and eukaryotes and is involved in the formation
of disulphide bridges. It is in equilibrium with the dimer GSSG, which contains a disulphide
bridge. Glutathione reacts with cysteines R-SH and R'-SH from two polypeptides or from a
single polypeptide in a disulphide exchange reaction:
R-SH + GSSG-+ R-S-S-G + GSH.
RSSG is called a mixed disulphide. It is reacted with a further cysteine of a polypeptide, so
that as a result a disulphide bridge is obtained between two cysteines:
R-S-S-G + HS-R'-+ R-S-S-R' + GSH.
Glutathione is kept enzymatically in the reduced form (GSH) in the cytosol. "Reducing
conditions" in the cytosol are therefore referred to. Conditions are established in the
solubilization buffer so that the disulphide compound it comprises catalyses the formation of
disulphide bridges in accordance with the reactions described above. The GSSG is employed,
for example, in a concentration of from 10 mM to 0.5 M.
Alternatively, as reducing agent (reductant), Cysteine might be used.
In a preferred embodiment of the invention, the denaturation solution is a Tris buffer.
The denaturation solution can comprise further conventional additives, for example EDTA or
salts. The pH of the solubilization buffer is, for example, between 7 and 10, preferably pH 8.
The solubilization is preferably assisted mechanically, for example with conventional
homogenization apparatuses or by means of ultrasound. After the solubilization, solids which
remain are preferably separated off. The supernatant comprises the solubilized pro-NGF.
In one embodiment of the invention, the denaturing solution comprises
1. Guanidinium-HCl, 1 - 8 M, preferably 4-6 M, most preferred 4 M,
GSH or Cysteine, 1 - 100 mM, preferably 5 mM
11. Tris, 0.01 - 1 M, preferably 0.1 M,
111. EDTA, 1 - 50 mM, preferably 10 mM
IV. pH 7.0 - 10.0, preferably pH 8.0
A concentration of 4 M Guanidinium-HCl IS m most cases sufficient for a complete
denaturation of proNGF mutein.
Step b: Refolding of the proNGF mutant
After the solubilization of the proNGF mutant from inclusion bodies it is necessary to refold
the protein in its native conformation. For the refolding process it is important to minimize the
competing reactions misfolding and aggregation. To prevent aggregation the refolding IS
performed at very low protein concentrations because aggregation of the protein IS
predominant at high protein concentrations. In step b), transferring the proNGF mutant into a
refolding buffer occurs where the denatured proNGF assumes a biologically active
conformation. A biologically active conformation can be determined by the presence of
disulfide bridges occurring in natural beta-NGF.
In a preferred embodiment of the invention, the solubilized proNGF mutant is renatured in a
refolding solution which contains at least one chaperone, at least one a metal chelator, and a
redox shuffling system.
In a preferred embodiment, the method according to the present invention uses a refolding
solution in step b) comprising
1. a chaperone, preferably Arginine, 0.5-1.0 M, preferably 0.75 M,
11. a metal chelator, preferably EDTA, 1-10 mM, preferably 5 mM,
111. a redox shuffling system, at 0.1-10 mM, preferably 1 mM L-Cystine and 5 mM L
Cysteine, or 1 mM GSSG (oxidized glutathione) and 5 mM GSH (reduced glutathi
one).
1v. pH 8.0 - pH 11.0, preferably pH 9.5
Alternative redox shuffling systems such as Cystamin/Cysteamin could be used.
In a preferred embodiment of the invention, the folding assistant is Arginine. Compounds
which promote the folding of proteins can generally be employed as "folding assistants". Such
compounds are known to the person skilled in the art. They can assist the folding in various
ways. It is assumed that arginine destabilizes incorrectly folded intermediates, so that these
are at least partly unfolded again (from a thermodynamic dead-end) and therefore can be
correctly folded again. On the other hand, glycerol usually stabilises proteins. Compounds
which increase the absolute yield of folded pro-NGF mutein in the method according to the
invention by more than 5 %, in particular by more than 10 % or more than 20 % (based on the
total amount of pro-NGF employed for the folding), compared with a method without using
the folding assistant, are suitable in particular as folding assistants.
The refolding is preferably carried out at a pH of between 8 and 11, in particular pH 9.5.
To increase the protein concentration in the refolding vessel, a pulse renaturation was carried
out. Limiting for the number of pulses is the Guanidinium-HCl concentration which should
not exceed 0.3 M. The protein concentration per pulse should not exceed 50 µg/ml in relation
to the final refolding volume.
In a preferred embodiment of the invention, the solubilisate is added to the folding batch in
several fractions or continuously over several days. Preferably, the solubilisate is added in a
"pulse renaturing" by rapid dilution to the solubilisate. In this context, for example but by no
means limited to, at least 6 pulses could be performed in a time interval of, for example, 24
hours. The number of pulses is set such that after the addition of the solubilization batch the
concentration of protein which has not yet been folded is not too high, since otherwise
aggregates are obtained. For example, with each pulse 0,05 g/l to 0,2 g/l, preferably 0.1 g/l of
protein is newly transferred into the folding batch (based on the protein concentration in the
folding batch after addition of the solubilisate). For example, each refolding step takes at least
1-2 h.
After refolding, the refolding reaction needs to be clarified before loading onto a column. This
can be done by any methods known in the art, for example, by filtration.
In a preferred embodiment, the method for producing a correctly folded pro-NGF mutant
includes the following steps: a) Inclusion bodies which comprise insoluble proNGF mutant
are solubilized in a denaturing solution as described above, and b) the solubilized pro-NGF is
then renatured in a refolding solution buffer as described above.
In a preferred embodiment of the invention, the denaturing solution and/or the refolding
solution consequently contains no detergent. It has been found, that the use of detergents is
not necessary for the solubilization and/or folding of pro-NGF mutein. This is advantageous,
since certain detergents are comparatively aggressive chemical substances which
pharmaceutical products should not comprise or should comprise in only small amounts and
therefore must be removed in an expensive manner. The method according to the invention is
therefore advantageous compared with the method of Soejima et al., 2001, in which such
aggressive detergents (Triton X-100 or Brij-58) are employed for folding the protein. In other
words, no detergents are used in the entire production method according to the invention, and
the production method is therefore detergent-free.
Step c: Purification of proNGF mutant by chromatography
By carrying out the method according to the invention with the denaturation and subsequent
refolding, an aqueous solution of folded pro-NGF mutein is obtained. The folded pro-NGF
mutein can subsequently be purified further by known methods.
In a preferred embodiment the proNGF mutant is purified from the refolding (e.g. non- or
weak denaturing) solution via chromatographic purification, in particular by means of a mixed
mode chromatography (step c of the method of production of beta NGF from a proNGF
mutant of the invention). The most preferred column for the chromatography is a column with
a synthetic affinity ligand, preferably 4-mercapto-ethyl-pyridine (MEP Hypercell; Pall).
Advantages of this medium are that the binding is independent of the ionic strength, salt
stacking is not necessary and higher flow rates to fasten the process are possible. Further, the
elution is done by a pH-shift.
Other mixed mode material columns are known and could be used. For example, but not
limited to, MEP (Pall; affinity ligand is 4-Mercapto ethyl pyridine), HEA (Pall; affinity
ligand: Hexylamino ), PP A (Pall, affinity ligand: Phenylpropylamino ), MBI (Pall; affinity
ligand: 2-Mercapto-5benzamidazole sulfo acid), Capto MMC (GEHC), Capto adhere (GEHC;
affinity ligand: N-benzyl-N-methyl ethanolamine), CHT hydroxyapatite (BioRad), CHT
fluoroapatide). The MEP, HEA, PPA, and MBI columns have a hydrophobic binding, where
Capto MMC is a cation exchanger with mixed mode functionality and Capto adhere is an
anion exchanger with mixed mode functionality. The BioRad columns are ion exchange
columns with hydrophobic components. Any other mixed mode material column not listed
here could also be used to purify the proNGF mutant.
Step d: Cleavage of proNGF to beta-NGF
proNGF is the precursor of beta-NGF. Thus, in step d) of the method of production of beta
NGF from a proNGF mutant of the invention, the pro-sequence of the proNGF mutant is
cleaved in order to obtain an active beta-NGF.
Proteases having trypsin-like substrate specificity cleave the protein without digesting the
active portion of the protein molecule. Trypsin-like proteases cleave peptide bonds following
a positively charged amino acid such as Arginine or Lysine. As trypsin-like proteases, several
serine proteases (serine endopeptidases) are considered for processing of the proNGF to result
beta-NGF. Preferably, the serine protease Trypsin is used for the cleavage of the pro-sequence
but other proteases could be used instead.
It is noted that cleavage is not restricted to trypsin itself, but may involve other proteases
having trypsin-like substrates as well. Generally, if the ratio of proNGF to trypsin (or other
protease) is appropriately adjusted, the correctly folded, mature beta-NGF will not be cleaved
by this protease. In contrast, denatured proteins as well as folding intermediates expose
sequences which are susceptible to an attack by the protease.
Preferably for the cleavage of proNGF mutant to beta-NGF, the ratio of trypsin (or other
protease) to proNGF mutant is from 1 : 200 - 1 : 100.000, more preferably from 1 : 5.000 - 1
: 20.000 per weight, most preferred is a ratio of 1 : 10.000 (w/w). In a most preferred
embodiment, the cleavage occurs for 8-23 hours at room temperature, most preferred 18
hours. Under the conditions used in this invention, proNGF mutant is cleaved completely and
almost no by-products are formed. No aggregation was observed.
As clearly described in the Examples, the present inventors have found that the amino acid
modifications introduced in the proNGF mutant of the invention not only avoid cleavage of
the protein at undesired cleavage sites but also unexpectedly result in a great increase in the
efficiency of the cleavage of Trypsin compared to that of the wild type proNGF, which allows
to carry out the cleavage under very selective conditions to obtain a very pure product.
In details, the experimental data clearly show that at already a very low Trypsin/Protein ratio
such as 1:100.000, the proNGF mutant of the invention (SEQ ID NO: 5) results in very high
purity recombinant human beta-NGF with a high cleavage yield (about 85%). Furthermore,
wild type proNGF (SEQ ID NO: 1) at the same Trypsin/Protein ratio shows a low cleavage
yield (about 5% ). A satisfactory yield is only obtained at much higher trypsin/protein ratios
(1/250), but this is accompanied by low selectivity and a high product degradation due to
overdigestion.
Step e: Further purification of beta-NGF
The beta-NGF produced from a proNGF mutant of the invention is further purified, for
example, by several chromatographic methods. Further purification steps are required to
separate Trypsin and product related impurities of the tryptic digestion from beta-NGF.
Purification steps should reduce HCPs, Endotoxins, and DNA. Any methods known in the art
for protein purification can be used. Most preferred are chromatographic purifications, for
example with Sepharose columns (e.g. SP Sepharose HP, Q Sepharose FF).
The final product beta-NGF produced from a proNGF was analyzed regarding its purity by
SDS-PAGE, rp-HPLC, SE-HPLC, and IEX-HPLC. HPLC analyses revealed a purity of beta
NGF of at least 97%.
In a preferred embodiment of the invention, the method for the production of a pro-NGF
mutein suitable for obtaining beta-NGF includes the following steps:
a) expression of a recombinant pro-NGF mutant with substituted protease cleavage site
in prokaryotic cells
b) isolation of the pro-NGF mutein-containing inclusion bodies,
c) mixing of the inclusion bodies with a suitable denaturing buffer comprising at least (i)
a chaotropic substance, (ii) a chelator, (iii) a buffer, and (iv) a reducing agent
d) refolding in a refolding solution comprising at least a chaperone, a metal chelator,
and a redox shuffling system,
e) purification of the refolded pro-NGF mutant,
f) cleavage into the active form of beta-NGF with proteases such as trypsin
g) isolation and purification of the beta-NGF.
Use ofproNGF for the production of beta-NGF
In a third aspect, the invention is directed to the use of the proNGF mutant of the present
invention for producing human beta-NGF.
Pharmaceutical composition of betaNGF obtained from proNGF mutants of the invention
In a further aspect, the invention is directed to a pharmaceutical composition comprising
betaNGF obtained from a proNGF-mutant being substituted at the native protease cleavage
1 3 4 3 1
site R SK R at positions 101 and 103 (K and R ) of the human wildtype proNGF sequence
(SEQ ID NO: 1) as described above and a pharmaceutically acceptable carrier.
In one embodiment of the invention, the pharmaceutically active beta-NGF is administered to
the patient by gene-therapeutical methods. In gene therapy, there are two basic methods
available, suitable for introducing a gene, in the present case a gene coding for a beta-NGF,
into the patient.
In the ex viva application, the pharmaceutically active gene encoding beta-NGF is introduced
in a body cell by a vector, where the body cell preferably is a glial cell, and the cell treated in
this way then is re-introduced into the patient, for example by micro- or nanoparticles.
Particularly preferred is a specific integration of the beta-NGF gene in the cellular genome.
In the in viva-gene therapy, the beta-NGF gene is transported to target cells in the body by
vectors, for example by means of viruses, which on the one hand may infect the target cell
und, thus, will be able to introduce the pharmaceutically active beta-NGF gene, but, on the
other hand, are not able to reproduce themselves within the target cell. In this approach, nano
or microparticles, for example liposomes, which may fuse with the cell membrane, may be
used a vectors as well.
As a vector for the beta-NGF gene, a virus or an antibody might be used as an example,
capable of specifically infecting the host cell or which immunoreacts with an antigen in the
target cell. As a viral vehicle, retroviruses might be used as an example. Furthermore, it is
possible to use adenoviruses or Vaccinia based vectors, for example, modified vaccinia virus
Ankara (MV A).
The skilled person will be able to select a suitable formulation based on routine considerations
and will chose a suitable form for administering the present pharmaceutical composition to a
patient. For example, the pharmaceutical composition might comprise one or more
pharmaceutically acceptable ingredients, for example carriers or diluents. Among these
classes of substances, one might name fillers, salts, buffers, stabilisators, penetration
enhancers and other well-known materials. Techniques for the formulation of pharmaceutical
compositions of the present invention may be found in well-known standard textbooks such as
"Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition.
The dosage of the betaNGF obtained by the method of production as described in the present
invention might be in a range of 0.1 µg/kg to 500 µg/kg body weight, if administered by
infusion, and from 2 µg/kg to 2 mg/kg body weight if administered by injection.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the sequence of pro-NGF and of the pro-NGF mutants of the invention.
Shown in bold letters is the sequence of the proform of human beta-NGF. Shown in bold and
underlined is the protease (trypsin) cleavage site (amino acids 101-104 of SEQ ID NO: 1;
trypsin cleavage sites are between amino acids 101-102 (R\ 103-104 (K ) and 104-105 (R )).
X in the sequence can be any amino acid.
Figure la shows a sequence of human proNGF (SEQ ID NO: 1) with protease cleavage site
RSKR (SEQ ID NO: 9).
Figure lb shows a sequence of a proNGF mutant of the invention (SEQ ID NO: 2) with the
protease cleavage site VSXR (SEQ ID NO: 10).
Figure le shows a sequence of a proNGF mutant of the invention (SEQ ID NO: 3) with the
protease cleavage site mutated to XSXR (SEQ ID NO: 11).
Figure ld shows a sequence of a proNGF mutant of the invention (SEQ ID NO: 4) with the
protease cleavage site mutated to XSAR (SEQ ID NO: 12).
Figure le shows a sequence of a proNGF mutant of the invention (SEQ ID NO: 5) with the
protease cleavage site mutated to VSAR (SEQ ID NO: 13).
Figure 1f shows a sequence of a proNGF mutant of the invention (SEQ ID NO: 7) with the
protease cleavage site mutated to XXXR (SEQ ID NO: 14).
Figure lg shows a sequence of a proNGF mutant of the invention (SEQ ID NO: 8) with the
protease cleavage site mutated to VXAR (SEQ ID NO: 15).
Figure lh shows sequences of protease cleaving sites (SEQ ID NOs: 6, 9-15).
Figure 2. Processing of proNGF or pro NGF mutants to beta-NGF
Figure 2a shows six beta NGF cleavage products after trypsin cleavage by using the wild type
proNGF having a native furin cleavage site RSKR. The drawing clearly shows that a cleavage
of wild type proNGF to betaNGF results in an inhomogenous mixture of many different
cleavage products.
Figure 2b shows native beta NGF cleavage products after trypsin cleavage by using a
proNGF mutant SP174-101 (SEQ ID NO: 5) with deletion of the native furin cleavage site.
The protease cleavage site RSKR (SEQ ID NO: 9) was substituted by two amino acids to
result a site VSAR (SEQ ID NO: 12). This site can only be cleaved by a protease after the
amino acid Arginine in position 104; Trypsin can only cleave at one cleavage site (instead of
three). The drawing clearly shows that a cleavage of mutant proNGF SP174-101 (SEQ ID
NO: 5) to beta-NGF results in only one homogenous cleavage product (beta-NGF).
Figure 3 shows the refolding of the proNGF mutant SP174-101 (SEQ ID NO: 5) compared to
wild type proNGF. The figure compares the refolding yield of the wild type proNGF
(continuous line) and the proNGF mutant (broken line) with the protease cleavage site
mutated to VSAR. It can be clearly seen from the figure that the refolding efficiency of wild
type and mutant proNGF is identical.
Figure 4 shows the purification of a proNGF mutant SP174-101 with the protease cleavage
site mutated to VSAR (SEQ ID NO: 5) by a MEP HyperCel column. The figure shows an
elution profile of MEP HyperCel purification of a refolded and filtrated proNGF mutant.
Figure 5 shows the cleavage of a proNGF mutant SP174-101 with the protease cleavage site
mutated to VSAR (SEQ ID NO: 5) by Trypsin. The figure shows a Coomassie stained SDS
PAGE gel of fractions of the tryptic cleavage. The tryptic cleavage product of the proNGF
mutant can be seen in lanes 4-7. The figures clearly show that the purified proNGF mutant
results in only one cleavage product (beta-NGF).
Figure 6 shows the purification of beta-NGF. The figure shows a profile of a SP Sepharose
HP column after the tryptic cleavage. The tryptic digestion reaction was loaded onto a SP
Sepharose HP column. The elution was done in three steps (a. 25 % 25 mM sodium phosphate,
1 M NaCl, pH 6.5 (buffer B), b. in a linear gradient from 25-50 % buffer B, and c. 100 % buffer
B (flow rate 60 cm/h)).
Figure 7 shows the structure of the proprietary expression vector pSCIL101.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a
complete disclosure and description of how to make and use the methods and compositions of
the invention, and are not intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect to numbers used but some
experimental errors and deviations should be accounted for. Unless indicated otherwise,
molecular weight is average molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1. Substitution of wild-type pro NGF at the protease cleavage site at positions
1 3 4
101 to 104 (R SK R )
Substitution of Arginine R and Lysine K corresponding to positions 101 and 103 of human
pro-NGF (SEQ ID NO: 1) was realized on DNA level using a synthesized gene by methods as
known to someone skilled in the art. Serine in position 102 either remained unchanged or
substitution of position 102 of human pro-NGF (SEQ ID NO: 1) was also realized on DNA
level using a synthesized gene by methods as known to someone skilled in the art. Lysine K
corresponding to position 104 was not substituted. Sequences are shown in Figure 1.
Example 2. Recombinant expression of proNGF mutant SP174-101 (SEQ ID NO: 5) in
prokaryotic cells
The bacterial host E. coli JM108 used for expression of rh-proNGF (DSMZ 5585; F thi Δ (lac-
proAB) end AI gyrA96 relAl phx hsdRl7 supE44 recA) is proline-auxotrophic, which was
neutralized by the use of the plasmid with the designation pSCIL101. The plasmid pSCIL101
is based on the plasmid pSCIL008 (see WO05061716). The strain cannot synthezise thiamine
(Vieira & Messing, 1982 Gene. Oct;19(3):259-68). The pro-NGF mutant shown in SEQ ID
NO: 5 is expressed under the control of the tac promoter located on pSCILlOl. The vector
pSCILlOl used here is a high copy plasmid with a kanamycin resistance. The expression is
carried out in defined mineral salt medium and is induced by the addition of IPTG. The pro
NGF mutant is deposited in the cytosol in the form of inclusion bodies (IBs).
Cell line:
• host strain, e.g. E.coli HMS174 (K12) or JM108 (K12)
• proNGF mutant SP174-101 (SEQ ID NO: 5)
• Tac promoter (IPTG induction)
• ColEl replicon
• Kanamycin resistance
• proBA selection
• Proprietary vector system pSCIL 101 (e.g. see W005/061716)
Example 3. Fermentation
The aim of this fermentation was to obtain product and biomass for subsequent process steps.
To monitor the over-expression of the target protein during the fermentation process, samples
were analyzed by means of SDS-PAGE before and after induction.
• Mineral salt medium without antibiotics
• Batch phaseµ;:::: 0.25 h- (0Dena=18)
• Fed batch phase I exponential feeding with µset= 0.18 h-
• Fed batch phase II: constant feed rate
• Point of induction ODind = 60 ±5
• 1.0 mM IPTG
• Time of induction 5 h
• Final OD = 82 ±4
• Process time 28.5 h ±1.25
• Plasmid Stability 100 %
• Yield: 40 mg/g proNGF; 1,2 g/L ± 0.2 g/L proNGF
Example 4. Primary Recovery of inclusion bodies containing SP174-101
In bacterial cells, the recombinant protein is present in the form of aggregates. The expression
of the pro-NGF mutein took place in the form of /Bs. The cell breakdown and the IB
preparation were carried out in accordance with standard protocols and can be conducted on
the laboratory scale up to a working up of approx. 200 g of biomass. The preparation of these
"inclusion bodies" containing the proNGF mutein was performed according to Rudolph, R., et
al. (1987); Folding proteins. In: Creighton, T. E. (ed.): Protein Function: A Practical
Approach. Oxford University Press, pp. 57-99, and according to EP0994188Bl. For cell
disruption, the cell pellets were resuspended in a suitable buffer and subsequently the cells
were disrupted using high pressure homogenization in 50 mM Natriumphosphat pH 7.0, 1
mMEDTA.
Example 5. Dissolving the proNGF mutant SP174-101 in a denaturating solution
(solubilization of inclusion bodies)
The inclusion bodies were solubilized in a denaturing solution which comprised a solution (i)
a chaotropic agent, (ii) a chelator, (iii) a buffer, and (iv) a reducing agent. For solubilization,
Guanidinium HCl (GuaHCl) was tested in a concentration range of 4.0-6.0 M. The
solubilization buffer was mixed in different ratios with a inclusion-body slurry (IB slurry). All
experiments had a final Cysteine concentration of 5 mM and were carried out at room
temperature. Results were analyzed by SDS-PAGE (data not shown). The experiments
revealed that a concentration of 4 M GuaHCL was sufficient for complete solubilization of
inclusion bodies. The ratio of inclusion body-slurry to buffer is 1 + 1.25 (v/v) (IB slurry
:buffer). The final conditions of the denaturing solution for solubilization of inclusion bodies
were:
1. 4 M Guanidinium-HCl,
11. 0.1 M Tris,
111. 10 mM EDT A
1v. 5 mM Cysteine
v. pH 8.0
The solubilisate is clarified by depth filtration according to standard procedures.
The protein concentration was then determined using the method of Bradford (Bradford, M.
M., Anal. Biochem. 72 (1976) 248). The protein concentration of proNGF mutein was
between 10-20 mg/ml.
Example 6. Transferring the proNGF mutant SP174-101 into a refolding buffer where
the denatured proNGF assumes a biologically active conformation
After solubilization, it is necessary to refold the protein in its native conformation and thereby
minimize misfolding and aggregation. To prepare biologically active proNGF mutein
according to the invention from solubilized materials, these were diluted into a refolding
solution wherein proNGF assumes a biologically active conformation.
The final refolding solution for the solubilizate based on IB-slurry comprised
1. 0.75 M Arginine
11. 5 mMEDTA
111. 1 mM L-Cystine and 5 mM L-Cysteine
IV. pH 9.5
The obtainment of NGF in the active conformation was confirmed by the presence of the
disulfide bridges occurring in mature human beta-NGF.
To increase protein concentration in the refolding process, a pulse renaturation was carried
out. A pulse was given every hour per 50 µg/ml proNGF mutant protein. The concentration of
Guanidinium-HCl in the solution should not exceed 0,3 M. In order to achieve this, 15pulses
were required. The clarified refolded fraction was filtered before loading to further columns.
The performance of the refolding reaction was analysed after every pulse by rp-HPLC. The
resulting peak area was blotted against the number of pulses. For the rp-HPLC, a reversed
phase column (e.g., 214MS54, 4.6 x 250 mm; 300 A, 5 µm, Vydac) with guard column (e.g.
214GK54; 300 A; Vydac) was used. The running buffers were H 0 with 0.05 %
trifluoroacetic acid (TFA) and Acetonitrile with 0.05 % TFA. The flow rate was 1 mL/min.
Results are shown in Figure 3. It can be seen from Figure 3 that the refolding efficiency of
wild type and mutant proNGF is identical.
Example 7. Purifying the proNGF mutant SP174-101 from the refolding solution via a
mixed mode material column
A column with a synthetic affinity ligand, 4-mercapto-ethyl-pyridine (MEP) was used. The
elution was done by shifting the pH-value. Further, elution was carried out with a low salt
concentration which is beneficial for an efficient process design.
The column was equilibrated with 0.75 M Arginine, 5 mM EDTA, pH 9.5. The clarified
refolding reaction was loaded onto MEP HyperCel column (Pall) with a maximal loading
capacity of 5 g proNGF mutant per L column media. In the washing step, most impurities and
unbound protein were depleted by using buffer 2 M GuaHCl, 0.1 M Tris-HCl, pH 8.0 and 10
mM Tris-HCl, pH 8.0. The elution was done in a linear gradient from 0-70 % 50 mM Acetate,
pH 4.0 (flow rate 120 cm/h). Figure 4 shows an elution profile of MEP HyperCel purification
of a refolded and filtrated proNGF mutant with the protease cleavage site mutated to VSAR
(SEQ ID NO: 5) of the invention. At the GuaHCL washing step"many impurities were
removed. At ,,pool", about 60-70% of the proNGF mutant was recovered.
Example 8. Cleaving the proNGF mutant SP174-101 to obtain active beta-NGF
For the tryptic digestion of proNGF mutant to beta-NGF, such a Phosphatebuffer was used,
which do not inhibit the activity of the protease,. Sodium phosphate buffer was added to the
MEP-eluate to a final concentration of 25 mM sodium phosphate. The pH-value was adjusted
to pH 6.5. For proteolysis, Trypsin (Roche, GMP grade) was added in a ratio of 1:10,000
(w/w) (trypsin:proNGF). The proteolysis was carried out using an incubation time of 18 h at
room temperature. Performance and yield of the tryptic digestion were analyzed by SDS
PAGE, rp-HPLC and UV/VIS280nm. Figure 5 shows an SDS-PAGE of fractions of the
tryptic cleavage. A 4-12 % Bis/Tris-Gel, 1 mm, lx MES as running buffer (Invitrogen) was
used. Lanes 5-7 show the tryptic cleavage products compared to the uncleaved proNGF
mutant (rhproNGF*, see lane 3) and to the mature beta-NGF (NGF; see lane 8). The figures
clearly show that the purified proNGF mutant results in only one cleavage product (beta
NGF). A complete digestion of proNGF mutant to beta-NGF could be observed.
Example 9. Purification of active beta NGF
After the tryptic digestion, beta-NGF was loaded onto a SP Sepharose HP column to deplete
Trypsin, by-products of the cleavage and further impurities. The SP Sepharose HP
purification is shown in Figure 6.
The column was equilibrated with 25 mM Na-phosphate buffer (pH 6.5). The tryptic digestion
reaction was loaded onto a SP Sepharose HP column (2 g beta-NGF/L medium) and unbound
protein washed with the equilibration buffer. The elution was done in three steps (3 cv 25 %
mM Na-phosphate pH 6.5 I 1 M NaCl (buffer B), 10 cv in a linear gradient from 25-50 %
buffer B, and 3 cv 100 % buffer B (flow rate 60 cm/h)).
Figure 6 shows the purification of beta-NGF. The figure shows a profile of a SP Sepharose
HP column after the tryptic cleavage. The yield of beta-NGF was 85-95 % (peak "sample
elution").
Example 10. Cleavage efficiency of Trypsin on mutant SP174-101 and wild type proNGF
This procedure was applied in parallel for both proNGF-mutant SP174-101 (SEQ ID NO: 5)
and human wild-type proNGF (SEQ ID NO: 1; rhProNGF).
mL of purified rhProNGF were dialyzed against 25 mM phosphate buffer pH 6.5. Following
dialysis, a protein concentration of 0.08 mg/mL was measured by HPLC-UV. Per digestion
sample, 80 µg of proNGF were employed. After proteolysis, all samples were analyzed by
HPLC-UV.
Mass ratio 1/10.000 w/w of trypsin/rhProNGF mutant was used, while different mass ratios of
trypsin/rhProNGF wild type were used (see Table 3). As per trypsin solution 1.0 µg/mL and
µg/mL were used. After an overnight incubation (about 17 hours) at room temperature, all
samples were analysed. For control porpoises rhProNGF mutant without added protease was
also incubated.
Table 3
Trypsin/rhProNGF Trypsin Trypsin rhProNGF rhProNGF rhProNGF
ratio Volume (µL) Amount (µg) Type Volume (µL) Amount (µg)
Control Mutant 1000 80
1/10000 8 (1 µg/mL) 0,008 Mutant 1000 80
1/10000 8 (1 µg/mL) 0,008 Wild Type 1000 80
1/5000 16 (1 µg/mL) 0,016 Wild Type 1000 80
1/1000 8 (10 µg/mL) 0,08 Wild Type 1000 80
1/250 32 (10 µg/mL) 0,32 Wild Type 1000 80
Pelformances and yields of all tryptic digestions were analysed by HPLC-UV using a Vydac
214MS C4 column.
Table 4 shows the cleavage yields obtained after tryptic digestion. The experimental data
clearly show that cleavage of the proNGF mutant SEQ ID NO: 5 with Trypsin results in only
one product (beta-NGF) at high cleavage yield (about 85%) using a very low trypsin/protein
ratio (1/10.000). This can be compared to the cleavage of the wildtype proNGF (SEQ ID NO:
1) which shows a low cleavage yield (only about 5%) at low trypsin/protein ratio (1/10.000)
and a high product degradation (overdigested) at high trypsin/protein ratio (1/250).
Table 4
Amount Trypsin/ProNGF % ProNGF betaNGF % betaNGF
µg ratio Overdigested Forms
ProNGF Standard 80 100
NGF Standard 42 100,0
SEQ ID NO:
ProNGF 5 80 1/10000 1,9 84,5
ProNGF Wild Type 80 1/10000 67,1 4,6
ProNGF Wild Type 80 1/5000 21,5 18,6 6,6
ProNGF Wild Type 80 1/1000 0,0 77,9 12,9
ProNGF Wild Type 80 1/250 0,0 67,9 25,7
Example 11. Test for the biological activity of proNGF via stimulation of the
proliferation of TFl cells
TFl cells (ATCC, catalog nr. CRL2003) were cultivated according to standard procedures. A
test medium (90% medium RPMI 1640, 10% foetal bovine serume FBS, 50 U/ml Penicillin
und 50 µg/ml Streptomycin) was added to the cells and centrifuged. The pellet was
of 1,5· 10 cells/ml in test medium at 37 °C. The cell suspension was
resuspended at a density
10 9 9
mixed with different concentrations of proNGF protein (10- M, 3.10- M, 10- M, 3.10- M,
8 8 7 7 6 6 5 5
- M, 3· 10- M, 10- M, 3.10- M, 10- M, 3· 10- M, 10- Mund 3.10- M) and analyzed in 96-
well-plates. After incubation for 48 hat 37 °C, cell proliferation reagent (e.g. WST-1, Roche
Applied Science, cat no. 1644807) was added and the plates again incubated for 4 hat 37 °C.
The absorption was measured at 450 nm and the EC -value determined by using suitable
programs (z. Bsp. Sigma-Plot 2000).
PATENT
Claims (36)
1. A proNGF mutant wherein the protease cleavage site R SK R is substituted at least at positions R and K corresponding to positions 101 and 103 of the human wildtype proNGF sequence (SEQ ID NO: 1) by any amino acid selected from a non-basic amino acid and Histidine.
2. The proNGF mutant according to claim 1, wherein the protease cleavage site IS substituted in positions 101 and 103 by any amino acid but not Arginine or Lysine.
3. The proNGF mutant according to claim 1, wherein the protease cleavage site is substituted in positions 101 and 103 by any amino acid selected from Alanine, Glycine, Valine, Serine, Threonine, Methionine, Tyrosine, Histidine, Asparagine, Aspartic Acid, Glutamine, Glutamic Acid, Phenylalanine, Isoleucine, Leucine, Tryptophan, Cysteine, and Pro line.
4. The proNGF mutant according to claim 1, wherein the protease cleavage site is substituted in positions 101 and 103 by any amino acid selected from Alanine, Valine, Glycine, Serine, Threonine, Methionine, Tyrosine, Histidine, Asparagine, Aspartic Acid, Glutamine, Glutamic Acid.
5. The proNGF mutant according to claim 1, wherein the native protease cleavage site is substituted in positions 101 and 103 by any amino acid selected from Alanine and Valine.
6. The proNGF mutant according to claims 1 to 3, wherein the protease cleavage site is substituted in position 101 by Valine.
7. The proNGF mutant according to claims 1 to 6, wherein the protease cleavage site IS substituted in position 103 by Alanine.
8. The proNGF mutant according to claims 1 to 7 wherein the amino acid at position R corresponding to position 104 of human wildtype proNGF sequence (SEQ ID NO: 1) is selected from Arginine or Lysine.
9. The proNGF mutant according to claims 1 to 8, wherein the substituted amino acid in position 102 of human wildtype proNGF sequence (SEQ ID NO: 1) is selected from amino acids Serine, Glycine, Cysteine, Asparagine, Tyrosine, Threonine, Aspartic Acid, Glutamine, Alanine, Valine, Glutamic Acid, Histidine, Isoleucine, Leucine, Phenylalanine, Praline, Tryptophane, Methionine.
10. The proNGF mutant according to claims 1 to 9, wherein the amino acid in position 101 of human wildtype proNGF sequence (SEQ ID NO: 1) is substituted by Valine, in position 102 by Serine, in position 103 by Alanine, and wherein the amino acid at position 104 is Arginine.
11. The proNGF mutant according to claims 1 to 10 having SEQ ID NO: 5.
12. The proNGF mutant according to claims 1 to 11, wherein the mutant is obtained by recombinant expression in prokaryotic cells.
13. The proNGF mutant according to claim 12, wherein the mutant 1s obtained by recombinant expression in E. coli.
14. A method of preparing a biologically active human beta-NGF comprising (i) providing a proNGF mutant according to any of claims 1 to13, and (ii) cleaving the proNGF mutant in order to obtain active human beta-NGF.
15. The method of claim 14 comprising the steps of: a. dissolving the proNGF mutant according to any of claims 1-13 by solubilisation of inclusion bodies in a denaturating solution; b. transferring the proNGF mutant into a refolding solution where the denatured proNGF assumes a biologically active conformation, c. purifying the refolded proNGF mutant, d. cleaving the pro-sequence of the proNGF mutant to obtain the active beta-NGF.
16. The method of claim 15, wherein the denaturing solution compnses a solution containing (i) a chaotropic substance, (ii) a chelator, (iii) a buffer, and (iv) a reducing agent.
17. The method of claim 16, wherein the denaturing solution comprises 1. 1 - 8 M Guanidinium-HCl, preferably 4-6 M, 11. 0.01 - 1 M Tris, 111. 1 - 50 mM EDTA, IV. 1 - 100 mM selected from Gluthione (GSH) or Cysteine, v. pH 7.0 - 10.0
18. The method of claim 17, wherein the denaturing solution comprises 1. 4 M Guanidinium-HCl, 11. 0.1 M Tris, 111. lOmMEDTA IV. 5 mM GSH or Cysteine v. pH 8.0
19. The method according to claim 15, wherein the refolding solution comprises 1. 0.5-1.0 M of a chaperone, 11. 1- 10 mM of a metal chelator, 111. 0.1 - 10 mM of a redox shuffling system, IV. pH 8.0 - pH 11.0.
20. The method according to claim 18, wherein the refolding solution comprises 1. 0. 7 5 M Arginine, 11. 5 mMEDTA iii. 1 mM L-Cystine and 5 mM L-Cysteine, or 1 mM GSSG (oxidized glutathione) and 5 mM GSH (reduced glutathione), iv. pH 9.5.
21. The method according to claims 15 and 17 - 20 wherein the refolding is carried out as a pulse renaturation.
22. The method according to claim 21 wherein in relation to the final refolding volume during pulse renaturation, the concentration of Guanidinium HCl does not exceed 0.3 Mand the protein concentration per pulse should not exceed 50 µg/ml.
23. The method according to any of claims 15, wherein the proNGF mutant is purified via mixed mode chromatography.
24. The method according to claim 23, wherein the chromatography column is a mixed mode material column with a synthetic affinity ligand.
25. The method according to claim 24, wherein a mixed mode material column is a column with a 4-mercapto-ethyl-pyridine (MEP), Hexylamino (HEA), Phenylpropylamino (PPA), 2-Mercapto-5benzamidazole sulfo acid (MBI), Capto MMC (GEHC), N-benzyl-N methyl ethanolamine (GEHC)), CHT hydroxyapatide or CHT fluoroapatide.
26. The method according to claim 25, wherein a mixed mode material column is a column with a 4-mercapto-ethyl-pyridine (MEP).
27. The method according to claims 14 to 26, wherein the pro-form of the proNGF mutant is cleaved by a protease.
28. The method according to claim 27, wherein the pro-form of the proNGF mutant is cleaved by a serine protease.
29. The method according to any of claim 28, wherein the pro-form of the proNGF mutant is cleaved by trypsin.
30. The method of claims 27 to 29, wherein the ratio of trypsin to proNGF mutant is from 1 : 200 - 1 : 100.000.
31. The method of claim 30, wherein the ratio of trypsin to proNGF mutant is from 1 5.000 - 1 : 20.000 per weight.
32. The method of claim 31, wherein the ratio of trypsin to proNGF mutant is a ratio of 1 : 10.000 (w/w).
33. The method of claims 14 to 32 further comprising an additional step of purifying beta- NGF.
34. The method of claim 33 further comprising an additional step of purifying beta-NGF by column chromatography.
35. The method of claim 34 further comprising an additional step of purifying beta-NGF by SP Sepharose HP column.
36. Use of a proNGF mutant according to any of claims 1-14 for producing human beta- NGF.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11194208 | 2011-12-19 | ||
EP11194208.2 | 2011-12-19 | ||
PCT/EP2012/076251 WO2013092776A1 (en) | 2011-12-19 | 2012-12-19 | Novel prongf mutants and uses thereof in the production of beta-ngf |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ627054A NZ627054A (en) | 2015-06-26 |
NZ627054B2 true NZ627054B2 (en) | 2015-09-29 |
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