Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2000-03-22
2003-03-11
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S069100, C435S091530, C435S220000, C435S252330, C435S320100, C530S303000
Reexamination Certificate
active
06531294
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the preparation of carboxypeptidase B and procarboxypeptidase B and to the use of procarboxypeptidase B for the preparation of carboxypeptidase B which is active in biotechnological processes.
BACKGROUND OF THE INVENTION
Carboxypeptidases are a group of zinc-containing enzymes (proteases) which cleave proteins and peptides, by means of which individual amino acids are hydrolytically removed stepwise from the carboxyl terminus of the proteins or peptides to be degraded. Carboxypeptidases therefore belong to the exopeptidases. Animal carboxypeptidases are formed in the pancreas as precursors (procarboxypeptidases) and are converted in to the active forms by trypsin in the intestine, where they are of great importance for the digestion of peptides, the primary cleavage products of the proteins. According to the substrate specificity, a differentiation is made between various carboxypeptidases.
Carboxypeptidase B releases, for example, exclusively basic amino acids, such as arginine, lysine and ornithine. Carboxypeptidases are useful for determining the sequence of peptides and proteins, because they aid identification of the amino acids on the carboxyl termini of digested polypeptides. Moreover, proteins can be made to measure by use of carboxypeptidase B.
An industrial example of the use of carboxypeptidase B is the preparation of the important pharmaceutical product insulin. The preparation of this peptide hormone is described, inter alia, in European Patent Application EP-A 0 489 780. In this application, carboxypeptidase B is used in an important step in the conversion to insulins of proinsulin structures which have been opened with trypsin.
Carboxypeptidase B, which is used in industrial processes of this type, as a rule originates from carboxypeptidase B preparations, which are commercially obtainable. These are preferably obtained from porcine pancreases (Folk, J. E.,
Meth. Enzym
.: 19, 504, 1970).
Enzymes of animal origin, however, have the disadvantage that they can be afflicted by the risk of contamination with animal viruses. The detection of freedom from viruses is complicated and enters as an essential factor into the calculation of the preparation costs. If the enzyme is used in large industrial processes, such as the industrial production of insulin, a further disadvantage lies in high logistics costs of obtaining and storing frozen pancreases.
Biotechnological preparation offers itself as an alternative to the production of carboxypeptidase B by means of the extraction of pancreas tissue. In this process, there are a number of known preparation routes, such as direct intracellular expression or expression via a fusion protein in bacteria, e.g. in the form of a &bgr;-galactosidase-carboxypeptidase B fusion protein (
J. Biol. Chem
., 267:2575-2581, 1992) or corresponding expression in yeast. A further alternative to this is the expression of a carboxypeptidase B precursor, procarboxypeptidase B, which consists of the amino acid sequence of the carboxypeptidase B plus a signal sequence which leads to the secretion of the expression product. Suitable expression systems have been described for
Bacillus subtilis
, Streptomyces,
E. coli
and the yeasts Saccharomyces, Candida,
Hansenula polymorphus
and
Pichia pastoris
. Some of these expression systems are commercially obtainable.
The prerequisite for the use of expression systems for the preparation of carboxypeptidase B is that the growth of the host strain chosen in each case is not inhibited by the presence of active carboxypeptidase B, such that expression is possible in the first place. Host strains which have this property are usually difficult to ferment, such that a relatively poor yield arises over space and time.
One problem with intracellular expression of carboxypeptidase B (direct expression or as a fusion protein) lies in the fact that the protein is initially not present in the correct spatial structure and is thus inactive. It must then be folded in vitro during the purification and processing. In this process, defective folding can occur, which has an adverse effect on the activity and specificity of the enzyme and makes use in the pharmaceutical preparation difficult.
The preparation of carboxypeptidase B by secretion of the mature, active form of the enzyme also shows disadvantages. During the fermentation, carboxypeptidase B is constantly inactivated by the reaction with peptide-like fragments of cellular constituents or constituents of the nutrient medium which are recognized as substrate, such that yield losses occur.
SUMMARY OF THE INVENTION
The invention provides a way out of the aforementioned dilemma, namely the expression and secretion of an inactive carboxypeptidase B form in spatially correct form. By reaction with an enzyme, the active carboxypeptidase B can be prepared from this precursor in vitro. The precursor mentioned can be natural procarboxypeptidase B or a derivative thereof, e.g. an isoform or a mutein or carboxypeptidase B which has been inactivated by addition of a peptide sequence. The nature of the protein sequence added is described in greater detail below.
Accordingly, one subject of the invention is a process for the preparation of pancreatic carboxypeptidase B or of an isoform or a mutein of carboxypeptidase B, where
(a) a natural or unnatural precursor form of carboxypeptidase B or an isoform or a mutein of carboxypeptidase B is expressed in a microorganism by secretion,
(b) the precursor form expressed by secretion is purified and
(c) the purified precursor form is converted into the active carboxypeptidase B or an isoform or a mutein of the carboxypeptidase B by means of an enzymatic treatment;
in particular a process of the type in which the pancreatic carboxypeptidase B or an isoform thereof is human in origin, preferably a process of the type in which the natural precursor form corresponds to procarboxypeptidase B or an isoform thereof.
A particularly preferred process is one of the type in which the unnatural precursor form has the following structure:
S—(As)
x
—E—CB, (1)
where
S is a signal peptide which brings about the secretion, from the respective microorganism, of the fusion protein formed during expression;
As is any desired genetically encodable amino acid;
E is a peptide linker consisting of an endopeptidase recognition sequence;
CB is the amino acid sequence of carboxypeptidase B or an isoform or a mutein of carboxypeptidase B; and
x is an integer from 0-100.
In addition, the invention relates to a process of the type in which a peptide sequence is attached to the natural or unnatural precursor form which makes possible the purification of the precursor form by affinity chromatography, particularly prefer ably a process of the type in which the sequence attached for the purification by affinity chromatography is 1 to 6, preferably 4, histidine residues.
The invention likewise relates to a process of the type where the yeast
Pichia pastoris
is used as a microorganism for expression.
An additional subject of the invention is a process of the type in which the enzymes trypsin, elastase, factor Xa, chymotrypsin or collagenase, preferably trypsin, are used for the enzymatic treatment.
Moreover, the invention relates to a process of the type in which step (c) proceeds under suitable reaction conditions in the presence of an insulin precursor form comprising the B, C and A chains of insulin or of an insulin derivative and in this process mature insulin or a mature insulin derivative is formed, which is isolated from the reaction mixture.
A further subject of the invention is the use of procarboxypeptidase B and carboxypeptidase B in a process of this type.
In addition, the invention relates to a nucleic acid construct for use in a process of this type, comprising a DNA sequence coding for a natural or unnatural precursor form of carboxypeptidase B or an isoform or a mutein of carboxypeptidase B, where the coding sequence mentioned is operatively linked to a promoter which makes possible the expres
Aventis Pharma Deutschland GmbH
Fronda Christian L.
Heller Ehrman White & McAuliffe LLP
Nashed Nashaat T.
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