Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-02-07
2003-05-06
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S069700, C435S252300, C435S320100, C435S325000, C530S303000, C530S350000, C536S023100, C536S023500
Reexamination Certificate
active
06558924
ABSTRACT:
RELATED APPLICATION
This application claims priority to British Application No. 9716790.2, filed Aug. 7, 1997, the content of which is incorporated herein by reference.
The present invention relates to the production of insulin C-peptide from recombinant DNA molecules comprising multimeric copies of a gene sequence encoding said insulin C-peptide.
Insulin is a protein hormone involved in the regulation of blood sugar levels. Insulin is produced in the liver as: its precursor proinsulin, consisting of the B and A chains of insulin linked together via a connecting C-peptide (hereinafter this C-peptide derived from the proinsulin molecule is referred to as “insulin C-peptide”). Insulin itself is comprised of only the B and A chains. Several recent studies indicate that the C-peptide has a clinical relevance (Johansson et al., Diabetologia (1992) 35, 121-128 and J. Clin. Endocrinol. Metab. (1993) 77, 976-981). In patients with type 1 diabetes, who lack endogenous C-peptide, administration of the peptide improves renal function, stimulates barrier function (Johansson et al., 1992 and 1993 supra).
Although not yet widely recognised, there is a growing awareness in the medical field,of a therapeutic utility for the insulin C-peptide. Accordingly, there is a need for a method for the ready synthesis of insulin C-peptides, economically and efficiently. Whilst methods for the chemical synthesis of peptides, e.g. by stepwise addition of amino acids on a solid support, are now well developed, they remain, despite automation, time-consuming and, more significantly, costly to perform, and may also be limited in terms of the maximum peptide length economically and reliably synthesisable. As an alternative, methods for peptide production by expression of recombinant DNA have been developed, although these too are not without their drawbacks e.g. in terms of yield.
Current production schemes for insulin C-peptide are based on the processing of proinsulin
1
the precursor molecule for insulin and C-peptide, normally by the use of trypsin and carboxypeptidase B (Nilsson et al., (1996), J. Biotechnol. 48, 241-250); Jonasson et al., (1996) Eur. J. Biochem. 236, 656-661). Proinsulin was produced as a fusion protein that was capable of expression at high levels in
E. coli
, and the fusion protein was engineered in such a way that the fusion partner could be cleaved off simultaneously with the processing of proinsulin to insulin and C-peptide. Proinsulin was produced as a fusion protein with ZZ, a synthetic affinity fusion tag derived from staphylococcal protein A which binds IgG (Immuno-globulin) (Nilsson et al., (1987) Prot. Eng. 1, 107-113). This fusion tag was selected due to its stability to proteolysis, its IgG-binding capacity, its high expression levels and solubilizing properties. The chosen production strategy allowed the use of an affinity tag for efficient purification, after solubilization of inclusion bodies and subsequent renaturation, without the inclusion of additional unit operations for cleavage and removal of the ZZ affinity tag. The tag was demonstrated to be simultaneously cleaved off with the trypsin/carboxypeptidase B digestion of proinsulin to insulin and C-peptide. However, production of small peptides via the expression of large fusion proteins generally gives rather low yields, as the final product constitutes only a small part of the expressed gene product.
Shen in Proc. Natl. Acad. Sci. USA, 81, 4627-4631, 1984 describes a method for preparing human proinsulin by expression of a fused or unfused gene product comprising multiple tandemly linked copies of the proinsulin polypeptide domain. This gene product can be cleaved into single proinsulin units by cyanogen bromide treatment. It is proposed that human insulin can be prepared by cleavage of the proinsulin units with trypsin/carboxypeptidase. However, the problem of improving the yield of insulin C-peptide is not addressed.
There remains, therefore, a need for a recombinant expression method which improves the yield of insulin C-peptide, as an unfused product. The present invention addresses this need.
The present invention seeks to improve on existing methods for recombinant expression of peptides and essentially is based on the concept of increasing the amount of expressed target peptide (in this case an Insulin C-peptide) by expressing, as a single gene product, a multimer (i.e. a multimeric polypeptide) having multiple copies of the target peptide (insulin C-peptide), and then cleaving such a multimeric gene product (i.e. the multimeric polypeptide) to release the target peptide as individual monomer units.
In one aspect, the present invention thus provides a method of producing an insulin C-peptide, which comprises expressing in a host cell a multimeric polypeptide comprising multiple copies of a said insulin C-peptide, and cleaving said expressed polypeptide to release single copies of the insulin C,-peptide (i.e. to release the insulin C-peptide monomers from the multimer).
The multimeric polypeptide (gene product) is encoded by a genetic construct (in other words a nucleic acid molecule) comprising multiple copies of a nucleotide sequence encoding an insulin C-peptide. The multiple copies, or repeats, are linked in the construct in such a manner that they are transcribed and translated together into a single, multimeric gene product (i.e. a multimeric polypeptide) i.e. in “read-through format” e.g. the multiple nucleotide sequences are linked in matching reading frame in the construct. In essence, the genetic construct (nucleic acid molecule) advantageously comprises a concatemer of the insulin C-peptide encoding nucleotide sequence. Preferably, the genetic construct comprises tandem copies of the encoding nucleotide sequence. Such a genetic construct is thus prepared and is then introduced into a host cell in a standard manner, and expressed. The expressed gene product (polypeptide) may then be recovered and cleaved to release the insulin C-peptide monomers.
In a further aspect the invention thus provides a method for producing an insulin C-peptide, which comprises culturing a host cell containing a nucleic acid molecule comprising multiple copies of a nucleotide sequence encoding a said insulin C-peptide, under conditions whereby the multimeric polypeptide of said nucleic acid molecule is expressed, and cleaving said expressed polypeptide to release single copies of said insulin C-peptide.
As used herein the term “multiple” or “multimeric” refers to two or more copies of an insulin C-peptide or the nucleotide sequence which encodes it, preferably 2 to 50, 2 to 30 or 2 to 20, more preferably 2 to 15, or 2 to 10. Further exemplary ranges also include 3 to 20, 3 to 15 or 3 to 10.
Conveniently, the construct comprises 3 or more copies e.g. 3 to 7, or 5 to 7, copies of the nucleotide sequence encoding a insulin C-peptide. Ranges of 7 or more, for example 7 to 30, 7 to 20 or 7 to 15 may also be useful.
The term “insulin C-peptide” as used herein includes all forms of insulin C-peptide, including native or synthetic peptides. Such insulin C-peptides may be human peptides, or may be from other animal species and genera, preferably mammals. Thus variants and modifications of native insulin C-peptide are included as long as they retain insulin C-peptide activity. The insulin C-peptides may be expressed in their native form, i.e. as different allelic variants as they appear in nature in different species or due to geographical variation etc., or as functionally equivalent variants or derivatives thereof, which may differ in their amino acid sequence, for example by truncation (e.g. from the N- or C-terminus or both) or other amino acid deletions, additions or substitutions. It is known in the art to modify the sequences of proteins or peptides, whilst retaining their useful activity and this may be achieved using techniques which are standard in the art and widely described in the literature e.g. random or site-directed mutagenesis, cleavage and ligation of nucleic acids etc. Thus, functionally equivalent variants or d
Jonasson Per
Nygren Per-Ake
Stahl Stefan
Uhlen Mathias
Creative Peptides Sweden AB
DeBerry Regina M.
Fish & Richardson P.C.
Kemmerer Elizabeth
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