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
1999-07-08
2001-10-16
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S069100, C435S069300, C435S069200, C435S006120, C435S007100, C435S320100, C435S173300, C435S189000, C435S195000, C530S350000, C536S023100, C536S023400, C536S024500, C514S002600, C514S012200
Reexamination Certificate
active
06303340
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and reagents useful in the production of peptides. In particular, the invention relates to the production of peptides which have pI's below about 5 or above about 8. A preferred embodiment of the invention relates to the production of b-type natriuretic peptide.
BACKGROUND OF THE INVENTION
The present invention is directed to efficient methods for expressing and recovering a peptide, particularly when the peptide one desires to produce has a high pI or a low pI, which makes it desirable to employ ion exchange chromatography as a step in the purification of the peptide. The invention will be exemplified with respect to b-type natriuretic peptide, which has a relatively high pI. Nonetheless, those skilled in the art will appreciate that the methods and reagents disclosed herein will have applicability to the production of other peptides having either a high or low pI.
It is well known in the art that the production of peptides of less than about 50 amino acids in length by expression of peptide-encoding DNA in a recombinant host cell such as
E. coli
is commonly plagued by the problem of enzymatic degradation of the expressed peptide within the host cell, resulting in partial or complete loss of the peptide. The most commonly employed means to overcome this problem is to insolubilize the peptide within the host cell. This can be effected by expressing the peptide as a fusion protein in which the peptide is linked to a fusion partner. Normally, the fusion partner will be fused to the N-terminus of the peptide. The fusion protein forms inclusion bodies within the cell, within which the peptide is protected from degradation by proteolytic enzymes.
Once the inclusion bodies are recovered from the host cell, the peptide must be separated from the leader sequence, purified and recovered in an active form. Separation from the leader sequence may be accomplished by placing a sequence of amino acids at the junction of the leader and the peptide which are specifically recognized and cleaved under appropriate conditions, e.g. acid cleavage or enzymatic cleavage. Enzymatic cleavage is frequently impractical for commercial scale production due to the enzyme's high cost and limited useful lifetime, even when employed on an immobilized column.
Acid cleavage can be accomplished by placing a specific dipeptide at the junction of the leader sequence and the peptide, wherein the first amino acid in the dipeptide is aspartic acid. Selection of the second amino acid will determine the rate at which the dipeptide bond is cleaved under acidic conditions. Of course, if the desired peptide contains any internal dipeptide sequences that are acid cleavable, then the cleavage site at the junction of the leader and the peptide must undergo acid cleavage at a substantially greater rate than the internal cleavage in order to avoid unacceptable loss of yield. The relative reaction rates of acid cleavable dipeptides are as follows:
Dipeptide
Rel. rxn. rate
Asp-Pro*
10x
Asp-X
1x
X = Gly*, Ser*, Leu,*Ile, Val
Asp-Lys*
0.5x
Asp-Arg**
0.2x
*F. Marcus, Intl. J. Peptide and Protein Res. (1985) 25: 542-546
**Our observations
Prior to enzymatic or acid cleavage, the fusion protein in the inclusion bodies is normally solubilized by treatment with a chaotropic agent which causes unfolding of the protein structure. The solubilized fusion protein is then cleaved, the desired peptide is isolated and purified, and the peptide is subjected to conditions which cause it to refold into a biologically active conformation, such as by removal of the chaotrope and oxidation, if necessary to cause formation of internal disulfide bonds.
The peptide known as b-type natriuretic peptide or BNP occurs in humans as a 32-amino acid peptide which is produced in vivo by the cleavage of a 134-amino acid precursor protein. The DNA sequence encoding the human b-type natriuretic precursor has been isolated (U.S. Pat. No. 5,114,923). B-type natriuretic peptide has been shown in human clinical trials to improve heart function without direct cardiac stimulation (which may cause harmful side effects such as arrhythmias) and to decrease levels of neurohormones associated with increased mortality and acceleration of the progression of heart failure. Accordingly, it is useful in the treatment of congestive heart failure patients.
While b-type natriuretic peptide offers certain clinical advantages over other drugs used to treat congestive heart failure patients, the relatively high cost of production of peptide drugs compared with non-peptide drugs could present an impediment to its acceptance in clinical practice. Consequently, there is a need to provide a highly efficient means of producing b-type natriuretic peptide in order to minimize its cost. Recombinant production of the peptide in the form of inclusion bodies presents several problems. The use of enzymatic cleavage of a fusion protein to yield b-type natriuretic peptide is undesirable because of the high cost of the enzymes that would be required. If one wishes to take advantage of the relatively high pI of b-type natriuretic peptide (≧10) by using ion exchange chromatography as a purification step, then the use of an ionic chaotrope such as guanidine hydrochloride is to be avoided, since the ionic chaotrope will interfere with the ion exchange chromatography. On the other hand, urea, the most commonly used non-ionic chaotrope, is problematical if one is to employ acid cleavage of the fusion protein. Under high temperature acidic conditions, the presence of urea causes the degradation of the peptide.
SUMMARY OF THE INVENTION
In the present invention, acid cleavage of the fusion protein is conducted in the absence of chaotropes, resulting in soluble b-type natriuretic peptide. The soluble peptide can be separated from the fusion partner and other contaminants, which remain insoluble, using any convenient method known for separating soluble proteins from insoluble proteins, e.g. by ultrafiltration, diafiltration or centrifugation. Since ultrafiltration and centrifugation are suboptimal steps from a commercial point of view, a preferred embodiment of the invention employs solubilization of the insoluble materials, following cleavage, by treatment with a non-ionic chaotrope such as urea, and isolation of b-type natriuretic peptide by ion-exchange chromatography.
The present invention provides an efficient method for producing a peptide having a pI above about 8 or below about 5. A preferred embodiment of the method of the invention comprises the steps of:
(a) expressing said peptide, in a recombinant host cell as a fusion protein in which the desired peptide is fused at its N-terminus to a fusion partner comprising an amino acid sequence having an Asp residue at its C-terminus, wherein (i) the C-terminal Asp residue of the fusion partner and the N-terminal residue of the peptide form a bond which is cleavable under acidic conditions, (ii) the peptide has a sufficiently different net charge from that of the fusion partner and any undesired fragments produced by acid cleavage of the fusion protein that the peptide is capable of being separated from the fusion partner and any undesired acid cleavage fragments of the fusion protein by ion-exchange chromatography and (iii) the fusion protein forms inclusion bodies within the recombinant host cell;
(b) recovering the inclusion bodies from the recombinant host cell;
(c) cleaving the peptide from the fusion partner by subjecting the fusion protein to acidic conditions in the absence of a chaotrope;
(d) solubilizing the insoluble cleavage products by treating them with a non-ionic chaotrope under conditions which cause them to become solubilized without degrading the primary structure of the peptide; and
(e) isolating the peptide by ion-exchange chromatography.
There is also provided herein an expression vector which is useful for expressing a fusion protein that is cleavable under acid conditions to yield a desired peptide having a pI above 8 or below 5, s
Buckley Douglas I.
Hartman Taymar E.
Pollitt N. Stephen
Stathis Peter A.
Zhong Ziyang
Carlson Karen Cochrane
Knobbe Martens Olson & Bear LLP
Robinson Hope A.
Scios Inc.
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