Preventing endogenous aminopeptidase mediated n-terminal amino a

Details Preventing endogenous aminopeptidase mediated n-terminal amino a Preventing endogenous aminopeptidase mediated n-terminal amino a

1995-05-26

2000-12-26

Nashed, Nashaat T.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 695, 435 697, 530300, 530350, C12P 2106

Patent

active

061657468

DESCRIPTION:

BRIEF SUMMARY
This invention relates to the recombinant production of mammalian proteins and peptides in prokaryotic host cells.
When a protein of mammalian origin is expressed in a bacterial host, for example E. coli, it is normally expressed as a precursor protein which carries a methionine residue (Met) at its N-terminus. This Met residue must be removed to obtain the protein in its natural, mature form. This can be done by use of certain aminopeptidase enzymes, for example aminopeptidase-1 (AP-1) which is obtained from Aeromonas, which are capable of sequentially cleaving N-terminal amino acids from proteins. This cleavage process is halted by certain amino acids or combinations of amino acids which act as "stop signals".
Stop signals for AP-1 include aspartic acid (Asp), glutamic acid (Glu), and the combination X-Pro where X is any amino acid other than proline. Consequently, as described in International Patent publication WO86/01229, this enzyme may be used to cleave specifically the N-terminal Met from a protein, expressed in a bacterial host, that has the N-terminal sequence:
However bacterial hosts such as E. coli contain endogenous peptidase enzymes which can cleave the N-terminal residues of certain proteins produced by the bacterium in a non-specific manner. This gives rise to a mixture of products which is often very undesirable. For example, when a protein having Pro in the 2-position is expressed in E. coli, the nascent Met-X-Pro- protein is processed by the N-terminal methionine amino peptidase (MAP) enzyme of E. coli. This enzyme does not recognize X-Pro as a stop signal and not only the Met residue but also the X may be removed.
One example of such a protein is IL-6. IL-6 is a secretory lymphokine which in mammalian cells is generated by the synthesis and processing of a precursor protein having a signal sequence, at the N-terminus, of 28 amino acids. Removal of this signal sequence gives the mature protein of 185 amino acids that has the N-terminal sequence Ala-Pro-Val-. When the recombinant protein is produced in E. coli, a protein coding sequence is used which does not contain codons coding for the signal peptide, but has a start codon preceding the codons coding for the mature protein. On expression, this start codon generates a Met residue so that the initial translational product has the N-terminal sequence:
However partial processing of this product in vivo by the MAP enzyme of E. coli results in a heterogeneous mixture of IL-6 species with different N-termini, as shown below for certain fermentation conditions:
Thus only 16% of the protein product obtained is the desired, mature protein. Further, treatment of this mixture with AP-1 to cleave the Met from the first of these products causes further cleavage of the third product to Val.sup.3 -Pro.sup.4 - - - IL-6, which further increases the heterogeneity.
Another example of such a protein is LIF (Leukaemia Inhibitory Factor) which is described in EP 0 285 448. When this protein is produced in E. coli, the initial translational product has the N-terminal sequence:
Partial processing of this product by the MAP enzyme of E. coli results in the following heterogeneous mixture of LIF species for certain fermentation conditions:
Hence only 12% of the proteins obtained are the desired, mature protein. Again treatment of the mixture with AP-1 to cleave the Met from the first of these products would cause further cleavage of the third product.
Similar problems occur in the recombinant production in bacterial hosts of many other useful proteins.
Thus it is an object of this invention to provide a process for the production of proteins and peptides in prokaryotic host cells in which processing of the product by endogenous MAP enzymes is reduced.
German patent publication DE 40 39 415 discloses a method of specifically cleaving the N-terminal Met from proteins by causing the bacterial host to express additional amino acids at the N-terminus. An IgA-protease is then used to cleave the Met-Y to leave the protein X-Pro-A. However this German patent publication

REFERENCES:
Rall, et al., J. Biol. Chem., 257, Table VII, 1982.
Biological Abstracts, vol. 93, No. 3, Feb. 1, 1992, abstract No. 27458.

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