Deformylation of f-met peptides in bacterial expression system

Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor

Reexamination Certificate

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C530S399000

Reexamination Certificate

active

06190902

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to materials and methods useful in production of recombinant proteins in transformed bacteria. In one important embodiment, the present invention is directed to materials and methods useful in diminishing the retention of N-formyl groups on the N-terminal methionine residues of recombinant proteins produced at high levels in transformed bacterial host cells. In another embodiment, novel methods for increasing the levels of peptide deformylase in transformed bacterial host cells are disclosed.
BACKGROUND OF THE INVENTION
Methionine is the universal starting amino acid residue for growing peptide chains (e.g., protein synthesis) in most living systems. In order for methionine to function as the initiating amino acid for nascent peptide synthesis in bacteria, it must undergo a series of transformations both before initiation of protein synthesis and after the protein has been made (see review by Meinnel, T., Mechulam, Y., and Blanquet, S., 1993). In
E. Coli
, all of the enzymes involved in these transformations and their respective genes have been isolated and/or sequenced (Meinnel, T. and Blanquet, S., 1994).
Two of the most notable transformations in the initiating amino acid methionine amino acid involve the addition of an N-formyl group to methionine molecules prior to the initiation of messenger RNA (mRNA) translation and the subsequent removal of the N-formyl group from the amino (N-) terminal methionine of the nascent peptide. The removal of the N-formyl group is accomplished by the enzyme peptide deformylase (EC 3.5.1.27 according to the IUB nomenclature as published in “
Enzyme Nomenclature Recommendations
” (1992) Academic Press, San Diego). Peptide deformylase (PDF) cleaves the formyl group from most nascent formyl-methionine-peptides in a substrate specific reaction. However, there are exceptions to the routine action of PDF. For example, some
E. coli
proteins remain either wholly or partially formylated. (Hauschild-Rogat, P., 1968; Marasco, W. A., et al., 1984; and Milligan, D. L. and Koshland, Jr., D. E., 1990). It has also been observed that several recombinant proteins which are normally free of N-formyl-methionine (f-Met) exhibit a significant retention of f-Met when they are overproduced in recombinant
E. coli
strains. Examples of this phenomenon include
E. coli
tryptophan synthase alpha- and beta-subunits (Sugino, Y., et al., 1980; Tsunasawa, S., et al., 1983); bovine somatotropin (BST) (Bogosian, G., et al., 1989); eel growth hormone (Sugimoto, Y., et al., 1990);
E. coli
1-acyl-sn-glycerol-3-phosphate acyltransferase (Coleman, J., 1992); human granulocyte colony-stimulating factor (Clogston, C. L., et al., 1992); bovine fatty acid-binding protein (Specht, B., et al., 1994); bovine cytochrome P450 (Dong, M. S., et al. 1995); methanothermus fervidus histone A (Sandman, K., et al., 1995); human interleukin-5 (Rose, K., et al., 1992); human parathyroid hormone (Rabbani, S., et al., 1988; Hogset, A., et al., 1990); human gamma-interferon (Honda, S., et al., 1989);
E. coli
threonine deaminase (Eisenstein, 1991); and
E. coli
To1Q membrane protein (Vianney, A., et al., 1994).
Retention of the formyl group on a protein expressed in and purified from bacterial expression systems is undesireable when preparing recombinant pharmaceuticals. As a result, complex and expensive purification procedures are required to purify the deformylated protein of interest to a degree sufficient to qualify it for pharmaceutical use. In addition, costly analytical methods to quantify the formylated isoform must frequently be devised in order to insure that the level of such isoform in the final product is below a desired level. Therefore, there exists a need to achieve the efficient removal of undesirably retained N-formyl groups on recombinant proteins without interfering with the level of recombinant protein production in bacterial expression systems.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of methods and materials useful in removing retained N-formyl groups from nascent recombinant N-formyl methionine peptides produced in transformed bacterial hosts (e.g.,
E. coli
). In a general and overall sense, the invention provides methods which are capable of reducing the occurrence of retained N-formyl groups on recombinant, bacterially-expressed recombinant peptides or proteins (collectively referred to as “recombinant proteins”), without substantially decreasing the level of recombinant protein production, by causing the bacterial host cells to increase expression and/or activity of PDF, for example by genetic or epigenetic manipulations. Preferably, this result is achieved by transforming an expressible PDF gene into suitable bacterial host cells so that the level of PDF is increased in the bacterial host cells. Also included within the invention are the host cells so transformed and the substantially deformylated recombinant protein so produced.
In one preferred embodiment, the present invention provides a method for production, in transformed bacterial host cells, of recombinant protein having a diminished retention of N-formyl methionine. This method includes the step of transforming bacterial host cells with DNA comprising a first expressible DNA sequence encoding a peptide deformylase enzyme also operably linked to a promoter operable in the bacterial host cells and a second expressible DNA sequence encoding a recombinant protein also operably linked to a promoter operable in the bacterial host cells. The expressible DNA sequences may be present on a single DNA segment or, alternatively, on different segmentswhich may be co-transformed into the bacterial host cells. One may then identify transformed bacterial host cells having both the first and second expressible sequences and culture the transformed bacterial host cells under conditions which cause coexpression of the recombinant protein and the PDF enzyme, resulting in substantial deformylation of the coexpressed recombinant protein.
In a more preferred embodiment, each expressible DNA sequence is on a DNA molecule containing a marker gene so that transformed bacterial host cells may be identified by selecting bacteria having a marker trait conferred by the marker gene.
In another preferred embodiment, the recombinant protein produced in the transformed host cell has both a diminished retention of N-formyl methionine and a level of recombinant protein production which is substantially equivalent (i.e., at least about 80%) of that of otherwise identical host cells transformed only with a DNA molecule comprising the gene encoding the recombinant protein.
In yet another embodiment, the present invention provides a novel method for increasing the level of PDF in transformed bacterial host cells which includes transforming the host cells with an expressible DNA sequence encoding a peptide deformylase enzyme operably linked to a promoter operable in the host cells. In a preferred embodiment, the DNA used for transformation will also contain a marker gene. The transformed host cells may then be identified by virtue of having a marker trait conferred by the marker gene. The transformed host may be used as a universal expression system for expression of proteins for which retention of N-formyl methionine is desired to be decreased.
Additional embodiments relate to cells coexpressing genes encoding PDF and the recombinant protein of interest and to vectors encoding PDF and the recombinant protein of interest.


REFERENCES:
patent: 5834243 (1998-11-01), Bogosian
Warren et al.,Gene, vol. 174, 1996, pp. 235-238.
Bogosian et al.,J. Biol. Chem., vol. 264, 1989, pp. 531-539.

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