Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-05-15
2001-08-14
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S212000, C435S219000, C435S252300, C435S254110, C435S325000, C435S320100, C536S023100, C536S023200
Reexamination Certificate
active
06274366
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to a method of making enzymatically-active human &bgr;-tryptase in a genetically-engineered microbial host, expression constructs encoding human &bgr;-tryptase, and genetically-engineered eukaryotes which express enzymatically-active recombinant human &bgr;-tryptase.
BIBLIOGRAPHY
Complete bibliographic citations to the references noted herein are included in the Bibliography section, immediately preceding the Sequence Listing.
DESCRIPTION OF THE PRIOR ART
Human mast cell &bgr;-tryptase is a neutral serine protease of presently unknown biological function in vivo. However, it has been implicated in angiogenesis and tissue remodeling. It constitutes up to 20% w/v of the total granule protein of mast cells. &bgr;-tryptase is selectively stored in mast cell granules and is released upon mast cell degranulation. Because &bgr;-tryptase is unique to mast cells, it has gained favor as a specific marker of mast cell-mediated pathology. For a complete discussion regarding mast cell heterogeneity, structure, and mediators, see Nilsson and Schwartz (1994).
Purified native human &bgr;-tryptase is a tetrameric endoprotease of approximately 134 kDa. Each of the four subunits is approximately 31 to 34 kDa in size. As noted in Schwartz (1995), human tryptase was first purified to apparent homogeneity from dispersed and enriched lung mast cells in 1981. However, further research has shown that there are at least two different types, or groups, of human tryptase. These tryptase isoforms are designated herein as &agr;-tryptase and &bgr;-tryptase. For purpose of brevity, the unadorned term “tryptase” as used hereinbelow shall refer solely to the &bgr; isoform.
Human tryptase is isolated conventionally from cadaveric lung tissue, as described by Smith et al. (1984).
A number of researchers have reported cloning cDNAs which encode human tryptase, see Miller et al. (1990), Vanderslice et al. (1990), and Blom and Hellman (1993), as well as rat mast cell tryptase, see Ide et al. (1995).
However, previous attempts at cloning human tryptase using either bacterial or baculovirus expression systems are plagued with a myriad of problems, including protein folding problems that result in a lack of enzymatic activity. These failures are due, at least in part, to the fact that the tryptase enzyme is extensively modified post-translationally to yield the active form of the enzyme. Consequently, the specific activity of a recombinant tryptase produced in a prokaryote would be expected to be low due to the lack of post-translational glycosylation. As a further consequence, previous attempts to produce enzymatically-active recombinant human tryptase have proven to be far less than ideal because the methods require post-expression chemical modifications to activate the enzyme precursor.
For instance, Sakai et al. (1996) report the expression and purification of recombinant human &agr;-tryptase and &bgr;-tryptase precursors in a baculovirus system. However, the tryptase precursors formed are inactive.
Regarding the use of methylotrophic yeasts (e.g.,
Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis
and
Yarrowia lipolytica
) as hosts for the production of heterologous proteins, the characteristics of these organisms and their suitability for such use has been extensively reviewed in the relevant literature. See, for example, Faber et al. (1995) and Buckholz and Gleeson (1991).
SUMMARY OF THE INVENTION
According to the present invention, DNA encoding human tryptase is cloned, incorporated into a eukaryotic expression vector, and transformed into a suitable eukaryotic host cell. Successfully transformed host cells express, post-translationally process, and secrete enzymatically-active human tryptase. The tryptase so formed has an N-terminal amino acid sequence that is identical to that reported by Vanderslice et al. (1990). Further still, the tryptase produced according to the inventive method has tryptic activity which is comparable to tryptase isolated from cadavers.
In particular, the invention is directed to a DNA expression construct comprising, in 5′ to 3′ order: a promoter, the promoter operationally linked to a signal sequence, and the signal sequence operationally-linked to a DNA sequence encoding human &bgr;-tryptase.
In the preferred embodiment, the DNA expression construct comprises, in 5′ to 3′ order: a promoter selected from the group consisting of GAP, AOX1, MOX, FMD, ADH, LAC4, XPR2, LEU2, GAM1, PGK1, GAL7, GADPH, CYC1, and CUP1, the promoter operationally linked to a signal sequence, the signal sequence operationally-linked to a DNA sequence encoding human &bgr;-tryptase, and the DNA sequence operationally linked to a terminator sequence. Suitable hosts transformed to contain these expression constructs express and secrete enzymatically-active human tryptase.
The invention also is directed to a method of producing enzymatically-active human &bgr;-tryptase. The method comprises transforming a yeast host cell with an expression construct as described herein, whereby the host cell expresses enzymatically-active (mature) human &bgr;-tryptase. The invention also is directed to the glycosylated tryptase produced and secreted by the transformed host.
A third embodiment of the invention is directed to a genetically-engineered yeast cell which expresses enzymatically-active human &bgr;-tryptase. In the preferred embodiment, the genetically engineered yeast cell comprises a
Pichia pastoris
host cell transformed to contain and express an expression construct as described herein.
The invention entails operationally linking DNA sequences that encode the mature form of tryptase immediately downstream (i.e., in the 3′ direction) and in-frame to a secretion signal sequence to yield an expression construct. The expression construct, the preferred embodiment of which is a plasmid designated pPIC9-HumTry, then is transformed into a suitable host, preferably a strain of
Pichia pastoris.
Hosts so transformed express and secrete the tryptase encoded by the expression construct. The tryptase expressed is correctly processed by the host cell and secreted into the cell medium as enzymatically-active human tryptase.
Another embodiment of the invention is directed to the enzymatically-active, glycosylated, recombinant protein having tryptase activity which is produced by organisms transformed to contain and express the expression construct.
The invention is further drawn to a method of generating polyclonal or monoclonal anti-human tryptase antibodies comprising inoculating an animal with the enzymatically-active tryptase produced by organisms transformed to contain and express the expression construct. The invention is also drawn to polyclonal or monoclonal anti-human tryptase antibodies produced thereby.
Another embodiment of the invention is a method of screening a substance, such as a chemical compound or mixture of compounds, for its effect on the enzymatic activity of tryptase enzymes. Here, the method comprises contacting the substance with an enzymatically-active recombinant tryptase produced by an organism transformed to contain and express the construct described herein and then measuring the enzymatic activity of the recombinant tryptase.
The invention takes advantage of the fact that native tryptase is synthesized as a proprotein. The modified tryptase amplicon described herein lacks the sequences that encode the N-terminal amino acid prosequence. By cloning this sequence as an in-frame fusion to an N-terminal yeast secretion signal sequence, there is no need to subject the secreted tryptase protein to an activation process. It is secreted as an active enzyme without any further exogenous manipulation required.
To accomplish this result, the signal peptide cleavage site is positioned immediately adjacent to the N-terminus of mature tryptase protein. Cleavage of the signal peptide by action of a host cell protease then removes the signal peptide from the tryptase as
Haak-Frendscho Mary
Maffitt Mark A.
Niles Andrew L.
DeWitt Ross & Stevens S.C.
Hutson Richard
Leone, Esq. Joseph T.
Promega Corporation
Prouty Rebecca E.
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