Preparation of factor XIIIa by gene manipulation

Details Preparation of factor XIIIa by gene manipulation Preparation of factor XIIIa by gene manipulation

1995-06-02

2004-06-08

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C435S252300, C536S023100, C530S384000

Reexamination Certificate

active

06746866

ABSTRACT:

Coagulation factor XIII is the final member of the “coagulation cascade” in the natural process of blood coagulation in vertebrates. The enzymatically active form of factor XIII, factor XIIIa, also called “activated fibrin-stabilizing factor”, “fibrinoligase” or “plasma transglutaminase” and, hereinafter, “F XIIIa”, catalyzes the fusion of fibrin units in preexistent thrombi by intramolecular crosslinking (Lorand et al., Methods in Enzymology 80 (1981), 333-341; Curtis et al., Annals New York Academy of Sciences 1983, 567-576). The molecular weight of factor XIII from plasma is about 300 kD (Loewy et al., J. Biol. Chem. 236 (1961) 2634). The molecular weight of the active subunit F XIIIa is about 80 kD (Bohn and Schwick, Arzneimittelforschung 21 (1971) 1432). During the activation of factor XIII, thrombin splits off from the precursor a peptide which is about 4 kD in size and has a known sequence of 36 amino acids (Takagi and Doolittle, Biochemistry 13 (1974) 750-756). In addition, a sequence embracing four amino acids is known (Holbrook et al., Biochem. J. 135 (1973) 901-903).
The invention relates to a process for the preparation of F XIIIa by gene manipulation, to the mRNA necessary for this, to the cDNA obtained therefrom, to DNA structures and vectors containing all or part of this cDNA, to cells transformed with DNA of this type, and to the polypeptide expressed by these cells. The invention also relates to part-sequences of the amino acid sequence of F XIIIa, to specific antibodies obtained therewith, to diagnostic aids and antibody columns produced from these antibodies, and to a polypeptide obtained with the aid of such columns. Another aspect of the invention relates to diagnostic aids which contain all or part of the DNA or RNA coding for F XIIIa, and to diagnostic methods with which body fluids and tissues are examined using diagnostic aids of this type. Further aspects of the invention and its preferred embodiments are illustrated in detail hereinafter and defined in the patent claims.
The drawings, in which the numbers coincide with those in the examples, illustrate the invention:
FIG. 1
shows the cDNA coding for F XIIIa (the coding region being shaded) and, below this, the DNA regions of the isolated and characterized clones.
FIG. 2
shows the construction of the expression plasmid pFXIII-13. For clarity, in this figure the starting plasmids pIC19H-12.1 and pIC19H-11.1, as well as the DNA fragments located immediately below them, are represented by double lines, as is the product pFXIII-13 constructed from the single-stranded fragments.
FIG. 3
is a diagram of the construction of the plasmid pTrc97A,
FIG. 3
a
that of pFXIII-C4 from pTrc97A and pFXIII-13, and finally
FIG. 3
b
the construction of pMB259 from pFXIII-13 and the known plasmids pIC20H and pBD2.
FIG. 4
is a diagram of the construction of the plasmid pMB240 from pFXIII-13 and the known plasmid pAAH5.
FIG. 5
shows the construction of pZET4 from the known plasmid pSV2dhfr and the plasmid pSVA STOP1,
FIG. 5
a
shows the construction of pSVF13 from pSVA STOP1 and pFXIII-13,
FIG. 5
b
shows the construction of pZF13 from pZET4 and pFXIII-13, and finally
FIG. 5
c
shows the construction of pHSF13 from pSVF13 and the known plasmid pSP6HS9.
FIGS. 6-1
to
6
-
9
show the nucleotide sequence encoding factor XIIIa, and the amino acid sequence depecting factor XIIIa.
The amino acid sequence of F XIIIa fragments was determined for the construction of suitable probes. The corresponding peptide fragments were obtained by proteolysis or cleavage with cyanogen bromide. Based on knowledge of the amino acid sequences of such fragments, two oligonucleotide probes were synthesized, one 20 mer and one 66 mer.
In the 20 mer probe all theoretically possible codons for the amino acid sequence
Met-Met-Asp-Ile-Thr-Asp-Thr
were taken into account, with, in the case of the last amino acid, the third position in the codon being omitted. The 20 mer probe is thus 48-fold degenerate, i.e. a mixture of all 48 theoretically possible oligonucleotides coding for the said amino acid sequence (Table 1; Appendix).
The 66 mer probe was selected on the basis of the following amino acid sequence
Tyr-Gly-Gln-Phe-Glu-Asp-Gly-Ile-leu-Asp-Thr-Cys-Leu-Tyr-Val-Met-Asp-Arg-Ala-Gln-Met-Asp
and with the assistance of statistical data (Lathe, J. Molec. Biol. 183 (1985) 1-12) (Table 2, Appendix).
These probes were used to screen a cDNA bank. The cDNA was prepared from mRNA from a mature human placenta, the mRNA being isolated from the latter, and the cDNA being prepared therefrom. The cDNA was provided with EcoRI ends and ligated into the EcoRI cleavage site of the phage vector &lgr;gt10. A positive clone, &lgr;gt10-12, which was identified with the abovementioned probe, was further analyzed (FIG.
1
). The sequencing, by methods known per se, resulted in the DNA sequence which codes for F XIIIa.
Rescreening of the cDNA bank with this DNA sequence resulted in isolation of further clones which expand both towards the 5′- and towards the 3′-end.
FIG. 1
shows the restriction map of the DNA sequence which codes for F XIIIa. “N” designates the N-terminal end and “C” designates the C-terminal end of the coding region, and “A(
89
)” designates the poly(A) sequence of 89 bases. This sequence represents the whole of the coding sequence of F XIIIa. Table 3 (Appendix) shows the DNA sequence found (coding strand) and, deduced therefrom, the amino acid sequence from the cloned cDNA fragments from &lgr;gt10-11 and &lgr;gt10-12. The total length of the cDNA is 3905 base-pairs. The N-terminal sequence embracing 36 amino acids found by Takagi and Doolittle (loc. cit.) is present in the sequence which was found. This sequence is indicated in Table 3 with an unbroken line between nucleotide positions 88 and 198. In addition to the sequence found by Takagi and Doolittle, the cDNA codes for a valine in nucleotide positions 187-189. The sequence embracing four amino acids found by Holbrook et al. (loc. cit.)—Gly-Gln-Cys-Trp—is coded for by the cDNA in positions 1021-1032. This sequence is likewise indicated by an unbroken line. In addition, the positions of the 20 mer and 66 mer oligonucleotide probes are indicated by broken lines. The 20 mer probe hybridizes between positions 1507 and 1526, and the 66 mer probe hybridizes between positions 766 and 831.
It is possible according to the invention to use the coding cDNA for the preparation of modified genes which code for proteins having altered biological properties. It is possible for this purpose to undertake, in a manner known per se, deletions, insertions and base-exchanges.
It is also possible, by the choice of the host, to influence the nature of the modification to the F XIIIa. Thus, there is no glycosylation in bacteria, while that taking place in yeast cells differs from that in higher eukaryotic cells.
Knowing the amino acid sequence of F XIIIa, it is possible to prepare, by conventional methods or gene manipulation, part-sequences of amino acids which can act as antigens for the preparation of polyclonal or monoclonal antibodies. Such antibodies can be used not only for diagnostic purposes but also for the preparation of antibody columns with which it is possible to remove F XIIIa from solutions which contain this factor in addition to other proteins.
It is also possible, by use of the cDNA or parts thereof, straightforwardly to isolate from a genomic bank the genomic clone which codes for F XIIIa and using which it is possible not only to express it in eukaryotic cells but also to gain further diagnostic information.
F XIIIa deficiencies can result in various syndromes which, to a large extent, are attributed to the inability to convert the precursors into the active form of the enzyme. Knowledge of the cDNA of F XIIIa now permits the preparation of diagnostic aids with which it is possible straightforwardly to establish whether genetic modifications are present.
Thus, it is possible according to the invention to prepare a highly pure factor XIIIa without any risk of contamination by, for exam

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