Highly active alkaline phosphatase

Details Highly active alkaline phosphatase Highly active alkaline phosphatase

1999-05-05

2002-06-18

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S252300, C435S320100, C536S023200, C530S350000

Reexamination Certificate

active

06406899

ABSTRACT:

The invention concerns a DNA coding a eukaryotic highly active alkaline phosphatase with a specific activity of more than 3000 U/mg. Furthermore the invention concerns a process for the production of the DNA according to the invention as well as a vector containing the DNA according to the invention as well as a cell line containing this vector. The invention additionally concerns a recombinant highly active alkaline phosphatase with a specific activity of more than 3000 U/mg which is coded by the DNA according to the invention.
Alkaline phosphatases (AP) are dimeric, zinc-containing, non-specific phosphomonoesterases which are found in all organisms from
E. coli
to mammals (McComb et al., 1979). Comparison of the primary structure of different alkaline phosphatases showed a high degree of homology (25-30% homology between
E. coli
and mammalian AP) (Millán, 1988; Harris, 1989).
In humans and higher animals the AP family consists of four members which are coded on different gene loci (Millán, 1988; Harris, 1989). The alkaline phosphatase family includes the tissue-specific APs (placental AP (PLAP), germ cell AP (GCAP) and intestinal AP (IAP)) and the non-tissue-specific APs (TNAP) which are mainly located in the liver, kidney and bones.
A decisive property of the previously known APs is the large variability of the catalytic activity of the mammalian APs which have a 10-100-fold higher specific activity than
E. coli
AP. Among the mammalian APs the AP from the bovine intestine (bIAP) exhibits the highest specific activity. This property makes the bIAP attractive for biotechnological applications such as enzyme conjugates for a diagnostic reagent or dephosphorylation of DNA. In 1985 Besman and Coleman proved the existence of two IAP isoenzymes in the bovine intestine, the IAP from the calf intestine and the IAP from the intestine of a mature cow (bIAPs), by amino-terminal sequencing of chromatographically purified AP fractions. A clear difference at the amino terminus was described between the bIAP of the mature cow (LVPVEEED) and the bIAP from calf intestine (LIPAEEEN). In 1993 Weissig et al. achieved an accurate biochemical characterization by cloning a recombinant bIAP (bIAP I) with a specific activity of ca. 3000 U/mg and the N-terminus LVPVEEED. However, bIAPs from calf intestine with specific activities of up to 8000 U/mg are also commercially available (Boehringer Mannheim, Biozyme, Oriental Yeast) which, however, have previously not been further characterized. All attempts at cloning these highly active alkaline phosphatases were unsuccessful. It was therefore not possible to produce a recombinant highly active alkaline phosphatase. However, the possibility of recombinant production is absolutely essential for an economic production of highly active alkaline phosphatase.
Consequently the object of the present invention was to provide highly active alkaline phosphatases by recombinant means which can also be cloned. Highly active within the sense of the present invention means that the alkaline phosphatase according to the invention has an at least 10% increased activity compared to previously known alkaline phosphatases.
The object was achieved according to the invention by the provision of a DNA coding a eukaryotic highly active alkaline phosphatase with a specific activity of more than 3000 U/mg, preferably of at least 3500 U/mg in which the amino acid residue at position 322 is smaller than aspartate. A eukaryotic DNA is preferred within the sense of the present invention. Eukaryotic cDNA is particularly preferred which means a DNA that no longer contains introns. The term “amino acid residue smaller than aspartate” is understood as any amino acid, preferably natural amino acids or amino acids derived therefrom, which has a smaller spatial dimension than the structure of the amino acid aspartate. A DNA according to the invention is preferred in which the amino acid residue 322 is glycine, alanine, threonine, valine or serine. A DNA according to the invention is particularly preferred in which the amino acid residue 322 is glycine or serine. It is quite especially preferred that the amino acid residue 322 is glycine. A DNA according to SEQ ID NO.: 1, 3 and 5 (FIGS.
1
,
3
,
5
) and the associated amino acid sequence according to SEQ ID NO.: 2, 4 and 6 (FIGS.
2
,
4
,
6
) are part of the present invention. The present invention also concerns those cDNAs which differ from the afore-mentioned only in that the N-terminus is longer or shorter in comparison to the cDNAs according to SEQ ID NO.: 2, 4 and 6. In such cases the name for position 322 according to SEQ ID NO.: 2, 4 and 6 changes correspondingly. If for example the N-terminus is x amino acids longer or shorter than SEQ ID NO.: 2, 4 and 6, the relevant position 322 is also shifted by x amino acids. SEQ ID NO.: 1 contains the DNA code for the sequence of the highly active bIAPII isoenzyme. The native enzyme was known but not characterized and not possible to clone. Hence the determination of the amino acid sequence of the highly active bIAP II isoenzyme is a subject matter of the present invention. A highly purified fraction with high specific activity from the calf intestine (Boehringer Mannheim) was used to determine the sequence. Peptide maps of the highly active AP were produced by cleavage with the endoproteinases LysC, AspN, GluC, trypsin and chemical cleavage by bromocyanogen. The peptides produced in this manner were separated and isolated by means of reversed phase HPLC. Each peptide was analysed by electrospray mass spectroscopy and sequenced by means of Edman degradation. The sequences obtained in this way were compared with the published sequence of bIAP I (Weissig et al., 1993). As expected the amino terminus of bIAP II has the start sequence LIPAEEEN as described by Besman and Coleman (
J. Biol. Chem
. 260, 11190-11193 (1985)). The complete amino acid sequence of bIAP II is shown in SEQ ID NO.: 2 (FIG.
2
). According to this the bIAP II has a total of 24 amino acid substitutions compared to bIAP I. The number of amino acids in the isolated highly active bIAP II isoenzyme is 480 amino acids. The nucleotide sequence of 1798 bp (
FIG. 1
) includes a coding region of 514 amino acids. The amino acids that are possible from position 481 to 514 inclusive can vary within wide limits.
In the following the present invention describes the cloning and complete characterization of two new previously unknown bIAPs (bIAP III and bIAP IV). Northern blot analyses were carried out on RNA samples from different sections of the bovine intestine. A cDNA bank of the probes with the strongest hybridization signal was set up with an oligo dT primer (Stratagene, San Diego, Calif., USA) in the vector IZAP II (Stratagene, San Diego, Calif, USA). The complete bank (1.0×10
6
recombinant clones) was screened with the 1075 bp HindIII fragment of bIAP I which covers a region from exon I to VIII of the bIAP I gene. 65 Clones were isolated and sequenced. In this process two new bIAPs were identified (bIAP III and bIAP IV) whose characterization is described further below and were neither completely homologous to bIAP I nor to bIAP II. The nucleotide sequences of bIAP III and IV are shown in
FIGS. 3 and 5
. The sequence differences of bIAPs I IV are shown in FIG.
7
. However, none of the new bIAPs has the expected N-terminus LIPAEEEN but rather new previously not described N-termini (see FIG.
7
). The cDNA of the two new bIAP isoenzymes was recleaved with appropriate restriction enzymes and inserted by ligation into the CHO expression vector pcDNA-3 (e.g. from the Invitrogen Co. San Diego, Calif., USA). The clones which contained the new bIAP isoenzymes were brought to expression according to the method described by Invitrogen and the isoenzymes were characterized. The expression of a bIAP gene in various hosts is described in WO 93/18139 (CHO cells,
E. coli
, baculovirus system). The methods, vectors and expression systems described in this document are part of the disclosure of the present application. The present inve

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