AU781876B2 - Beta-hexosaminidase, DNA sequence from ciliates for coding the same and use thereof - Google Patents

Description

SMB

1-Hexosaminidase and a DNA Sequence Coding it Obtained from Ciliates and Use Thereof The present invention relates to a nucleic acid coding for a p-hexosaminidase.

The expression of foreign proteins in microorganisms, such as bacteria, yeasts or mammal cells, is of great importance to the biotechnological preparation and production of recombinant proteins. Thus, bacterial expression systems based on E. coli or B. subtilis are used for the production of recombinant peptides or proteins, such as insulin, interleukin-2, tissue plasminogen activator, proteases and lipases. In Gram-negative bacteria, the expression systems are mostly based on the use of genetic elements such as the lac operon or the tryptophan operon.

The proteins foreign to the host are produced either into "inclusion bodies" within the cell, or when expression systems based on p-lactamase genes are used, into the periplasmic space. The production of recombinant proteins into the surrounding fermentation medium has not been established. In Gram-Positive bacteria, to date, almost exclusively cell-inherent proteins are introduced and expressed.

Yeasts, such as S. cerevisiae, Kluyveromyces lactis or Pichia pastoris, are also employed for the heterologous expression of recombinant proteins, such as human factor XIIIa, bovine pro-chymosin, or surface antigens. In yeasts, the expression systems are based on shuttle vectors (vectors having both yeast and bacterial portions) which are constructed from the genetic elements of galacto-kinaseepimerase, acid phosphatase or alcohol-dehydrogenase. As a rule, the recombinant protein is produced into the cytoplasm of the cell. When yeast-inherent signal sequences, such as the alpha factor, are used, the expressed proteins may also be secreted into the fermentation medium. The glycosylation of secreted proteins is effected according to the "high mannose" type.

-2- Mammal cells, such as various cell types from rodents (CHO cells, C127 cells) or simians (vero, CV-1 or COS cells), are also employed for the heterologous expression of recombinant proteins. Here, the expression systems are based on recombinant viruses (BPV vector) or on shuttle vectors. To regulate the expression, viral enhancer/promoter systems or cellular enhancer elements are employed.

The recombinant proteins, such as erythropoietin, are secreted into the fermentation medium because the foreign genes usually bring their own signal sequences, which are understood by the expression system and used for targeting.

Further, for the biotechnological production of glycosylated extracellular enzymes, ciliates such as Tetrahymena are employed. Ciliates will grow on inexpensive fermentation media using standard fermentation methods. For the transformation of such ciliates, vectors are available which are based on the rDNA elements of the ciliate Tetrahymena. For the heterologous expression of bacterial proteins in ciliates, DNA constructs consisting of genes from Tetrahymena are employed.

When suitable genetic elements for the regulation of the transcription, targeting and glycosylation of foreign proteins are available, ciliates are an ideal expression system for the inexpensive production of therapeutic recombinant proteins.

The Gram-negative bacterial expression systems used to date usually lead to the formation of "inclusion bodies" in the cell, accompanied by a denaturing of the proteins. To recover the recombinant protein, the cells must be lysed, and the denatured inactive protein must be folded back to function. This causes additional cost-intensive process steps and reduces the yield of the desired protein. Glycosylation, which is important to eukaryotic proteins, is completely omitted. When Gram-positive bacterial expression systems are used, degradation of the target protein due to high proteolytic activities in the fermentation broth is an additional problem.

When yeasts are used for heterologous expression, the desired target protein is often produced into the cell only from where it must be removed by cell lysis. As in bacterial expression systems, this causes additional time- and cost-intensive process steps. When yeast-inherent signal peptides are used, the foreign proteins are not correctly spliced and glycosylated for secretion.

PAOPERbp ZMAmot=413153IO 0rzl dcIS93005 -3- In contrast, when mammal cell systems are employed for the production of recombinant proteins, the desired proteins are found in the fermentation medium in an extracellular state, correctly spliced and glycosylated. However, what is disadvantageous here is, on the one hand, the low expression rate due to the defective processing and inefficient translation of genes which have been introduced into the genome of the production cell line via viral vectors. On the other hand, the serum-containing fermentation media for mammal cells are extremely cost-intensive. In addition, the fermentation technology for the shear-sensitive cell lines is complicated and similarly expensive due to construction for bubblefree aeration. Further problems arise from the high infection risk for the cell lines from mycoplasmas and viruses. All in all, the use of mammal cells for the *biotechnological preparation of recombinant proteins results in very high costs, safety demands and low yields.

15 To the use of ciliates, such as Tetrahymena, the above mentioned drawbacks in the production of proteins do not apply. Thus, for example, some acid hydrolases which are involved in the digestion of food particles are exported from the cell in high quantities and with complex glycosylation.

20 In J. Euk. Microbiol. 43 1996, pages 295 to 303, Alam et al. describe the cloning of a gene which codes for the acid a -glucosidase of Tetrahymena pyriformis. However, only a small portion of the protein is exported from the cell.

However, to date, it has not been possible to bring other foreign glycosylated eukaryotic proteins to expression in ciliates which are also secreted into the fermentation medium. This is due to the fact that the DNA sequences from ciliate-inherent secreted acid hydrolases which are necessary for the construction of expression vectors have not been known to date.

In one aspect, therefore, the present invention provides a DNA sequence for the expression of secreted proteins of ciliates. The DNA sequence is to enable heterologous proteins in an expression system to be exported into the fermentation medium after transformation in ciliates. This system also enables a P'IOPER =Amft%-_2tU5 A 2S35I m L dc-ISM -4high expression rate of the heterologous protein, which is exported in large amounts from the cell under culture conditions.

This aspect is achieved, more particularly, by a system in which a nucleic acid having a sequence with Seq. Id. No. 1 and coding for P -hexosaminidase is used.

In particular, the inventive DNA sequence of the /-hexosaminidase includes a signal peptide and a propeptide, and optionally further genetic elements for the targeting of proteins. The use of these sequences in a vector enables heterologously expressed proteins to be exported from the cell and thus to be purified from the fermentation broth without a cell lysis.

Figure 1 shows a nucleic acid coding for /J-hexosaminidase from ciliates. Figure 2 shows a corresponding expression product of the nucleic acid according to Seq.

S* 15 Id. No. 1. The invention also relates to this protein according to Seq. Id. No. 2.

In particular, the invention relates to the signal sequence of the protein according to the invention. This is preferably amino acids 1 to 17 of the protein according to the invention. The invention also relates to a nucleic acid which 20 codes for the N-terminal fragment. This is preferably a fragment of the nucleic acids according to the invention, especially with the nucleic acid sequence from 1 to 51 according to Figure 1.

A further aspect of the invention is the use of a nucleic acid sequence of acid hydrolases according to the invention or parts thereof for the homologous or heterologous expression of recombinant proteins and peptides, and for homologous or heterologous recombination ("knock-out, "gene replacement").

The invention also relates to a method in which the nucleic acid or parts thereof according to the invention which code for /-hexosaminidase is combined with the usual, in homologous or heterologous expression, enhancers, promoters, operators, origins, terminators, antibiotic resistances, or other nucleic acids or DNA fragments, or sequences of any kind from viroids, viruses, bacteria, archezoans, protozoans, fungi, plants, animals or humans.

P1DPER. eAbzln ctA42c Acxntmmt s2453510reW 1.6 1d In particular, the nucleic acid or parts thereof according to the invention is inserted into a vector, a plasmid, a cosmid, a chromosome or minichromosome, a transposon, an IS element, an rDNA, or any kind of circular or linear DNA or

RNA.

The skilled person will understand that nucleic acids having at least homology with the nucleic acid according to Seq. Id. No. 1 can also be employed according to the invention. The protein according to Seq. Id. No. 2 can also be modified without losing its function. Thus, for example, so-called conservative exchanges of amino acids may be performed. Thus, for example, hydrophobic amino acids can be interchanged.

For the purification and isolation of P/-hexosaminidase from ciliates and for 15 determining its sequence, the following methods can be used. This is illustrated by the following non-limiting Examples.

Example 1 *o From a total of 3.2 I of cell culture, cells of the ciliate Tetrahymena in late logarithmic growth phase were washed into 400 ml of starving medium (10 mM Tris-HCI, pH and the cells were incubated for another 4 hours with shaking.

Then, the cell-free culture supernatant was harvested and filtered through a number of filters with decreasing pore diameters to remove any particular material. The filtrate was concentrated with an Amicon ultrafiltration cell and rebuffered into the starting buffer for the subsequent ion-exchange chromatography (IEC).

The collected chromatographic fractions were tested with specific 4-nitrophenyl substrates for acid hydrolases, acid phosphatase (aPAse) and -hexosaminidase (fp-Hex), and the fractions having the highest Bl-Hex activity were combined.

They were concentrated by another Amicon ultrafiltration and rebuffered into the starting buffer for affinity chromatography. The collected chromatographic P'AO =5A-xarni24S351O MqW I .CI 5A3 fractions were tested for enzymatic activities as described above, and the fractions having the highest activity for an acid hydrolase were combined and rebuffered into phosphate buffer From a total of eight purifications, the chromatographic

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-6fractions containing hydrolases were pooled, concentrated, portioned and frozen until further characterization.

Part of the thus purified hydrolase was separated by two-dimensional SDS gel electrophoresis (a total of eight gels), the main spot was respectively punched out and collected. The protein present in these gel pieces, p-Hex, was digested with the protease trypsin. The thus obtained peptide fragments were leached out of the gel pieces and separated by reverse-phase HPLC (RP-HPLC). The purity of the HPLC fractions was tested with a mass-spectroscopical method (MALDI-MS), and the pure fractions, those which contained only one peptide species of a defined mass, were sequenced from their N-terminus using Edman degradation.

From the sequence of the peptide fragments obtained by the trypsin digestion, p-hexosaminidase-specific PCR primers were established. By a sequence comparison with the existing p-Hex sequences, a preselection of the primer combinations for RT-PCR could be made. With this information, the first specific cDNA fragments were amplified and sequenced, using RT-PCR, from isolated whole RNA whose quality was previously tested by Northern hybridization. Using these fragments, sequence-specific primers were constructed which were employed for further PCR experiments. Each primer and each partial sequence obtained was checked by data base alignment, and prior to further experiments, it was also checked for any overlooked vector sequences, inter alia. By and 3'-RACE, the cDNA sequence was elongated at the and 3'-termini. The thus completed sequence of the p-Hex cDNA then served as the basis for further sequence analyses.

The thus established sequence of a p-hexosaminidase from the ciliate reads as set forth in Figure 1. In total, this sequence comprises 1836 base pairs including and 3'-non-translated regions of p-hexosaminidase with an open reading frame having a length of 1656 base pairs. The complementary amino acid sequence has a length of 551 amino acids and reads as set forth in Figure 2.

The sequence of the p-hexosaminidase has a total of 9 glycosylation sites and contains a signal peptide and a pro sequence for targeting the enzyme through the sorting mechanism of the cell.

P4PE~v~mdcMWD-2SAm~ftxnt3435IO 1. 1MW -7- The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

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EDITORIAL NOTE APPLICATION NUMBER 32856/00 The following Sequence Listing pages 1 to 5 are part of the description. The claims pages follow on pages to SEQUENCE LISTING <110> Cilian AG <120> Beta-hexosaminidase, DNA sequence from ciliates for coding the same and use thereof <130> <140> <141> <160> 3 <170> Patentln Ver. 2.1 <210> 1 <211> 1656 <212> DNA <213> Tetrahymena thermophila.

<400> 1 atgcaaaaga tacttttaat. tactttcctt cttggaatag ctctcgctca aattactcct ggcgttgac ttgagctta aaagtaccc, aacgaaaacl atgcgtagal ttggccact< tat tggaaat caattattcE attcaAgatt ttatcagttc ctccattggc attactaaat attgtagact cacgcttttt tataatggat gaagatatga gaagaataat tctacatata attaatgcta gatgatatta aacaaaataa tatggtggaa gttcctggaa gatgattCta ctatttcagi ttgtcactgi accatgtcti t cttgtgctat -tacaacattc 3 cagacacact -tgactgctaa kCttaagacga aacctgacta aaactatttt acatcactga.

atggagccta aagctctcaa catgggctag agttagaccc atacttaatt gctggaataa ctgatttgta ctaagcctgc t tcaatggtg ttttatcttt gttatggcag taaggttatg cctaaaccta agaattacac ttatggagat 120 tccttgcgga gtctcttaca gaccttctgt tgggtcagga 180 :gtaaagagaa "ctaaaatata :cgaagacgaa Lcaaatatgtt agacactgaa.

catctacaga aaaaactatt tactgaatcc Ctctaagaag caagggtatt atctccttaa aacactaaat ctacactgct acgccctgaa gaattattac tattttctg gggatctact ctatgataat catgtataact ggattctaca ttgtataaga gtcttcgata tat tatgatt ggtttactcc gattggtatt ggtcttatga.

gattctatgt ttCCCCttcc aaacaataca taagttattc ttctctagta ttaacttaca.

aagtatgttc attaaggaat agaaagaact gcagattcaa z catgattttt c Icttatttgg a :gggatgtct t 3gcgaaacat 9 Icaagaaata g ctttgaatat atgaaacaac tttttatcta tataaattta gtggtttaga tgaataacat tagattcagc tattcaacaa ctcttaaatc gcttcgaaga ctgaagtcga ttggtctatt ctgctgttaa attttggtgg tcatgaatta aagttaacat1 ltactttgaa :ttcaatcaa Ltgttggtga

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aaactcttt c cttatggag t tttcaattct 240 cattgaaaag 300 agacgctgct 360 taataccaca. 420 aacttactct 480 ccctatttct 540 cagacatttc 600 gttgaatgtt 660 attccctaat 720 catttaataC 780 ttctccagga 840 atgtgattaa 900 gggtattatg 960 tgatgaagtt 1020 aaataacatc 1080 ttggaaatca 1140 atatggtc ct 1200 agatcttcct 1260 3ggaaataga 1320 ~aatcctaga 1380 ttaagggtga aattcttggt cttaattcta aagactttgg tgcatttgc tgaaagactt 1500 tggaacactg atgctgctaa caatgaaacttaaac agtattgaat 16 tacaaaacta gagctttagt tagcagaatg 1560 gtctttatgc aacaccgttt aactgctaga ggaatccctg cttctcctgt aacagttggt 1620 atttgtgaat aaaacctttc tctctgctac aattga 1656 <210> 2 <211> 549 <212> PRT <213> Tetrahymena thermophila <400> 2 Met Gin 1 Lys Ile Leu Leu Ile Thr Phe 5 Le u 10 Leu Gly Ile Ala Leu Ala Gin Ile Thr Pro Lys Asn Pro Gly Val Asp Pro Ser Ala Lys Vai Met Pro Lys Thr Asp Pro Tyr Thr Tyr Gly Leu Ser Leu Leu Cys Gly Val Ser Tyr Arg Pro 55 Ser Val Gly Ser Gly Lys Val Pro Asn His Val Tyr Gin Ile Ile Gly Phe Tyr Thr Asn Ile Phe Asn Asn Glu Asn Ser Cys Ala Met Gin Arg Glu 90 Tyr Lys Asn Glu Thr Thr Ile Giu Lys Ile Phe Ile 115 Met 100 Arg Arg Leu Gin His 105 Ser Gin Asn Ile Val Phe Asp 110 Leu Giu Asp Gin Asp Ala Ala Leu 120 Ala Thr Ala Asp Thr 125 Giu Tyr 130 Tyr Asp Leu Gin Tyr Asn Thr Thr Tyr 140 Trp Lys Leu Thr Ala 145 Asn Lys Tyr Val Gly 150 Leu Leu Arg Gly Giu Thr Tyr Ser Leu Phe Thr Gin Asp 165 Giu Asp Thr Giu Asp 170 Trp Tyr Leu Asn Asn Ile 175 Pro Ile Ser Ile Asp Ser 195 Ile 180 Gin Asp Gin Pro Asp 185 Tyr Ile Tyr Arg Gly Leu Met 190 Leu Lys Thr Ala Arg His Phe Leu 200 Ser Val Giu Thr Ile 205 Ile Asp Ser Met Leu Phe Asn Lys Leu Asn Val Leu His Trp His Ile 210 215 220 Thr 225 Thr Asp Thr Glu Ser Lys Tyr Gly Ala 245 Phe 230 Pro Phe Pro Leu Ser Phe Pro Asn Tyr Ser Lys Lys Gin Tyr Ser Phe Giu Asp 255 Ile Gin Tyr Pro Giu Val 275 Ile 260 Val Asp Gin Ala Leu 265 Asn Lys Gly Ile Gin Val Ile 270 Arg Ser Pro Asp Ser Pro Gly His 280 Ala Phe Ser Trp Ala 285 Gin Phe 290 Ser Ser Ile Gly Leu 295 Leu Cys Asp Gin Tyr Asn Gly Gin Leu 300 Lys Gly Ile Met Giu Asp 305 Pro Thr Leu Asn Leu 310 Thr Tyr Thr Ala Asp Met Asn Thr Gin 325 Phe Tyr Thr Ala Tyr Vai His Phe Gly Gly 335 Asp Giu Val Phe Met Asn 355 Giu 340 Giu Gin Cys Trp Asn 345 Lys Arg Pro Giu Ile Lys Giu 350 Gin Asn Tyr Gin Asn Asn Ile Ser 360 Thr Tyr Thr Asp Leu 365 Tyr Arg 370 Lys Asn Gin Val Asn 375 Ile Trp Lys Ser Ile 380 Asn Ala Thr Lys Pro 385 Ala Ile Phe Trp Asp Ser Asn Thr Lys Tyr Gly Pro Asp 400 Asp Ile Ile Gin Trp 405 Trp Gly Ser Thr Asp Phe Ser Ser Ile Lys 415 Asp Leu Pro Asp Val Gly 435 Asn 420 Lys Ile Ile Leu Ser 425 Phe Tyr Asp Asn Thr Tyr Leu 430 Giu Gly Asn Arg Tyr 440 Gly Gly Ser Tyr Gly Ser Met Tyr 445 Asn Trp 450 Asp Val Leu Asn Ser 455 Phe Asn Pro Arg Val1 460 Pro Gly Ile Lys Gly Glu Ilie Leu Gly Giu Thr Cys Leu Trp Ser Giu Met Asn Asp Asp Ser Thr Gin Phe Gin 485 Arg Leu Trp Thr Asn Ser Ala Phe Ala Glu 495 Arg Leu Trp Ala Leu Val 515 Thr Asp Ala Ala Asn 505 Asn Giu Thr Tyr Lys Thr Arg 510 Thr Ala Arg Ser Arg Met Val Phe 520 Met Gin His Arg Leu 525 Gly Ile 530 Pro Ala Ser Pro Val1 535 Thr Val Gly Ile Cys 540 Glu Gin Asn Leu Ser Leu Cys Tyr Asn 545 <210> 3 <211> 1837 <212> DNA <213> Tetrahymena thermophila <400> 3 cagcagtaat actttccttc aaggttatgc ccttgcggag caaattattg taaagagaat taaaatatag gaagacgaat aaatatgttg gacactgaag atctacagag aaaactattg actgaatcct tctaagaaga aagggtattt tctccttaat acactaaatt tacactgcta cgccctgaaa aattattaca attttctggg aaaaaattct ttggaatagc ctaaacctaa t ct ct ta cag gattctacac tgtataagaa tcttcgatat attatgattt gtttactccg attggtattt gtcttatgat attctatgtt tccccttccc aacaatacag aagttattcc tctctagtat taacttacac agtatgttca ttaaggaatt gaaagaacta cagattcaaa aaatatattg tctcgctcaa gaattacact accttctgtt tttgaatatt tgaaacaacc ttttatctaa ataaatttat tggtttagaa gaataacatc agattcagcc attcaacaag tcttaaatca cttcgaagac tgaagtcgat tggtctatta tgctgttaag ttttggtggt catgaattaa agttaacatt tactttgaaa attgtagcta attactcctg tatggagatt gggtcaggaa ttcaattcta attgaaaaga gacgctgctt aataccacat acttactctc cctatttcta agacatttct ttgaatgttc ttccctaata atttaataca tctccaggac tgtgattaat ggt at ta tgg gatgaagttg aataacatct tggaaatcaa tatggtcctg tgcaaaagat gcgttgaccc tgagcttact aagtacccaa acgaaaactc tgcgtagatt tggccactgc attggaaatt aattattcac ttcaagatta tatcagttga tccattggca ttactaaata ttgtagacta acgctttttc ataatggata aagatatgaa aagaataatg ctacatatac ttaatgctac atgatattat acttttaatt tatttcagct tgtcactgat ccatgtctat ttgtgctatg acaacattcc agacacactc gactgctaac ttaagacgaa acctgactac aactatttta catcactgat tggagcctac agctctcaac atgggctaga gttagaccca tacttaattc ctggaataaa.

tgatttgtag taagcctgct tcaatggtgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 ggatctactc atgatttttc ttcaatcaaa gatcttccta acaaaataat tttatctttc 1320 tatgataata atgtataact attcttggtg agactttgga aatgaaactt actgctagag ctctgctaca gaatatttta attgaatttt cttatttgga gggatgtctt gcgaaacatg caagaaatag acaaaactag gaatccctgc at Lga t tc a gtataaaaac agctaaaaaa tgttggtgag aaactctttc cttatggagt tgcatttgct agctttagtt ttctcctgta aatataaara tgtattttaa aaaaaaaaaa ggaaatagat aatcctagag gaaatgaatg gaaagacttt agcagaatgg acagttggta ttaaataaat ttgataaaaa aaaaaaa atggtggaag ttcctggaat atgattctac ggaacactga tctttatgca tttgtgaata attttaagaa aaatataaat ttatggcagc taagggtgaa ttaattctaa tgctgctaac acaccgttta aaacctttct atatttttaa attat tat ta 1380 1440 1500 1560 1620 1680 1740 1800 1837

Claims (9)

1. Use of an isolated nucleic acid or parts thereof coding for a peptide having the sequence MQKILLITFLLGIALAQ for the homologous or heterologous expression of recombinant proteins and peptides.

2. Use of an isolated nucleic acid or parts thereof coding for a peptide having the sequence MQKILLITFLLGIALAQ for homologous or heterologous recombination.

3. Use of an isolated nucleic acid according to claim 1 or 2 in a method in which said nucleic acid is combined with enhancers, promoters, operators, origins, terminators, antibiotic resistances, or other nucleic acids or DNA fragments, or sequences of any kind from viroids, viruses, bacteria, archezoans, protozoans, fungi, plants, animals or humans.

4. Use of an isolated nucleic acid according to claim 3 in a method in which the nucleic acid is incorporated or inserted into a vector, a plasmid, a cosmid, a chromosome or minichromosome, a transposon, an IS element, an rDNA, or any other kind of circular or linear DNA or RNA.

5. An isolated peptide of the sequence MQKILLITFLLGIALAQ.

6. An isolated nucleic acid coding for the peptide according to claim

7. Use of the isolated peptide according to claim 5 for the homologous or heterologous expression of recombinant proteins and peptides.

8. Use of the isolated peptide according to claim 5 for homologous or heterologous recombination. P.'DEnltV-d.-UL1O(M-2005 AmcemtsuI2453510,tImro dm.I9)4O5 -9-

9. An isolated peptide according to claim 5 or an isolated nucleic according to claim 6 or a use according to any one of claims 1 to 4, 7 and 8 substantially as described hereinbefore with reference to the examples and/or figures. DATED this 1 9 t h day of April, 2005 Cilian AG by DAVIES COLLISON CAVE Patent Attorneys for the applicant *.I