CN1553919A - Shrimp Alkaline Phosphatase - Google Patents

Abstract

The present invention relates to a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID Nos. 1 or 20 or having at least 55 % sequence identify therewith and/or capable of hybridising under medium stringency conditions to the complementary sequence of SEQ ID Nos. 1 or 20, or the complementary sequence of said sequences, wherein said nucleotide sequence encodes or is complementary to a sequence which encodes a heat labile alkaline phosphatase and to a recombinant heat labile alkaline comprising: (a) all or a significant part of an amino acid sequence as shown in SEQ ID No. 2; or (b) all or a significant part of an amino acid sequence which has at least 60 % sequence identify with SEQ ID No. 2 as well as methods for the manufacture thereof.

Description

Shrimp Alkaline Phosphatase

The present invention relates to alkaline phosphatase, in particular to the enzyme derived from the northern prawn (Pandalus borealis) and a nucleic acid molecule encoding the enzyme.

Alkaline phosphatase (EC3.1.3.1), also known as alkaline phosphate monoesterase, phosphate monoesterase or glycerol phosphatase, is an orthophosphoric acid-monoester phosphate with the most suitable enzyme activity under alkaline conditions hydrolase. The substrates of alkaline phosphatase (ALP) are DNA, RNA, and riboside and deoxyriboside triphosphates. Hydrolysis produces alcohol and orthophosphate. From another perspective, ALP dephosphorylates DNA, RNA, rNTP, and dNTP. Protein dephosphorylation by different ALPs has also been reported. ALP is ubiquitous in nature and can be found in a variety of organisms from bacteria to humans. Complex organisms generally contain both tissue-specific and non-specific ALPs. These polypeptides range in size from 15 kDa to 170 kDa. Some of these proteins are bound or "anchored" to the cell membrane. Most commonly, these enzymes require divalent metal cations, such as Mg ²⁺ , or Zn ²⁺ .

Current applications of ALP in molecular biology focus on, but are not limited to, three main methods of analyzing or preparing DNA: 1) Dephosphorylation of vector DNA following restriction enzyme digestion in order to eliminate cloning vectors 2) dephosphorylate the dNTP after PCR amplification, combined with the application of single-stranded exonuclease to hydrolyze the primer to make it into dNTP, thereby omitting 3) dephosphorylate the DNA terminus, which facilitates subsequent 32P labeling with [γ-32P]NTP and T4 polynucleotide kinase. The described ALP enzyme reaction is an intermediate step in the DNA analysis process. Other important applications known in the art include those using enzyme activity as a marker, for example in enzyme-linked immunosorbent assays (ELISA), in gene fusion or gene delivery systems, or in conjugation with oligonucleotides When doing hybridization probes.

The three main ALPs used in commercially available products are;

I) Calf intestinal alkaline phosphatase (CIAP)

II) Shrimp Alkaline Phosphatase (SAP) from the northern prawn from the Arctic

III) Bacterial Alkaline Phosphatase (BAP)

CIAP and SAP in animals have similar specific activities of 2000-4000 units/mg protein, while the specific activity of BAP protein is 50 units/mg, so the first two enzymes are more attractive as high-efficiency enzymes. As described below, SAP is inactivated by moderate heat, so it is preferred for applications where enzyme activity needs to be removed prior to subsequent steps.

A genetically engineered temperature-sensitive BAP variant has been reported [Shandilya, H. AndChatterjee, D.K., 1995. A genetically engineered heat-sensitive alkaline phosphatase for DNA dephosphorylation. Focus, 17(3):93-95], but did not give its engineering details. This mutant enzyme (TsAP), sold by Life Technologies, Inc., is inactivated (95% or more) by heating (65°C, 15 minutes) in the presence of EDTA alone. The recommended reaction temperature for this mutant and wild-type enzyme is also 65 °C. The enzymatic activity of TsAP is 40-fold higher than that of wild-type BAP. Due to the high specific activity of TsAP, it is almost comparable to other ALPs such as CIAP and SAP.

The heat-sensitive ALPs of two psychrophilic microorganisms have been purified and characterized [de Prada, P., and Brenchley, J.E., 1997, Two extracellular alkaline phosphatases from a psychrophilic Arthrobacter isolate Purification and Identification, Appl.Env.Microbiol., 63(7):2928-2931]. The two enzymes differed in their substrate specificity and kinetic characteristics, and they showed different heat sensitivities, with the most heat-sensitive one having a heat sensitivity similar to that of SAP. There is no report on their specific activity expressed in units/mg and primary structure.

A cold-adapted ALP of Atlantic cod was isolated and characterized [Asgeirsson, B., Hartemink, R., and Chlebowski, J.F., 1995, Atlantic cod (Gadus morhua) alkaline phosphatase. Kinetic and structural properties that can show its adaptation to low temperature. Comp. Biochem. Physiol., 110B(2):315-329]. This enzyme shows similar thermosensitivity to SAP. The primary structure of its gene/protein is not provided.

A study on the ALP isozyme of salmon [Whitmore, D.H., and Goldberg, E., 1972, Trout intestinal alkaline phosphatase II.The effect of temperatures upon enzymatic activity in vitro and in vivo.J.Exp.Zool., 182 :59–68] showed that ambient temperature affects isoenzyme patterns and that some isoforms are thermosensitive.

Shrimp in warm waters around Taiwan contain several ALPs [Lee, A.-C., and Chuang, N.-N., Characterization of different molecular forms of alkaline phosphate in the hepatopancreas from shrimp Penaeus monodon (Crustacea: Depacoda). Comp .Biochem.Physiol., 99B(4):845-850], these enzymes are concluded to be thermostable. No primary structure is provided.

K.and Myrnes，B.，1991，Alkalinephosphatase from hepatopancreas of Shrimp(Pandalus Borealis)：a dimericenzyme with catalytically active subunits.Comp.Biochem.Physiol.99B(4)：755-761]。这种纯化蛋白的表观分子量为65kDa(每个亚单位)，且显示为一种具有催化活性亚单位的二聚体酶，而其它大多数动物性ALP与它相反，它们必需经过二聚化才能发挥活性。根据该报道，SAP的等电点为3.7。虽然5’突出末端比平末端或5’凹陷末端反应性更强，这种虾酶可以从限制性内切酶产生的任何DNA链末端(5’和3’突出末端或平末端)有效地去除5’末端磷酸。A type of alkaline phosphatase from the hepatopancreas of northern longhorn shrimp was found in the treatment wastewater of the shrimp industry [Olsen, RL, Johansen, A., and Myrnes, B., 1990, Recovery of enzymes from shrimp waste. Process Biochem.25:67-68], the enzyme was later purified from hepatopancreas [Olsen, RL,

K. and Myrnes, B., 1991, Alkalinephosphate from hepatopancreas of Shrimp (Pandalus Borealis): a dimericenzyme with catalytically active subunits. Comp. Biochem. Physiol. 99B(4): 755-761]. The purified protein has an apparent molecular mass of 65 kDa (per subunit) and appears to be a dimeric enzyme with catalytically active subunits, in contrast to most other animal ALPs, which must undergo dimerization to be active. According to the report, the isoelectric point of SAP is 3.7. Although 5' overhangs are more reactive than blunt or 5' recessed ends, this shrimp enzyme can efficiently remove any DNA strand ends (5' and 3' overhangs or blunt ends) generated by restriction enzymes 5' terminal phosphate.

Relative to CIAP, slightly lower temperature is required for SAP activity, but it is not considered to be a true low temperature activation. Enzyme activity peaked at about 40°C (45°C for CIAP), and almost 40% of enzyme activity was retained at 10°C or 50°C, compared to 10% and 90% for CIAP, respectively. Although SAP activity peaks at temperatures close to 40°C, the enzyme begins to lose activity after 15 minutes of preheating above 37°C. After preheating at 65°C, the enzyme activity will lose 95% or more, and after preheating at 70°C, the activity cannot be detected. In contrast, CIAP retained 40% and 20% of its activity after similar heat treatment.

So, relative to its commercial competitors, SAP is heat sensitive and activated at low temperature, making it particularly suitable for those multi-step laboratory protocols where a simple heating step can inactivate SAP , so that it does not participate in later method steps.

Norway，从捕虾工业废水中生产商用SAP。在捕虾船上，刚捕的鲜虾被冻成大块。船靠岸后，这些虾被再循环的冷水小心地解冻。每4000千克虾大约用1000升水。在冷冻与解冻的过程中，虾的肝胰脏破裂，里面的物质就释放到水中。然后这些废水被回收，再经过几次层析步骤来纯化SAP。BIOTEC ASA,

Norway, production of commercial SAP from industrial effluents from shrimp fishing. Freshly caught prawns are frozen into large chunks on a shrimp fishing boat. After the boat docks, the prawns are carefully defrosted by recirculating cold water. About 1000 liters of water is used for every 4000 kg of shrimp. During the freezing and thawing process, the shrimp's hepatopancreas ruptures, releasing the contents into the water. The wastewater is then recycled and subjected to several chromatographic steps to purify the SAP.

Manufacturers supply SAP to the global market through well-known companies such as USB, Boehringer-Mannheim or Amersham Pharmacia Biotech.

Because of its high specific activity and versatility of DNA ends, and its relative heat sensitivity, SAP is often used to dephosphorylate cloning vectors prior to ligation reactions, and to dephosphorylate PCR amplification product mixtures prior to DNA sequencing reactions. Processing, as described in US Patent Nos. 5,741,676 and 5,756,285.

The current SAP production is plagued by unstable quality of shrimp fishing wastewater, which affects production efficiency. Factors that produce quality variability are, I) the natural seasonal variation of enzyme production in shrimp, and ii) manipulation of the shrimp source before or during freezing and manipulation of the shrimp or water during or after thawing.

There are also concerns about the future availability of wastewater; I) shrimp, being a natural resource, is not guaranteed to always be available; II) the shrimp industry is looking for a new way to freeze shrimp, i.e., Freezing method, by which enzymes in wastewater, such as SAP, can be detected in very small amounts.

In addition to these practical concerns, there is a demand for recombinant SAP products, as recombinant products are often preferred for use in molecular biology techniques, especially when product purity becomes an issue, e.g. in the production of DNA-based therapeutics pharmaceuticals or in the field of forensic science. SAP has great advantages in enzymatic and physicochemical properties, so it is the preferred enzyme in the laboratory operation in which it is applied. Therefore, there will be considerable demand in the market for synthetic or recombinant SAP in a single purified form. However, the DNA and amino acid sequences of SAP have not been elucidated before.

In order to isolate the gene and then clone and produce recombinant SAP, several attempts have been made to obtain the N-terminal sequence of the purified protein. But these attempts were unsuccessful. Sequence analysis revealed multiple (4 or more) amino acid substitutions for each specific N-terminal position. Therefore, even if highly degenerate oligonucleotide probes were used, no protein sequence information that could be used to obtain the SAP gene was obtained. The reason for the presence of non-homologous N-termini in apparently homogeneous enzyme preparations is only speculative. It may be because these SAP isoforms are produced by different genes, undergo different splicing, or undergo different protein post-translational modifications, or SAP has been attacked by proteases, but no reduction in molecular weight can be detected.

The present inventors have also found that the limited identity between the alkaline phosphatases of the hepatopancreas of the northern prawn and already sequenced alkaline phosphatases of other species in the Arctic precludes the use of conventional methods to isolate the alkaline phosphatase encoding the northern prawn Nucleic acid sequence of sex phosphatase.

Although there are difficulties in obtaining the relevant consensus amino acid sequence of SAP, the inventors surprisingly isolated the cDNA encoding SAP from the cDNA library of the northern prawn, thereby elucidating the coding sequence of SAP. The present invention relates to the preparation of SAP cDNA and its use in providing another new source of this enzyme.

SEQ ID NO.1 corresponds to the complete cDNA sequence of the enzyme after removing a small part of the 5' end sequence. However, expression products of nucleic acid molecules comprising this sequence are considered to possess all or at least a substantial portion (ie, at least 60%, 70%, 80% or usually at least 90%) of the activity of the native enzyme. The missing N-terminal amino acids at the end of this sequence do not include binding sites for substrates or cofactors.

Therefore, it is generally considered that the SAP or heat-sensitive alkaline phosphatase involved here also includes those molecules smaller than naturally occurring ALP but having the same or substantially the same enzymatic activity. More advantageously, these expression products of the present invention and other recombinant ALPases may have stronger activity than isolated naturally occurring enzymes. Preferably the specific activity is higher than the SAP published value of 1900 U mg-1. The applicable method for measuring enzyme activity is as described in [Olsen et al.].

Thus in one aspect the invention provides an (isolated) nucleic acid molecule comprising (preferably consisting of) the nucleotide sequence shown in SEQ ID No. 1, or having at least 55% sequence identity thereto sequence, and/or a sequence that can hybridize with the complementary sequence of SEQ ID No.1 under medium stringency conditions, or a complementary sequence with the above-mentioned sequence, wherein the nucleotide sequence encodes a thermosensitive alkaline phosphatase or A sequence that hybridizes to the complement of the enzyme coding sequence.

A nucleic acid molecule according to the invention may thus be single- or double-stranded DNA, cDNA, or RNA.

A nucleic acid molecule of the invention may be an isolated nucleic acid molecule (or in other words, isolated from various components commonly found in nature), or may be a recombinant or synthetic nucleic acid molecule.

This relates to sequences consisting essentially of SEQ ID No. 1 and variants thereof. There may therefore also be flanking regions at both ends. In particular, the 5' flanking region may have 1-81 or 1-60 nucleotides, more particularly 1-30 nucleotides, and these flanking regions are used to direct the activity of the enzyme during transcription/translation Signal sequence for processing in the mature form. The 3' flanking region typically comprises 1-60, such as 1-30 additional nucleotides.

The complement of any given sequence will be the only definitive sequence that can be determined according to the principles of base pairing.

Preferably, the above-mentioned nucleotide sequence has at least 65%, more preferably at least 75%, such as at least 85% or at least 95% sequence identity with SEQ ID No. 1 or its complementary sequence. Likewise, the nucleotide sequence preferably hybridizes to SEQ ID No. 1 or its complementary sequence under high stringency conditions. The present invention is based on the identification of the amino acid sequence and the cDNA sequence of SAP, but it is recognized in the art that variants of these sequences, such as allelic variants and single or multiple base substitutions, insertions or deletions Related sequences that are modified and functionally equivalent to the newly identified sequences exist and can be generated.

A particularly preferred embodiment of the invention is an isolated nucleic acid molecule encoding eukaryotic thermosensitive alkaline phosphatase and derivatives of these enzymes, preferably said enzyme is from shrimp. The derivatives may contain conservative amino acid substitutions, wherein amino acids of the native enzyme may be substituted with amino acids having similar functional properties, such as size, lipophilicity, polarity. Functionally relevant groups on amino acids are known to those skilled in the art. Additional insertions, deletions or substitutions may be included that do not significantly affect the catalytic activity of the enzyme. Generally speaking, the enzyme derivative will have 1-50, preferably 1-30, eg 1-15 non-conservative changes compared with the wild-type enzyme.

The term "functional equivalent" means that the polypeptide encoded by the nucleic acid molecule has alkaline phosphatase activity, preferably their specific activity is the same or higher than that of the wild-type enzyme isolated from northern prawn. In other words, functionally equivalent nucleic acid sequences encode polypeptides that can catalyze the removal of terminal 5' phosphate groups at the ends of DNA strands (5' and 3' overhangs or blunt ends) produced by, for example, restriction enzymes. reaction.

Variations in the nucleotide sequence encoding alkaline phosphatase can occur between different populations of the northern prawn species, between similar populations in different regions, or within the same organism. Such variations are included within the scope of the present invention.

To define the degree of stringency, medium stringency includes hybridization and/or washing under conditions of 42°C to 65°C, 0.2xSSC-2xSSC buffer, 1% (w/v) SDS, high stringency Hybridization and/or washing at least above 55°C, 0.1xSSC-0.2xSSC buffer, 0.1% (w/v) SDS are included. Conditions for hybridization and washing are well known to those skilled in the art.

The scope of the present invention also includes the ability to hybridize with SEQ ID No.1 or its complementary sequence, or its partial sequence (that is, the hybridization probe derived from SEQ ID No.1) under high stringency conditions and encode alkaline phosphatase or a nucleic acid sequence of a portion thereof. Conditions of high stringency can be readily selected according to techniques well known in the art, as described by Sambrook et al., 1989, A Laboratory Manual of Molecular Cloning, Second Edition. Hybridizing sequences included within the scope of the invention are bound under non-stringent conditions (room temperature, 6xSSC/50% formamide) and washed under high stringency conditions (eg, 0.1xSSC, 68°C). Wherein SSC=0.1 5M NaCl, 0.015M sodium citrate, pH 7.2.

The hybridization probe can be a partial sequence of SEQ ID No.1 (or complementary sequence), and its base length and composition should be sufficient to enable it to hybridize with the nucleic acid sequence of the sample or test, thereby judging whether hybridization occurs at high stringency. Therefore, the probes are at least 15 bases in length, preferably at least 30, 40, 50, 75, 100 or 200 bases in length. Representative probe lengths thus include lengths of 30-500 bases, eg 30-300, 50-200, 50-150, 75-100 bases.

The judgment of nucleotide sequence identity can use the BestFit program of the Genetics Computer Group (GCG) Version 10 software package designed by the University of Wisconsin. The program uses Smith and Waterman's local homology algorithm and sets the following default values: Gap generation penalty value = 50, Gap extension penalty value = 3, average match value = 10,000, average mismatch value = -9.000 .

Methods of generating specific variants described herein, such as site-directed mutagenesis, random mutagenesis, or methods of enzymatic cleavage and/or nucleic acid ligation, and by, for example, hybridization, determining whether a nucleic acid so engineered has significant identity to the subject sequence Genetic methods are well known in the art.

From another perspective, the present invention provides an isolated nucleic acid molecule that encodes or is complementary to the coding sequence of thermosensitive alkaline phosphatase, wherein said alkaline phosphatase comprises the amino acid sequence of SEQ ID No.2 or a variant thereof A variant, the sequence of the variant is at least 50%, preferably at least 60%, such as at least 70% or 80%, identical to SEQ ID No.2.

The present invention also provides a cDNA encoding a thermosensitive alkaline phosphatase comprising the amino acid sequence (SEQ ID No.2) in Figure 2, or a variant of the enzyme possessing thermosensitive and alkaline phosphatase activity or a fragment, preferably the cDNA is derived from northern shrimp.

The present invention also provides a cDNA, which encodes a heat-sensitive alkaline phosphatase, which is composed of a sequence of deletion, replacement or insertion of one or more amino acid residues in the sequence of SEQ ID No.2, preferably the The cDNA was derived from the northern spiny shrimp.

The present invention further provides a polypeptide encoded by the nucleic acid molecule defined in the present invention. The nucleic acid molecules defined according to the invention make it possible to obtain recombinant alkaline phosphatase, especially SAP and its variants, in previously unattainable quantities and in purity, thereby enabling a continuous supply of this enzyme, resulting in a complete Time, mass production under controlled conditions or under specified standard conditions.

Therefore, the contents of the present invention also extend to recombinant polypeptides, which include (or are basically composed of) the amino acid sequence that constitutes the heat-sensitive alkaline phosphatase encoded by the nucleic acid sequence (SEQ ID No.1) shown in Figure 1, or its function equivalent variant. Viewed from another perspective, the present invention also provides a recombinant polypeptide substantially free of other components of the northern prawn, which includes a thermosensitive base encoded by the nucleotide sequence (SEQ ID No.1) shown in Figure 1 The amino acid sequence of a sex phosphatase, or a functionally equivalent variant thereof.

The term "polypeptide" as used herein includes full-length proteins, as well as relatively short peptide sequences.

"Functional equivalent" as used above to describe the amino acid sequence of a polypeptide defines a polypeptide related to or derived from the above polypeptide sequence, but wherein the amino acid sequence has been modified and still retains catalytic activity, wherein the modification of the polypeptide includes a or multiple amino acid substitutions, additions or deletions, or chemical modifications such as glycosylation or deglycosylation. Such functionally equivalent variants can be produced by natural biological variation, or can be prepared by known techniques, for example, functionally equivalent recombinant polypeptides can be produced by known site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and /or prepared by amino acid linking technology.

The synthetic polypeptides of this aspect of the invention can be prepared by expression in a host cell comprising a recombinant DNA molecule comprising the nucleotide sequences discussed extensively above operably linked to expression control sequences, or a host cell It includes a recombinant DNA cloning vector carrying the above-mentioned recombinant DNA molecule, for example, a plasmid, a cosmid, a phage molecule or a yeast artificial chromosome. In addition, the naked DNA molecule of the present invention can also be injected directly into host cells to express polypeptides.

The synthetic polypeptide expressed as described above may be a fusion polypeptide comprising a catalytically functional portion of alkaline phosphatase and another polypeptide encoded by the DNA of the recombinant molecule fused thereto. For example, it may be desired to produce a fusion protein comprising synthetic alkaline phosphatase or another polypeptide of the present invention with, for example, β-galactosidase, phosphatase, glutathione-S-transferase, Protein fusion such as urease. The vast majority of fusion proteins are produced by the expression of a recombinant gene in which the two coding sequences are linked together and their reading frames are simultaneously coordinated. If desired, the synthetic peptide of the present invention can be fused with a protein or peptide (such as an antibody) having a ligand-binding function to generate, for example, an enzyme-linked reporter antibody, which is also well known in the art. All such fusion or hybrid derivatives of such alkaline phosphatase-encoding nucleic acid molecules and their respective amino acid sequences are included in the present invention. Such suitable techniques for recombinant DNA and polypeptide expression are described, for example, in Sambrook et al., 1989. Alternatively, polypeptides of the invention may be produced by chemical methods, such as the well-known Merrifield solid phase synthesis method.

More specifically, in this aspect the invention provides a recombinant polypeptide comprising (or consisting essentially of):

(a) all or part of the amino acid sequence shown in SEQ ID No.2; or

(b) all or part of an amino acid sequence having at least 60% sequence identity to all or part of SEQ ID No. 2.

The term 'recombinant' distinguishes the aforementioned molecules from native polypeptides and emphasizes that such molecules have been produced using 'recombinant DNA techniques' (a term well known in the art). The enzyme with the amino acid sequence of SEQ ID No. 2 is considered to be a truncated form of the native enzyme in the northern prawn, so if the corresponding nucleic acid is used in recombinant production, then the recombinant enzyme produced will not in any way differ from the naturally occurring The same non-recombinant molecule.

In particular, the above-mentioned amino acid sequence may show at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identity to the polypeptide of SEQ ID No. 2. Alternatively, the above-mentioned amino acid sequence may show at least 70%, 75%, 80%, 85%, 90%, 95% or 98% similarity to the polypeptide of SEQ ID No. 2.

Specifically, the above-mentioned mutations may occur at the N-terminus. The polypeptides of the invention may also comprise an additional 4-20 amino acid residues, eg 5-10, at the N-terminus. It should also be noted that the incomplete cDNA sequence of SEQ ID No. 1 corresponds to the amino acid sequence of SEQ ID No. 2, but only starts at the 7th residue, aspartic acid. In recombinant polypeptides, amino acid variations, especially substitutions, may occur in these first 6 amino acids (see the sequence in Figure 2). Furthermore, the N-terminal lysine in Figure 2 can be replaced by arginine.

A cDNA sequence corresponding to the amino acid sequence of SEQ ID No. 2 can be produced, which consists of SEQ ID No. 1 and the derived nucleotide sequence at the 5' end. Most of this region is provided by the forward primer SEQ ID No. 7, which is a sequenced fragment derived from native shrimp alkaline phosphatase (SAP). Due to the degeneracy of the genetic code, it is impossible to determine a more accurate sequence than AArGCnTAyTGGAAyAAr [SEQ ID No. 19] based on the amino acid KAYWNK, where r=A or G, n=T, C, A or G, y=C or T. The cDNA full sequence corresponding to SEQ ID No.2 (including SEQ ID No.19 and SEQ ID No.1) is hereby classified as SEQ ID No.20.

However, sequencing work as performed in Example 4 has nearly established the complete cDNA sequence of the northern shrimp (Pandalus brealis) alkaline phosphatase. Thus, the above-discussed problem of uncertainty regarding SEQ ID No. 19 was resolved, and the cDNA encoding KAYWNK was as follows: AAAGCATATTGGAACAAA [SEQ ID No. 21]. This sequence can replace SEQ ID No.19 in SEQ ID No.15, together with the part of coding amino acid sequence NPITEED in SEQ ID No.17, generate an accurate cDNA sequence encoding the recombinase. The full sequence is assigned to SEQ ID No.22.

Amino acid sequence identity or similarity can be determined using the Bestfit program of the Genetics Computer Group (GCG) Version 10 software package at the University of Wisconsin. The program uses Smith and Waterman's local identity algorithm, and its default values are: Gap generation penalty value=8, Gap extension penalty value=2, average matching value=2.912, average mismatching value=-2.003.

The "part" of the amino acid sequence of SEQ ID No. 2 above or a variant thereof comprises at least 20 contiguous amino acids, preferably at least 30, 40, 50, 70, 100, 150, 200, 300, 400 or 450 contiguous amino acids . Portions having more than 200, especially more than 300 amino acids are considered to be 'significant portions', and these portions generally carry the active site of the enzyme identified in the present invention, preferably the binding site for the cofactor.

The recombinant polypeptide preferably has a functional activity as defined above, eg enzymatic activity. Or it does not have functional activity itself, but it provides a region with functional characteristics for the whole, for example, representing the active site or cofactor binding site required for enzymatic activity.

As discussed above, SEQ ID No. 1 and SEQ ID No. 2 do not correspond to the complete cDNA or amino acid sequence of native SAP. A truncated polypeptide having the same or substantially the same enzymatic activity as native SAP and retaining the heat sensitivity of the enzyme forms a further aspect of the invention. Complete cDNA molecules or recombinant polypeptides inserted into the truncated sequences described herein, or functionally equivalent variants thereof, are further preferred aspects of the invention.

Yet another aspect of the invention provides a polypeptide having the same or substantially the same catalytic activity and thermal properties as SAP but lacking its N-terminal region. Preferably these polypeptides lack the N-terminal amino acid and 2-50 amino acids, especially 5-30, such as 5-10, in and adjacent to the native enzyme. By 'substantially the same catalytic activity' is meant that the polypeptide possesses at least 75%, preferably at least 85%, more preferably at least 90% of the enzymatic activity of native SAP. In addition to these missing N-terminal parts, the polypeptide is preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identical to the polypeptide shown in SEQ ID No.2. identity.

According to the present invention, when the catalytic activity of alkaline phosphatase cannot be detected after incubation at 65° C. for 15 minutes (preferably 10 minutes), it can be considered as thermosensitive.

The applicable method for measuring the catalytic activity of the enzyme of the present invention is according to Olsen et al. [RL Olsen, K. and B. Myrnes (1991) Alkaline phosphatase from the hepatopancreas of shrimp (Pandalus borealis): a dimeric enzyme with catalytically active subunits ), Comp. Biochem. Physiol., 998:755-761]. Alkaline phosphatase activity was determined by incubation with 6 mM p-nitrophenyl phosphate at 37°C in 0.1 M glycine/NaOH buffer, pH 10.4, which also contained 1 mM MgCl ₂ and 1 mM ZnCl ₂ in a total volume of 0.5 ml. After 15 minutes, the reaction was terminated with 0.5 ml of 2M NaOH, and the amount of p-nitrobenzene released was determined by measuring the absorbance at 405 nm (ε ₄₀₅ =18.5 mM ⁻¹ cm ⁻¹ ). One unit of enzyme activity can produce 1 μmol p-nitrobenzene per minute. SAP was not adversely affected by zinc, nor did it require significant amounts of magnesium above the minimum concentration.

Also included within the scope of the invention are variants of SEQ ID No. 1 or SEQ ID No. 2 that encode or comprise proteins that retain and exhibit the catalytic properties of SAP, but are thermostable, that is to say they are stable at 70 After heating at ℃ for 15 minutes, the activity will not be completely lost.

The invention also provides a method for preparing a recombinant nucleic acid molecule of the invention, comprising inserting a nucleic acid molecule comprising a nucleotide sequence of the invention into another nucleic acid molecule, eg, a vector nucleic acid, such as vector DNA.

Therefore, the present invention also provides a recombinant vector, which is a carrier DNA carrying a DNA fragment, and the DNA fragment comprises any cDNA of the present invention.

In recombinant vectors, the vector DNA is preferably an expression vector and is capable of propagation in host cells such as Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

Expression vectors of the present invention may include suitable regulatory sequences, such as translational regulatory elements (e.g. initiation and termination codons, ribosome binding sites) and transcriptional regulatory elements (e.g. promoter-operator regions, terminal termination sequences) , these elements are linked in matching reading frame to the nucleic acid molecule of the invention.

The vectors of the present invention can include plasmids and viruses (including phage and eukaryotic viruses) known in the art and documented in the literature, and can be expressed in different expression systems, which are also known in the art and documented in the literature .

A number of techniques are known for introducing such vectors into prokaryotic or eukaryotic cells for expression, or into germline or somatic cells for the production of transgenic animals. Suitable transformation or transfection techniques are well described in the literature.

A genetic construct comprising a nucleic acid molecule of the invention described herein operably linked to a promoter sequence forms a further aspect of the invention.

The invention also includes transformed or transfected prokaryotic or eukaryotic host cells, or transgenic organisms comprising the above-mentioned nucleic acid molecules of the invention. For convenience, transformation and transfection are not distinguished here. Such host cells may be prokaryotic cells such as E. coli, Streptomyces and other bacteria, or eukaryotic cells such as yeast (e.g. Pichia pastoris), or the baculovirus-insect cell system, transformed mammalian cells and genetically modified plants and animals. Prokaryotic host cells are preferred here.

Therefore, the present invention also provides a transformant, which is a cell transformed with the recombinant vector of the present invention.

In another aspect, the present invention provides a recombinant cell or organism having a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID No. 1 or 20, or a functionally equivalent variant of the molecule as defined herein, and Expresses or is capable of expressing heat-sensitive alkaline phosphatase. A 'recombinant' cell or organism means that the cell or organism contains genetic material (in this case encoding heat-sensitive alkaline phosphatase) that is not naturally produced by the organism, so the northern spiny shrimp is excluded because it expresses heat-sensitive Alkaline phosphatase, but its gene is naturally present in this species.

From another point of view, those cells or organisms expressing heat-sensitive alkaline phosphatase can be regarded as "modified", that is, cells or organisms with genotypes and phenotypes that do not exist in nature. Accordingly, the cell or organism may also be referred to as a "non-natural" cell or organism because it contains non-natural genetic material and is capable of producing proteins not produced by natural members of the species or cell type (thermosensitive alkaline phosphatase ).

The above-mentioned cells and organisms can be referred to as transformants, and they or their progenitors have been transformed by introducing the genetic material encoding the heat-sensitive alkaline phosphatase described herein.

The present invention also provides another transformant, which is Escherichia coli or Saccharomyces cerevisiae transformed with the recombinant vector. Suitable expression strains of bacteria, fungi and other cultured cells are well known in the art.

The present invention also provides a method for producing heat-sensitive alkaline phosphatase, comprising culturing the transformant of the present invention in a culture medium, and isolating the heat-sensitive alkaline phosphatase from the cultured transformant. More specifically, the present invention also provides a method for producing thermosensitive alkaline phosphatase, comprising transforming host cells with an expression vector containing the following sequence: the nucleotide sequence shown in SEQ ID No.1 or 20, or at least A sequence with 55% identity, and/or a sequence capable of hybridizing to the complementary sequence of SEQ ID No.1 or 20 under moderate stringency conditions, or the complementary sequence of the above sequence; maintaining the host cell in a culture medium, and from The cells were isolated to express a thermosensitive alkaline phosphatase. Methods of culturing cells are well known in the art and generally allow the cells to propagate and simultaneously or subsequently express the ALP gene. Gene products can accumulate intracellularly or extracellularly. Methods for recovering and purifying proteins produced by this pathway are well known in the art.

We carried out further work and obtained more accurate sequence information. The corrected cDNA and protein sequences have little change from the original. The corrected cDNA sequence is shown in Figure 4 and named as SEQ ID No.15. In addition to adding an additional 5' sequence (its equivalent amino acid sequence appears in SEQ ID No.2), the new sequence also lacks 9 nucleotides, as shown in the sequence comparison of Figure 7. The comparison revealed that the sequences were 99.4% identical.

There are slightly larger changes at the protein level because the corrections cause changes in the reading frame in a small fraction of the protein. The corrected amino acid sequence is shown in Figure 5 and named as SEQ ID No.16. A comparison with the starting amino acid sequence is given in Figure 8 . This alignment revealed that the sequences were 96% identical.

Therefore, all previous references to SEQ ID No. 1 and 2 can be replaced by SEQ ID No. 15 and 16, respectively. These sequences include those preferred embodiments described herein.

In addition, the full-length cDNA sequence of SAP has been elucidated recently, so its entire molecule corresponds to SEQ ID No.17 and then to SEQ ID No.15, and the full-length amino acid sequence of SAP (plus signal sequence) corresponds to SEQ ID No. .18 followed by SEQ ID No.2 or 16. It is believed that among the 47 amino acids of SEQ ID No. 18, the last 7 amino acids (NPITEED) or several of them, for example, PITEED or TEED, are found at the N-terminus of the mature SAP protein. The remaining amino acids are considered signal peptides. Molecules having these sequences form a further aspect of the invention. In a preferred embodiment of the present invention, the recombinant alkaline phosphatase has the amino acid sequence NPITEED at its N-terminus.

The present invention will be further described with some non-limiting examples below, wherein:

Figure 1 shows the cDNA encoding SAP from Pandalus borealis (with a small portion of the 5' end removed). The stop codon and the downstream sequence of the cDNA including the polyA tail are underlined.

Figure 2 shows the amino acid sequence encoded by the SAP cDNA shown in Figure 1 (with an additional N-terminal region added). Portions homologous to sequenced protein fragments are underlined and noted. The active site sequence of the enzyme (based on known alkaline phosphatases) is shown in bold; and

Figure 3 shows the alignment of the SAP sequence with some highly homologous tissue-nonspecific ALPs found from the Swiss-Port database, including mouse (accession number AnP09242), human (AnP05186), chicken (AnQ92058), and ALP of Bombyx mori (AnP29523).

Figure 4 shows the corrected cDNA encoding the northern long SAP. The polyA tail is indicated as (A)23. Due to the use of degenerate PCR primers for amplification and sequencing, errors occurred in the 5 nucleotide positions of the 5' sequence, so these positions are not specified.

Fig. 5 shows the amino acid sequence encoded by the SAP cDNA shown in Fig. 4 . Sequences homologous to sequenced protein fragments are underlined and indicated. Proposed enzyme active site sequences are shown in bold. Due to errors introduced by degenerate PCR primers as discussed in Figure 4, the N-terminal lysine residue (lower case letters) may be replaced with arginine.

Figure 6 corresponds to Figure 3 above, but with the corrected amino acid sequence from Figure 5 added.

Figure 7 shows the alignment of the corrected cDNA sequence (top column) in Figure 4 with the starting sequence in Figure 1 (with the addition of the 5' end which originally only appeared in the equivalent amino acid sequence of Figure 2). Alignment is performed using the Needle program, applying the Needle-Wunsch global alignment algorithm to find the optimal alignment of two sequences (including gaps) [Needle et al. J. Mol. Biol. (1983) 48, pp443-453]. The matrix Blosum is 62, the penalty for open gaps is 10.0, and the penalty for gap extension values is 0.5. Overall percent identity 99.4%.

Figure 8 shows the alignment of the corrected amino acid sequence (top column) in Figure 5 with the starting amino acid sequence in Figure 2. Alignments were performed using the Needle program described above, with a matrix Blosum of 62, a penalty of 10.0 for open gaps, and a penalty of 0.5 for gap extension values. Overall percent identity 96.3%.

Figure 9 shows the deduced signal sequence and the cDNA [SEQ ID No. 17] and amino acid [SEQ ID No. 18] sequences of the N-terminal region of northern long-fronted shrimp alkaline phosphatase. The SAP amino acid sequence is followed by KAYWNK... as depicted in Figures 2 and 4 .

Figure 10 is a photograph showing the SDS-PAGE of the total cell protein extract of the expression culture in Example 4, wherein: swimming lanes 1 and 10: wide-area standard protein, swimming lanes 2 and 9: SAP 1.5 μg, swimming lane 3: Negative control culture, not induced, lane 4: negative control culture, induced, lane 5: SAP-culture 1, not induced, lane 6: SAP-culture 1, induced, lane 7: SAP-culture 2, not induced, lane 8: SAP-culture 2, induced. The proteins in the wide-area standard protein are: myosin (200kD), β-galactosidase (116kD), phosphorylase b (97.4kD), serum albumin (66kD), ovalbumin (45kD), carbonic acid Anhydrase (31kD), trypsin inhibitor (21.5kD), lysozyme (14.4kD), aprotinin (6.5kD).

Figure 11 is a photograph showing the immunoblotting of total cell protein extracts expressed in Example 4, wherein: Lane 1 and Lane 8: SAP 50ng, Lane 2: negative control culture without induction, Lane 3: Negative control culture, induced, lane 4: SAP-culture 1, not induced, lane 5: SAP-culture 1, induced, lane 6: SAP-culture 2, not induced, lane 7 : SAP-culture 2, induced.

Example

The sequencing of embodiment 1-SAP protein and the separation of SAP gene

Protein Isolation, Fragmentation and Sequence Analysis

Shrimp alkaline phosphatase (SAP) was purified to homogeneity as described [R. Olsen et al.,]. After electrophoresis in 10% NuPAGE Bis-Tris gel system (Novex) with 50mM 2-(N-morpholine)ethanesulfonic acid electrophoresis buffer containing 0.1% SDS, a single A protein band of about 55kD.

1 mg of protein was lyophilized and submitted to a commercial sequence analysis service (Innovagen, Sweden). There, proteins were trypsinized into fragments, which were separated by reverse-phase HPLC. Over 30 fragments were generated, 4 of which were selected for further analysis. Sequences of selected fragments (H23:18, H23:30, 5ReRP6:26, 5ReRP6:17) were analyzed by mass spectrometry, yielding sequences of 12, 10, 8 and 5 amino acids, respectively.

Synthesis of oligonucleotides from protein fragment sequences

The standard codons were predicted according to the obtained amino acid sequence, and then a forward degenerate oligodeoxyribonucleotide primer and its reverse complementary sequence (Euggentec, Belgium) were made conventionally.

Synthesis of Shrimp cDNA

Freshly harvested northern prawns were dissected and individual hepatopancreas were stored in liquid nitrogen for later use. mRNA was isolated from individual hepatopancreas using PolyATract System 1000 (Promega). Isolated mRNA was used for first-strand cDNA synthesis prior to second-strand synthesis, and cDNA was amplified according to the Smart ^TM PCR cDNA Synthesis Kit (Clontech) and Advantage cDNA Synthesis Kit (Clontech) given Instructions to execute.

PCR Amplification and Sequence Analysis of SAP-gene Internal Fragment

PCR was performed using a small aliquot of the synthesized cDNA as a template and the aforementioned pairs of protein-derived oligonucleotides (forward and reverse) as primers. Amplification reactions with standard mixture compositions were performed in an Eppendorf gradient thermal cycler. PCR products were detected by agarose gel electrophoresis. The primer combination of oligonucleotides 17F and 26R yielded a 600 bp amplification product, which was sequenced using the ThermoSequenase radiolabelled terminator cycle Sequencing Kit (Amersham) and using the same PCR primers.

Rapid amplification of cDNA ends (RACE)

Based on the DNA sequence found in the PCR product, new specific forward and reverse primers were synthesized for 5' and 3' RACE reactions, and the method was described for amplification from a cDNA template using the Marathon ^TM cDNA amplification kit (Clontech) Same.

A 1.4 kb 3'-RACE fragment was generated using the SAP gene-specific forward primer MalpF and the AP1 primer contained in the kit, wherein the AP1 primer has sequence identity to the ligated adapter. Similarly, a 0.5 kb 5' RACE product was obtained using the reverse gene-specific primer MalpR. DNA sequence analysis of the 3' RACE product confirmed that it overlapped with the 600 bp PCR product. The 3' RACE product was subcloned into the pCR-Script vector (Stratagene) to prepare the pMalpF plasmid for further sequencing of the cDNA gene downstream of the sequence region contained in the 600bp PCR product.

The 600 bp PCR product and the pMalpF insert were sequenced multiple times in both directions with new gene-specific primers.

High-fidelity PCR amplification of complete cDNA genes

With primers SapF and SapR, Pfu polymerase (Stratagene), carry out final PCR reaction with cDNA as template, produce the SAP cDNA gene amplification product of 1.5kb. The 1.5 kb PCR product was analyzed to confirm the resulting sequence. As a result, it was found that the cDNA gene contained an open reading frame and a 3' sequence, the open reading frame encoded a polypeptide of 478 amino acids-see Figures 1 and 2, and the 3' sequence included a putative transcriptional polyadenylation signal.

Thermal cycling characteristics used in PCR

The following cycle properties are used for amplification reactions:

-Total cDNA amplification 1x: 95°C, 2 minutes

16x: 95°C, 5 seconds

65°C, 5 seconds

68℃, 6 minutes

-Internal 600bp fragment PCR 1x: 94°C, 2 minutes

36x: 94°C, 10 seconds

51°C, 10 seconds

72 ℃, 1 point

1x: 72°C, 5 minutes

-5’RACE 1x: 94°C, 30 seconds

5x: 94°C, 5 seconds

                                                                              

1x: 94°C, 30 seconds

5x: 94°C, 5 seconds

70℃, 4 minutes

1x: 94°C, 30 seconds

23x: 94°C, 5 seconds

68℃, 4 minutes

-3’RACE 1x: 94°C, 30 seconds

36x: 94°C, 5 seconds

68℃, 4 minutes

-High-fidelity PCR 1x: 94°C, 3 minutes

40x: 94°C, 10 seconds

55°C, 15 seconds

                                                                                                                                                          

1x: 72°C, 5 minutes

result

The SAP protein subunit was originally estimated to have a molecular weight of 65 kDa. A 59kDa SAP protein is described in the product table of USB. In the inventor's SDS-PAGE system, the SAP protein has a similar migration pattern to the standard protein with a molecular weight of 55kDa. These differences can be explained by the diversity of the different buffer systems and the quality of the gels used.

The isolated cDNA encoding a polypeptide having sequence identity with the analyzed SAP protein fragment is shown in FIG. 2 .

The SAP protein sequence was used as the query sequence for homology searches in public databases. Homology searches and multiple sequence alignments were performed using BLAST and ClustalW programs, respectively. The SAP sequence was aligned with its highest scoring homologues, the tissue-nonspecific ALP found in mouse (Accession No. P09242), human (A.n.P05186), chicken (A.n.Q92058) and the ALP of Bombyx mori ( A.n. P29523). See Figure 3 for a comparison arrangement. No equivalent sequence of the resulting cDNA was found by homology searches in public databases. However, the derived protein sequences had relatively high homology scores with some known ALPs, with 44% identity and 60% similarity in amino acid sequence. Protein homologies were searched in the SWALL database (non-repetitive protein sequence database; Swissport+Trembl+TremblNew) on the Internet server of the European Bioinforrnatics Institute by running the FastA3 program with default parameters (blosun50 matrix, gap opening penalty value =-12, gap extension penalty value=-2, KTUP=2). [W.R.Pearson and D.J.Lipman (1988), Improved tools for biological sequence analysis ("Improved tools for biological sequence analysis"), PANS 85: 2444-2448; W.R.Pearson, (1990), fast and sensitive Sequence comparison ("Rapid and sensitive sequence comparison with FASTP and FASTA"), Methods in Enzymology 183:63-98.]

Also, the cDNA-encoded protein contains the motif VTDSAASAT, which corresponds to the active site pattern of ALP defined by Prosite PSOO 123 in this sequence. Therefore, the isolated cDNA from the mRNA-transcript of the shrimp hepatopancreas represents the SAP gene of the northern shrimp (P. borealis).

There was one amino acid discrepancy between the sequenced protein fragment 5ReRP6:26 and the cDNA-derived sequence. It can be explained by allelic variation in the SAP gene or an error in protein sequence analysis.

Sequence used in Example 1

Protein fragments :

H23: 18 DINFRYASAAP(V)K[SEQ ID NO.3]

H23: 30 HLITDWLDDK[SEQ ID NO.4]

5ReRP6: 26 VIMGGERR[SEQ ID NO.5]

5ReRP6: 17 AYWNK[SEQ ID NO.6]

Oligonucleotides :

17F: GCIIAYTGGAAYAAR [SEQ ID NO.7]

26R: CKYTCICCICCCATDATIAC [SEQ ID NO.8]

                where D = A + G + T

                   I = Inosine

K=G+T

R＝A+G

Y＝C+T

MalpF: ATTTCGTGGGAAGAATTCGACTTTGC [SEQ ID NO.9]

MalpR: GATCTGCCAGCCTCCTGGAACCA [SEQ ID NO.10]

SapF: AARGCNTAYTGGAAYAARGAT [SEQ ID NO.11]

SapR: GAAGGTAATCATCTACATCTCCA [SEQ ID NO.12]

Repeat Example 1 to make the sequence information more accurate. As shown in Figures 4 and 5, the cDNA gene contains an open reading frame encoding a protein of 475 amino acids. The theoretical molecular weight of the cDNA in Fig. 4 is 53 kDa, and the result is compared with the molecular weight estimated value of 54-55 kDa of the purified native SAP on SDS-PAGE. According to the results of multiple sequence alignment, SAP contains 5-10 additional amino acids at its N-terminus, making its molecular weight close to 54kDa. As discussed earlier, this N-terminal region is believed to contain part or all of the heptapeptide NPITEED, see Example 4.

Example 2 - Amino acid composition of cDNA-derived protein sequences compared to native SAP

The lyophilized SAP protein was dissolved in 6M hydrochloric acid and hydrolyzed at 110°C for 24 hours. After evaporation of the hydrochloric acid, resuspended samples were resuspended in 0.2M sodium citrate buffer, pH 2.2, and subjected to HPLC analysis to identify and determine the molar percentages of the amino acids in the hydrolyzate.

NOTE: This system cannot differentiate between aspartate and asparagine or glutamate and glutamine. Therefore, the data for these amino acids in native SAP proteins are mixed. In addition, the number of cysteine residues may also be underestimated because the protein is not oxidized.

%

In cDNA-derived protein In native SAP

Alanine 8.3 9.6

Aspartic Acid + Asparagine (13.7) 13.6

Aspartic Acid 10.0 -

Asparagine 3.7 -

Glutamic Acid + Glutamine (9.0) 11.7

Glutamic acid 6.7 -

Glutamine 2.3 -

Cysteine 1.0 0.2

Phenylalanine 4.1 4.1

Glycine 8.4 8.4

Histidine 3.7 2.8

Isoleucine 5.2 5.4

Lysine 4.6 4.2

Leucine 7.3 8.0

Methionine 1.6 1.9

Proline 2.7 3.1

Arginine 5.2 5.2

Serine 4.6 4.6

Threonine 8.7 8.5

Valine 5.4 4.9

Tryptophan 1.6 -

Tyrosine 4.1 3.6

Conclusions: For most amino acids, the molar ratios of each amino acid in the cDNA-derived protein sequence are nearly the same as in the purified native protein. Significantly smaller differences are discussed above.

Therefore, compared with the SAP protein, the protein encoded by the isolated cDNA has a very similar or completely identical amino acid composition.

This example and analysis were repeated on the corrected cDNA sequence with the following results:

Amino acid composition of cDNA-derived protein sequences compared to native SAP.

%

In cDNA-derived protein In native SAP

Alanine 8.8 9.6

Aspartic Acid + Asparagine - 13.6

Aspartic Acid 10.1 -

Asparagine 3.4 -

Glutamic Acid + Glutamine - 11.7

Glutamic acid 7.1 -

Glutamine 2.3 -

Cysteine 0.8 0.2

Phenylalanine 4.0 4.1

Glycine 8.0 8.4

Histidine 3.3 2.8

Isoleucine 5.4 5.4

Lysine 4.6 4.2

Leucine 7.6 8.0

Methionine 2.1 1.9

Proline 2.7 3.1

Arginine 4.8 5.2

Serine 4.6 4.6

Threonine 9.0 8.5

Valine 5.4 4.9

Tryptophan 1.5 -

Tyrosine 4.0 3.6

Recombinant expression of embodiment 3-SAP in Pichia pastoris (Pichia pastoris)

The cDNA encoding SAP was used to express the enzyme heterologously in a suitable microorganism for the production of the recombinant enzyme by fermentation. Several expression systems are available, of which the eukaryotic expression system is preferred because it expresses comparable levels of the enzyme in its native state.

An example of such an expression system is the methylotrophic Pichia pastoris (Invitrogen), which has been reported to express recombinant proteins up to 12 g/L in cells [Clare, J.J. Raiyment, F.B., Ballantine, S.P., Sreekrishna, K. and Romans, M.A. (1991): High-level expression of tetanus toxin fragment c in Pichia pastoris strains containing multiple tandem integrations of the gene.Blo/Technology 9, 455-460], the highest level of protein secreted into the medium Up to 2.6g/L [Paifer, E., Margolles, B., Cremata, J., Montesino.R., Herrera, L. and Delgado, JM. (1994): Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences. Yeast 10.1416-1419].

The inserted gene is regulated by the AOX1 (alcohol oxidase) promoter, so expression is inducible by methanol.

The SAP cDNA was inserted into the commercial vector pPIC9K. Recombinant SAP is expressed as a secreted product by fusion in the vector to the signal peptide sequence of S. cerevisiae alpha factor. After integration into his- strains (KM71 or GS115), recombinant strains can be selected by selecting his+ clones in media without histidine, because the his genotype is complemented by the vector insert. In addition, multigene inserts in recombinants can also be screened for resistance to increased concentrations of geneticin (G418, Invitrogen), where the resistance is derived from the kanamycin resistance gene carried by the plasmid.

Specifically, the SAP cDNA generated by PCR was used as a template for a new round of PCR reactions to generate PCR products containing suitable restriction sites that enable integration of the product into the vector pPIC9K, with α The factor signal sequences are in the same reading frame. As a forward primer for the PCR reaction, an improved "17F" primer [SEQ ID No.13] was used, in which the ATG codon (Met) was replaced with a lysine codon (AAG) in order to recreate a trypsin-digestible Putative lysine residue. The reverse primer [SEQ.ID.No.14] was constructed to add a NotI restriction site at the 3' end of the stop codon of the SAP cDNA sequence.

The resulting PCR product was digested with NotI and then ligated into the pPIC9K vector which had been digested with SnaBI and NotI (thus generating plasmid pPIC9K-SAP). Because the junction site at the 5' end of the vector is blunt after SnaBI digestion, and both ends are in the reading frame, it is not necessary to modify the 5' end of the modified cDNA.

Then use the vector to transform a competent E. coli strain such as TOP10F' (Invitrogen) or an equivalent strain, propagate it, and select the recombinant with resistance to ampicillin. The isolated pP109K-SAP plasmid can then be restricted and linearized with ScoI to generate a vector cassette that can be inserted into the Pichia pastoris AOXI gene. After transformation of the linearized pPIC9K-SAP construct into Pichia pastoris cells (strain GS115 or KM71), recombinants were screened from His+ mutants according to the manufacturer's protocol.

The cells were then cultured in the presence of methanol using the method recommended by the manufacturer to detect the expression of recombinant SAP in the culture medium from isolated colonies. If necessary, clones containing multiple copies of the recombinant gene can also be screened from the recombinant strains to obtain a further increased expression level of the enzyme. This was achieved by culturing His+ mutants in the environment with increasing levels of the antibiotic G418 (Invitrogen). Clones growing in high G418 levels are likely to contain multiple copies of the gene.

All methods of cultivation, transformation, and screening were described in detail in the protocol provided by the supplier of the Pichia pastoris system (Invitrogen, The Netherlands).

A modified SAP construct produced a recombinant product in which 5 amino acids (EAEAY) from the remaining signal recognition sequence of the Pichia pastoris signal peptidase KEX2 were added. This N-terminal sequence results in a slightly truncated final product, but has considerable commonality with other alkaline phosphatases and still possesses the Pichia pastoris signal peptidase recognition sequence.

Primers used in Example 3

Forward:

AAGGCITAYTGGAAYAAR[SEQ.ID No.13]

in

I = inosine

R＝A+G

Y＝C+T

Reverse:

TACAGCGGCCGCCATCTCATTTTTCG[SEQ.ID No.14]

Expression of SAP in Example 4-Escherichia coli TOP10

Determination of the SAP signal sequence

The N-terminal signal sequence of SAP was determined by 5'-RACE using cDNA as a template. mRNA was isolated from 100 mg of shrimp hepatopancreas using the Oligotex Direct mRNA Mini Kit (Qiogen) following the manufacturer's protocol. Shrimp hepatopancreas cDNA was prepared using the SMART PCR cDNA Synthesis Kit (Clontech) following the manufacturer's protocol. The cDNA is blunt ended, by adding 40μg proteinase K to 50μl cDNA, incubating at 45°C for 45 minutes and 90°C for 8 minutes, then adding 15U T4-DNA polymerase (Promega), incubating at 16°C for 30 minutes and incubating at 72°C 10 minutes for purification. Finally, cDNA was extracted twice in phenol/chloroform and precipitated in ethanol. The cDNA was then ligated with RACE-adaptors as described in the Marathon cDNA Amplification Kit (Clontech). The 5'-RACE reaction was carried out as follows: in a final volume of 50 μl, with Advantage cDNA polymerase mix (Clontech), AP1-primer (provided from Marathon cDNA amplification kit), SAP-specific primer (5'-GCG TGG TGC ATA TGG TCA ATC CGTCC-3') and 5 μl of 50-fold diluted cDNA templates ligated with adapters were reacted. RACEPCR-reactions were performed in a Biometra TGradient thermal cycler with an initial denaturation step of 94°C for 30 seconds, followed by 5 cycles of 94°C for 5 seconds and 72°C for 3 minutes, and 5 cycles of 94°C for 5 seconds and 72°C for 3 minutes, Cycle 30 times at 94°C for 5 seconds and 68°C for 3 minutes. The resulting 1,200 bp SAP fragment was purified by agarose gel, reamplified by PCR and sequenced.

Cloning of SAP into pBAD/gIII A expression vector

In order to clone the SAP gene into the pBAD/gIIIA vector, the SAP gene was amplified by PCR with two primers containing SacI and HindIII sites (underlined) respectively. According to the obtained full-length SAP sequence (including main sequence and signal sequence), primers are designed to amplify the SAP gene whose N-terminus is NPITEEDKAYWNK... amino acid sequence. In addition, three bases in primer 1 were changed (C to A, A to C, and A to C) so that the three N-terminal codons were codon optimized (indicated in lowercase letters in the primer sequence) (Primer 1: 5'-ATG GAGCTC AAC CCa ATc ACc GAA GAA GAC AA-3' Primer 2: 5'-ATG AAG CTTTCATTT TTC GTC ACA GAAAGT G-3'). PCR reactions were performed as follows: in a final volume of 50 μl, containing 10 μM each of the two primers, 1.5 U of Pfu polymerase (Promega) with buffer provided, 0.2 mM dNTPs, and 10 ng of shrimp hepatopancreas cDNA as template. The PCR-initial denaturation step was 94°C for 30 seconds, followed by 30 cycles of 94°C for 15 seconds, 55°C for 1 minute, and 72°C for 3 minutes. PCR products were purified using Qiaquick PCR purification kit (Qiagen).

Both the PBAD/gIII A vector and the SAP PCR product were treated with 5U of SacI and HindIII restriction enzymes, respectively. In a 10μl reaction system, add 1U T4-DNA ligase (USB) and about 1mg of plasmid and SAP PCR products respectively to ligate the PCR products into the vector. The reaction mixture was incubated at 16°C for 16 hours. Then, 40 μl of the electrocompetent E. coli TOP10 cells were added with the ligation mixture, and electroporated at 1,800 V to transform the E. coli TOP10 cells with the ligation mixture. Immediately after electroporation, 1 ml of SOC-medium was added and incubated at 37°C for 1 hour. Cells were then plated on LB plates containing 100 mg/ml ampicillin to select for transformed cells.

Expression of SAP in Escherichia coli TOP10

Two E. coli TOP10 colonies containing the recombinant pBAD/gIII A vector (containing the SAP gene) were selected for expression. Escherichia coli TOP10 containing pBAD/gIII A empty vector was used as a negative control.

Inoculate 2.5 ml of an overnight culture in LB medium containing 100 μg/ml ampicillin in three Erlenmeyer flasks containing 50 ml of LB medium. The cells were cultured with shaking at 30°C until the cell concentration reached OD600≈0.5. Each culture was then divided into two (2 x 25 ml), one of which was induced by adding L-arabinose to a final concentration of 0.05% (w/v). Thereafter, all cell cultures were cultured with shaking at 30°C for 3 hours until the cells were recovered.

Analysis of expression cultures

200 μl of each expression culture was centrifuged, and the supernatant was discarded. Add 100μl of 1X SDS-PAGE sample buffer to the cell pellet, incubate at 100°C for 3 minutes, then centrifuge at 18,000g for 3 minutes. The cell lysates were then directly analyzed by SDS-PAGE gel electrophoresis. An additional SDS-PAGE gel was electroblotted to allow the protein to be transferred to a nitrocellulose blot (0.45um, Bio-Rad) and then treated with an antibody against SAP purified from shrimp processing wastewater and goat anti-rabbit IgG (H+L) (human IgG absorbed) horseradish peroxidase conjugate (Bio-Rad) was used for immunostaining. SDS-PAGE, electroblotting and immunostaining were all performed according to standard protocols.

As shown in Figures 10 and 11, recombinant SAP was expressed with the pBAD/gIII A vector using Escherichia coli TOP10 cells as a host. Both recombinant E. coli strains containing the recombinant pBAD/gIII A-SAP vector produced recombinant SAP, whereas the negative control gave no signal in western blot (Figure 11).

Claims (24)

1. A nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID No 1 or 20, or a nucleotide sequence having at least 55% sequence identity with SEQ ID No 1 or 20, and/or capable of combining with SEQ ID No 1 or 20 The complementary sequence of ID No 1 or 2 is a nucleotide sequence that hybridizes under medium stringency conditions, or the complementary sequence of said sequence, wherein said nucleotide sequence encodes or is complementary to a thermolabile alkali Sequence of phosphatase. 2. The nucleic acid molecule of claim 1, comprising a nucleotide sequence with at least 75% sequence identity to the nucleotide sequence of SEQ ID No 1 or 20. 3. The nucleic acid molecule of claim 1 or 2, comprising a nucleotide sequence with at least 85% sequence identity to the nucleotide sequence of SEQ ID No 1 or 20. 4. An isolated nucleic acid molecule encoding or complementary to a sequence encoding a thermolabile alkaline phosphatase comprising the amino acid sequence of SEQ ID No.2 or at least 50% identical to the amino acid sequence Source amino acid sequence variants. 5. The nucleic acid molecule of claim 3, wherein said alkaline phosphatase has an amino acid sequence at least 70% identical to SEQ ID No.2. 6. The nucleic acid molecule of claim 3, wherein said alkaline phosphatase has an amino acid sequence at least 80% identical to SEQ ID No.2. 7. The nucleic acid molecule of any preceding claim, wherein the alkaline phosphatase is northern shrimp alkaline phosphatase. 8. A genetic construct comprising the nucleic acid molecule of any one of claims 1-7 operably linked to a promoter sequence. 9. A recombinant expression vector comprising the nucleic acid molecule according to any one of claims 1-7 and capable of propagating in host cells. 10. The vector of claim 9, which is a plasmid or a viral vector. 11. A cell transformed with the construct or vector of any one of claims 9-11. 12. The cell of claim 11 which is a prokaryotic cell. 13. The cell of claim 11 which is part of a eukaryotic cell culture. 14. A recombinant cell or organism having a nucleic acid molecule comprising a nucleotide sequence as defined in any one of claims 1-3. 15. A recombinant thermolabile alkaline phosphatase, comprising: a) all or a major part of the amino acid sequence shown in SEQ ID No.2; or b) All or a substantial portion of an amino acid sequence at least 60% identical to SEQ ID No.2. 16. The recombinant alkaline phosphatase of claim 15, having the amino acid sequence shown in SEQ ID No.2 or the amino acid sequence having at least 75% sequence identity with the sequence of SEQ ID No.2. 17. The recombinant alkaline phosphatase according to claim 15 or 16, which is northern shrimp alkaline phosphatase. 18. A recombinant cell or organism capable of expressing a thermolabile alkaline phosphatase comprising the amino acid sequence of SEQ ID No. 2 or an amino acid sequence having at least 60% sequence identity to SEQ ID No. 2. 19. A recombinant cell or organism capable of expressing a thermolabile alkaline phosphatase comprising the amino acid sequence of SEQ ID No. 2 or an amino acid sequence having at least 75% sequence identity to SEQ ID No. 2. 20. A method for preparing thermolabile alkaline phosphatase, comprising transforming host cells with the expression vector of claim 9, culturing the cells in a culture medium, and isolating the thermolabile alkaline phosphatase expressed by the cells. 21. A nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID No.15, or a nucleotide sequence having at least 55% sequence identity with SEQ ID No.15, and/or capable of combining with SEQ ID No. The complementary sequence of 15 is a nucleotide sequence that hybridizes under medium stringency conditions, or the complementary sequence of said sequence, wherein said nucleotide sequence encodes or is complementary to a sequence encoding thermolabile alkaline phosphatase. 22. A recombinant thermolabile alkaline phosphatase comprising: a) all or a major part of the amino acid sequence shown in SEQ ID No.16; or b) All or a substantial portion of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID No.16. 23. The recombinant alkaline phosphatase of claim 22, having the amino acid sequence shown in SEQ ID No.16 or an amino acid sequence having at least 75% sequence identity with SEQ ID No.16. 24. A method of preparing the expression vector of claim.

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