JPH03505276A - Rearranged tissue plasminogen activator and its production method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Rearranged tissue plasminogen activator and its production method Background of the invention The present invention relates to the use of recombinant DNA technology to produce therapeutic proteins. Especially the novel modified human uterine tissue plasminogen activator (+lP^) gene. A gene and a plasmid containing the gene; a gene and a plasmid containing the gene; transformed or transfected (Htnslccted ) host cells and the plasminogen activator molecules produced therefrom. relates to the use of recombinant DNA technology for the purpose of

Tissue plasminogen activator (zPA) is a plasminogen activator It is a multidomain serine protease that catalyzes the conversion to itself, IPA has therapeutic value. When administered externally, IPA can cause blood clot lysis (thrombolysis). solution). (Clinical trials have shown that these two are effective in treating myocardial infarction.) It has been made clear. Indications tested include pulmonary embolism, severe venous thrombosis and cerebral thrombosis .

The FPA molecule has five distinct structural domains. Single-chain IP^ or zymogen The enzyme is activated in the presence of plasmin and cleaves into double-stranded form. The stalwarts are above "Finger" homologous to a part of fibronectin among the five domains mentioned above domain, a “growth factor” domain homologous to epidermal growth factor, and two different “growth factor” domains. Contains four regions of the ringle (k+ingle) J domain. Double chain IPA Plasmin cleavage to form occurs at the C-terminus of kringle 2 (arginine 2), Ru. This light chain is a serine protease homologous to trypsin and chymotrypsin. Contains the main.

IPA binds to blood clots due to its affinity for fibrin, which creates the clot matrix. It is a plasminogen activator with relatively high specificity for. This frame Fibrin affinity is due to the interaction between the finger and kringle 2 domains and fibrin. It is thought that this is due to usage. Lower resistance to fibrin with Kringle 1 Not much is known about their strong affinity.

One aspect of the invention is that high fibrin specificity, which is highly fibrin-specific than the wild-type IPA molecule. It is the production of lPA that achieves the elementary lysis phenomenon.

One aspect of the invention is to enhance the rate of fibrinolytic phenomena or to be present in human plasma. IPA domain to increase resistance to inhibition by endogenous IPA inhibitors. It is an object of the present invention to provide a method for widening the spacing between the ins.

1°PA secreted by human melanoma was refined by Rijksn et al. [Journal of Biological Chemistry] (J, Biol, Cbe+e,) Volume 256, Page 7035 (1982 ) The therapeutic utility of exogenous + PA has been demonstrated using melanoma-derived material. [Journal of Clinical Research by Collen et al. tion (J, Cl1n, Inv, ), vol. 71, p. 368 (191 2013), Journal of Ku by Kollinger (Ko+inget) et al. Linical Improvement, Volume 69, Page 573 (1982). black color There have been reports regarding the differences between IPA derived from tumor and IPA derived from normal uterine tissue. [FEBS Letters by Ball (POII+) et al., Vol. 168, No. 2] 9 (1984)].

Biocbem, by Hiken et al. Biopb7s, Ac1z, ), Volume 580, $140 page ( (1979), human tissue plasminogen activator (u A partial purification of lPA) has been described.

Described in European Patent Application No. 0093619 by Gocdel et al. As previously described, the cDNA encoding Bates IPA was recombinant to obtain m1lNA used to produce A from one line of cancer cells. DNA technology has been used. U.S., fully incorporated herein by reference. Pending U.S. Patent Application No. 7112, 686, assigned to Let et al. The issue describes the Dll^ sequence encoding iPA, and furthermore, the mammalian Encodes amino acids that are normally glycosylated in post-translational processing steps by living cells. [For site-directed mutagenesis of lNA sequences] It is described as follows. The produced modified l with the converted amino acid sequence The I'A molecule does not show glycosylation at the mutated site. About this research DNA by Wei et al., Volume 4, Page 76 (1985) and European Patent Application No. It is also reported in No. 178.105.

The expression vector for the expression of secreted tPA in mouse cells was then ready ( Journal of Cell Biochemistry (J, Ce1l Biocbe+a, ), Volume 100, Page 154 (1986) has been reported. European patent issued by Ptnnnekoek et al. Application No. 0.234.051 has rearranged domains but remains unchanged. IPA molecules with long light chains are discussed. Bowes melanoma cells are It was used as a source of IPA for research.

However, the application does not address the understanding necessary to design the actual production of IPA variants. Although it is described that the method provides the desired +lPA, the black does not provide a reproducible and predictable method for modifying cDNA from tumor cells.

Another aspect of the invention is that fPA cDNA is desired for producing desired m1PAs. It is an object of the present invention to provide a new method that predictably guarantees that changes will occur.

In European Patent Application No. 0,231.624 by MsroNi et al. are analogs of other human tissue plasminogens with rearranged or deleted natural domains. The body is described. The application states that oligonucleotides are completely chemically Complex and time-consuming process for producing tPA cDNA identified by synthesis Such methods have been described.

Moreover, its synthesis is based on natural IPA derived from human melanoma cells (Bows cells). Ru.

Another aspect of the invention is that ulPA-based IPA-like molecules have predictable domains. The objective is to provide a simpler and more direct method of reordering the ins.

Another aspect of the invention provides for specific restriction endonucleases at predetermined locations. A specific cDNA sequence encoding an IPA-like molecule with an enzyme site is provided, and the and to supply novel molecules that can be produced from it.

Other aspects of the invention provide biological activities associated with tissue plasminogen activator. The object of the present invention is to provide a new method for producing novel molecules having the following properties.

Summary of the invention In accordance with the principles and objectives of the present invention, domain deletions, rearrangements or duplications mfPA produced from the A molecule is supplied. Things that have been modified in this way become wild. still produce molecules with the biological activity associated with unmodified or unmodified IPA. It's amazing what it accomplishes. Some of these novel m1PAs are their The superiority of the parent molecule in terms of fibrinolysis is completely unexpected. It was.

A preferred method of the invention involves adding at least two restriction enzyme sites to the IPA cDNA. Including introducing. These sites may be located elsewhere, but the IPA protein It is logical that it is located at a location in the IPA cDNA that corresponds to the border of white matter domains. It's imaginative. The precise location of these sites preserves the desired activity of the parent IPA molecule. This is critical for this purpose. The most preferred embodiment of the invention is that one Avt II restriction site, one Nhe I restriction site, one Spe I restriction site , and introduces an Xba I restriction site. The preferred amino acid sequences to be produced are shown in Table 3. As stated in.

The above modified cDNA is then modified to contain the deletion or duplication of IPA as shown in Table 4. can be advantageously manipulated to occur.

These manipulations are described in additional detail below and are generally applicable to modified cDNAs. The resulting cDNA fragments are ligated to create a novel preferred cDNA. including forming cDNA.

The most preferred embodiment is to obtain molecules with extended half-lives in vivo, e.g. The above modified product for the purpose of converting asparagine to glutamine at Tide 451 has been combined with other modified ones.

Another preferred embodiment of the mutant invention is a parent organism for maintaining and replicating the sequence; Expression vector for expression of mjPA in CO3 cells, Cl27 cells, CIO cells and the mfPA protein derived from this expression system.

Brief description of the drawing A better understanding of the principles and objects of the invention may be gained by viewing the accompanying drawings.

Figure 1 shows the Avt II, Nhe I and Spe I sites in IPA cDNA. It indicates the location.

FIG. 2 is a schematic diagram of the structure of a vector expressing IPA with a deletion.

Figure 3 depicts the LK444BHS vector for temporary expression of mfPA. It is.

Figure 4 shows CLH3AXS2DHF for stable expression of mtPA in CHO cells. This represents the R vector.

Figure 5 shows CL)13AXBPV for stable expression of mtPA in Cl27 cells. It represents a vector.

Figure 6 is a schematic diagram of the construction of a vector containing m1PA with two duplications. Ru.

Figures 7a-1 and 7a-2 show SP, SN containing the υ11 rearranged mlP^ sequence. ^ Demonstrates vector construction.

Figures 7b-1 and 7b-2 show SP, υLT vectors containing the ULT rearranged 1P^ sequence. shows the construction of a vector.

The term "cell culture" refers to cells that are allowed to continue growing outside of their original plant or animal. refers to the containment of growing cells derived from either multicellular plants or animals. .

"Host cell" means a cell capable of being grown in cell culture and possessing IPA biological properties and molecules. transfected with a plasmid or vector containing a gene encoding a molecule with expression. Including infected or transformed yeast, bacterial and mammalian cells. microorganisms in the sense of

"Domain" refers to an independent continuous portion of an amino acid sequence that can have a specific function. . Regarding IPA, the literature [Common by BanHi, L. et al. ・Evolutionary Origin of the Fibrin-Pinding・ Thickness of fibronectin and tissue type plastic Sminogen activator (Common cvolultonxB) rigin of the Iib+in-binding sl+uefu+ ts of lib+oneclin xnd+ixsut-17peplx smfnogen *clivxlo+), FEBS Letters, Volume 163, No. 1 No. 37-41 (1983) and the book by Nye (NY), T., et al. Structurature of the human tissue type plasminogen Intron Activator Gene: Relation of Intron and ・Exxon Structural to Functional and Structure Natural Domains (The structure ol the) Iumtn Ti5sue-17pe Plssminolen＾cliytlo+ Ge ne: Co+relxlion oj l1ron xnd ErxonSt structures to Functional xnd 5luclursl Domsins), Proceedings of the National Academy e of Sciences (Proceedings ol the Ntjio+ slAcldem7 oI Sie’nces)] defines the domain area. The suitable locations of the domain regions are also shown in FIG.

The term "downstream" refers to sequences further in the direction of expression. For example, the code area The region is downstream of the start codon.

The term "interdomain" refers to the amino acid sequences of proteins that exist between domains. means.

The term "maintained" means that the plasmid is an autonomous replicator or part of the host genome. Refers to the stable presence of a plasmid in a transformed host that serves as an integration site. .

The term "microorganism" refers to unicellular prokaryotes and eukaryotes, such as actinomycetes and yeast. It refers to something that includes both things.

A "non-natural endonuclease restriction site" is a restriction site that is not normally present in natural cDNA. , an endonuclease restriction site synthesized at an existing restriction site in the natural cDNA sequence. means.

The term "operon" refers to structural genes, regulatory genes and regulatory gene products. The complete chain of gene expression and regulation, including control elements in DNA that are recognized by It refers to knitting.

The term "plasmid" refers to an autonomously replicating extrachromosomal circular DNA. This includes both expressed and non-expressed types. Cell culture that provides expression of such molecules When a recombinant microorganism is described as a host for an expression plasmid, the term “expression plasmid” is used. The term "lasmid" refers to extrachromosomal circular DNA and D. Includes both NA.

The term "promoter" refers to a promoter that binds RNA polymerase to initiate transcription. This refers to the DNA region that is involved in the fusion.

The term "DNA sequence" refers to adenosine, thymidine, cytosine and guanosine sequences. A single-stranded or double-stranded DNA molecule containing nucleotide bases consisting of It also includes genomic DNA and copy DNA (cDNA).

The term "suitable host" refers to a form of be compatible with the transformation plasmid, allow the plasmid to replicate, integrate into its genome, or refers to a cell culture or microorganism capable of expression.

The term "upstream" refers to sequences that proceed in the opposite direction of expression. For example, bacteria The promoter of is upstream of the transcription unit and the start codon is upstream of the coding region .

The term "restriction endonuclease", also known as restriction enzyme, is a enzyme that is specific for a particular enzyme. An enzyme that cuts double-stranded DNA (dsDNA) at a specific position or site. for example , restriction endonuclease EcoRI converts ds DNA to 5' GAATTC3' 3' CTTAAG5' at the site Cleavage produces 5'G and ^ATTC3' fragments.

3' CTTAA G5' Although many such enzymes are known, the most preferred embodiments of the invention are It primarily concerns selected restriction enzymes with the following characteristics.

The conventions used to represent plasmids and fragments are has the same meaning as that of the conventional representation of the fragment. Drawing rather than traditional annular shape The single line shown in represents both circular and linear double-stranded DNA, with initiation or transcription on the left. It occurs to the right (5' to 3').

Nucleotide numbering and amino acids corresponding to specific amino terminal forms are shown in Table 1. However, there are obvious modifications to the numbering for those with different NO2 termini. It will be easy to understand what is done.

Amino acid abbreviations Amino acid 3 letter abbreviation 1 letter code asparagine Asn N-aspartic acid ＾ill D-asparagine or Aspartic acid ^sx 33 cysteine C7s C Glutamine Gln Q Glutamic acid Glu E-glutamine or Glutamic acid GlxZ Glycine Glr G Histidine His H methionine Met M phenylalanine Phc F Proline Pro Pro P Serine Set S Threonine Tb+ T Tryptophan T +p W Tyrosine Ty+ Y Valine V! l　　　　　　　VGeneral method DNA preparation, restriction enzyme cleavage, restriction enzyme analysis, gel electrophoresis, DNA Fragment isolation, DNA precipitation, DNA fragment ligation, bacterial transformation methods for selection and growth of bacterial colonies are described. Molecular cloning (llolecultt CIoniB) by et al. 　　　　Ni Laboratory Manual [Cold Spring Harbor (Co 1d 5priBH+bor), New York, (1982) Ko (Honmei In the detailed description, it will be abbreviated as Maniatis hereafter. ) for details. in buffered medium RNA transcription in mama Hro and rabbit reticulocyte lysis in postpartum The method for protein translation with wire is described in the manufacturer's instructions [Promega Biochip The details are explained in [Promega Biolech]. DNA arrangement The sequence was determined using either single-stranded DNA or denatured double-stranded DNA using Sanger's dilution method. This was done using the deoxy method.

synthetic oligonucleotide linker The following oligonucleotide solution is available from BioLapse Inc. I got the linker.

1, d(CCTCGAGG) octamer (ller) 2. 6( CCCTCGAGGG) , decamer 3, d (CCCCTCGA GGGG) +2 each linker is restriction enzyme X) 101 (CTCGA G) contains the recognition sequence for IPA cDNA and 5P65 vector. is unique to These linkers play an important role in the generation of linker insertion mutants. The full-length cDNA of human uterus IPA was used for the subsequent generation of deletion mutants. Cloning is described by Re1dr et al. (1987). Essentially, mRN^ is produced by the guanidine triocyanate method, followed by CsCl gradient purification method and oligo-d1 affinity chromatography purification. Obtained from uterine tissue by method. using reverse transcriptase and Flenow fragment. to copy the information into double-stranded cDNA using introduced into the site. Oligonucleotide deduced from the sequence of Bowes melanoma IP^ tPA cDNA clones were screened. 2455 base pair cDNA When we isolated A and determined its sequence, we found that the sequence described in the publication [Penica (P Enici) et al., 1983.

Uterine 11'AcDNA was found in melanoma IPA and in several sites (mainly in (Reddy et al., 1987).

5lan1 site at the 5' end of the clone near the ATG start codon of IPA (nucleotide 16) and Bg111 site (2090), and the presence of dNTPs. The plant end is filled with Flenow, and the Sal linker is dissolved. Became. cDNA was reintroduced into BR322 as a Sal fragment, The 5111 site was then used to reintroduce into other vectors.

Generation of linker insertion mutants Other introduced ^ma + II, Nh! Can be used with l and Spe1 sites Add the SpeI linker to the Bgl l115) part of the IPA cDNA as shown in placed in the position.

Bgl 11115) Spel (nucleotide 8) for construction of mutants The detailed method is shown below.

5P6-IPA 1 μg was added to the unique Bg111 recognition site ( Cleaved with Bgl II at nucleotide 115). ethano linear DNA Precipitate with nick translation buffer [KPO-(pHL5) 4 0 mM, MgCl□6.61 amount M, mercaptoethanol 1. OLIIM 5dATP, dCTP.

5μ DNA Polylase I (Frenow) in dTTP and 250μM dGTP fragment). This step closes the 5′ attached end and A linear DNA with two ends was generated.

After incubation for 1 hour at room temperature, Flenow heat inactivation was performed at 65°C for 5 minutes. went. Add phosphorylated octamer Xho I linker (commercially available) to this mixture. 100 pmoje, ligation buffer [50mM] final concentration of Lis (pH 7,8) , DT720mM.

^TP lff1M and bovine serum albumin 50μgml-'] and T4DNA 200 u of ligase was added. This ligation was allowed to occur overnight at 22°C.

The ligated DNA was phenol:chloroform:extracted and ethanol precipitated. It was IAA. This was resuspended in restriction enzyme buffer, overdigested with Xho I, and % agarose gel (rIIllOn) to remove the multiple linkers and excess phosphorus. The car was removed from the relinearized DNA. Extract the relinearized DNA with agarose , ethanol precipitation was performed. Precipitated DNA was added to ligation buffer and T4 ligase. and binding was allowed to occur overnight at 16°C.

A small sample of the recombined DNA was transferred into E. coli strain DHS using standard protocols. Perform transfection and transfer the transfected bacterial plasmids onto LB agar i. Cultured on mp plates.

Bacterial colonies are collected, grown in LB medium, and DNA is produced on a small scale using standard methods. I let it happen. Plasmid DNA was analyzed by restriction enzyme analysis and a unique Bgl+1 site was identified. The deletion was confirmed.

Balls put out Synthesis of site-directed mutagenesis Using synthetic oligonucleotides generated by the solid-phase phosphorytriester method Human uterine tPA cDNA was modified by site-directed mutagenesis.

The following primer was synthesized and used for the mutagenesis described above. They are in antisense sequences existed in

1. Brymar EGAV For the introduction of the ＾mar　11 site (CCT-＾GG) at nucleotides 256-261 5' GCA, ACT, TTT, CCT, 1. CAC, TGA 3 “2, bu Reimer SWS For introduction of Spe I site (ACT, AGT) at nucleotides 379-384 5' GCA, CGT, GGC, ΔCT, ΔGT, ATC, TAT, TTC3 '3, Braimah KNI 5' for introduction of Spe1 site (ACTAGT) at nucleotides 409-444 CCCCTGTAACTACiTGCCCTG 3’4, Braimah KCI 5' for Nhe I (GCTAGC) introduction at nucleotides 619-624 GGGTGCTAIKGAACTCTGAG 3’5, Braimah KCAI 5' for introduction of Nbe I (GCTAGC) at nucleotides 613-618 GTGCTFCAG (JICTGAGCTGTAC3'6, Braimah KNB 2 For introduction of Nhcl (GCTAGC) site at nucleotides 673-678 5' GCCAC to GTAGCIAGCCCCATTCCC 3' nucleotide 8 5' GT for introduction of AtrIl (CCTAGG) site at 71-876 ACTCCCIAGGCAGCCTGiC3'8, primer KCA2 For the introduction of Atr II (CCTAGG) at nucleotides 862-867 5’　CGTCAGCCTCTGGTTCTTCAGC3’9, Primer SWN Nucleotide for introduction of Nhe I (OCTAGC) at nucleotides 90+-906 5' CC for introduction of Spe 1 site (ACTAGT) at Otide 943-948 Cding CCAcT embryo TGCGAAAACTG 3' nucleotide +693-1698 So ＾vt　l! 5’ CTGGTCACσ embryo for introduction of (CCTAGG) GCATGTTG 3' Site-directed mutagenesis M using the above oligonucleotide The I3-based oligonucleotide-directed mutagenesis method is essentially based on the Kunkel method. ) et al. Proceedings of the National: Academy Meobza Yences USA.

It is described in detail in Vol. 82, p. 488 (1985).

MI31F^3. MG^vector (said pending patent application and Wei et al. IPA eDN with Asn 451 gIn variant described in literature A) The phage containing E. coli strain C3236 (this is du+-υnl) The cells were grown in culture medium in the presence of 0.5 μg ml uridine. substitute for thymine Single-stranded DNA containing uracil residues was produced. Single strand MI31P^, MGA was produced by a conventional method.

The phosphorylated mutant oligonucleotide long was 1xssc (0.15M N1 Cl, 15mM sodium citrate) total amount of 20μ! Single strand DN inside A: Annealed at 1Rg. This annealing mixture was heated to 70°C, It was then gradually cooled down in a water bath for several hours. The annealed fragments are attached to the DNA port. It was converted into a covalently bonded circular DNA by the action of limerase and T4 ligase. a The kneeling mixture contained 20mM Hepes (p) 17.8), 2mM DTT, 1h+M MgC1, 500μiJ each dATP, dTTP, dCTP and dGTP, ^TP 1mM, 2.5 units of Freneau and 2 units It contained T4 DNA ligase of 100μi. 5 minutes at 0°C, then at room temperature Incubation was started for 5 minutes, 2 hours at 37°C and overnight at 16°C.

Add 10 μl of the above reactive mixture to 300 μl of competent DH, bacterial cells; It was left in water for 1 hour. Bacteria were heat shocked for 5 min at 37°C, followed by mid-phase J Dilute MIOI with 3mM soft agar (0.6%) containing 200ml and transfer to LB agar. Pour into a plate and allow to solidify. Incubate the plate overnight at 37°C. Ta.

Remove the nitrocellulose bump (1st I) from the plate and incubate at 80 degrees under vacuum for 2 o'clock. Tempering, hybridization with 6P″^TP-labeled mutant oligonucleotide I let it happen. Mutant positive isolates were measured by heat denaturation of DNA: oligodimers. and further for mgwi-p+ep R'f DNA isolation in IMIOI. Grown on a large scale. Contains Asn 4511111 variant and new carbon variant Further control of the IPA cDNA-containing Bti Hl-Hind III fragment SN5 vector and/or mammalian cell expression using conventional methods of restriction enzymatic cleavage and ligation. Re-cloned into vector.

As an example of this method, c Mutant KCA2 (R252P; N451Q) introduced at the 3' border of DNA There is generation.

559 57G AAC, CGC, AGG, CTG (251) N RRL (254) Site-specific oligonucleotide directed mutation Generation is ^wr If site (CCTAGG) occurs.

Other variants using this method are as follows.

EC, AV (V51R: N4SIQ) SWS (R92S; N4510) KNI (IIOIT : N451Q) KCI (C171S; N451Q) CA1 (E169A: F170S; N451Q) KNB2 (S1139A; A190S; N451Q) KC2 (T255P: H2 56R; N45+Q) KCA2 (R252P; N451 Q) SWN (S265A; T265S; N4510) OMT (I279T; 2 B0S; N451Q) Pen (R529P ; P530R: H45IQ) (Note: Specific restriction enzyme sites are mutant MGS (N451Q) and thus each contains this variant. )IPA protein The positions of the restriction enzyme sites with respect to the quality domain structure are as follows (see Figure 1). (see).

1111 II (115) 5R(8)-N-terminus and fission of mature tP^ protein The N-terminus of the index domain while EGAV - C-terminus of finger domain and growth factor domain N-terminus of between SWS - C-terminus of growth factor domain and Kringle 1 domain N-end of N between the edges KNI - N-terminus of Kringle 1 domain KCI - C-terminus of Kringle 1 domain KCAI - C-terminus of Kringle 1 domain KNB2 - N-terminus of Kringle 2 domain KC2-C-terminus of Kringle 2 domain end KCA2 - C-terminus of Kringle 2 domain SWN - C-terminus of Kringle 2 domain and protease main Between the N-terminus OMT - N-terminus of protease domain Pen - C-terminus of protease domain fPA cDNA manipulation Awr If, Nhe l, Spe I and Xba I restriction enzymes are Recognizes different hexanucleotide sequences. For example, person V + II CC TAGGNbg l GCTAGC 3pc l ACTAGT Xbt I Cut with a suitable restriction enzyme When used, the 5' protrusion is common to all four sites.

Awr II CCTAGG -” CCTAGGGGATCCGGA T.C.C. Nb5 I GCTAGC-” G CTAGCCGATCG CGATCGSpe I Ding＾GTTGATCA　　　TGATCAXbt　　I　　　　TCT AGA -T CTAGAAGATCT AGAT CT fragments with the above-mentioned ends are therefore easily ligated and have other benefits. As, when ends from differently cut sites are ligated, the resulting bibacterium The restriction enzyme site can be used to analyze correct ligation and/or fragment orientation. Even so, it is not recognized.

for example, Awr　If　S’Protrusion part　　NhelS’Protrusion part↓ cutting Since it has lost its batchwise character, either by x II or Nhe 1 The thighs are no longer severed. The same applies to other possible combinations.

Manipulation of the IPA cDNA is accomplished using the introduced sites described above to create deletions and duplications. occurs.

A) Deletion ^Mar　IL　Nhe　l and Spe　1 sites present in the following mutants: EG AL SWS 5KNI 5KCI SKC^1, KNB2, KC2, KCA2 , SWN, OMT and Pen to detect deletions of domains in the expressed protein product. IPA causes a deletion in the cDNA.

An example of such a manipulation is the creation of a Kringle domain using mutant KNI and KCAI. This is a deletion of cDliA, which encodes cDliA.

KNI　　　　　　　　　　　KCA1GAC-CAG-GGC-AC T-AGT-TAC-AGG AGC-TcA-GCT -AGC-DingGC-AGC-`CC-CCT asp gln gly thr ser tyr arg ser ser ala sir cys ser thr pr098 □ 105 167 174Spe I cutting 　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　↓GAC,C AG, GCC, ACT, AG C, TGC, AGC, AC C, CCT↓ join (Digested with Spe I and Nhe I and contaminating KNI or KCAI) remove. ) ↓ GAC, CAG, GGC, ACT, AGC, TGC, AGC, ACC, CCTa 5p gin gly thr ser cys ser thr pr.

98 99] oo 17+ 172173174 ie Kl del or del (1 (11-170); II) OTSI71; N45 Details of the 1QK1 deletion construction are as follows. 1 μg of plasmid 5P6KNI Hind III and Spe! to digest plasmid SP6 KCAl. Digested with Hind III and Nhe I. The generated DNA fragment separated and isolated by gel electrophoresis. SF3 Large fragment from KNI A small fragment from SF3 KCAI was ligated in a conventional manner, and SpeI and Nhe Re-digested with I, transfected into E. coli D)15 strain, and ampicillin The cells were cultured on LB medium plates containing 1. produced by resistance, Spe I and Nb e Deletion and loss of the I site were analyzed by restriction enzyme analysis. Schematic diagram of the process It is shown in Figure 2.

The same procedure is used to generate the series of mutants described below.

Mutant Region Deleted domain N del dgl (171-1911); Interkringle 170As] 9184 51Q Kl del del (lQI-110); Kringle 1100 TS]71;N45]Q ) (2del del (1119-256); Kringle 2 188AR257; N451Q KNI2 del del (101256); Kringle 1 and 2100TR257, N451Q F del Del (5-51) 4TR52; Finger 4 SIQ G del Del (51-92) 50S93; Growth factor FG d el del (5-92) 4T593; finger and growth N45 1Q factor Prote + lel (2-266); ITS2S7; Heavy chainase N451Q (Finger, growth factor, both kringles) In constructing mutants encoding deletions, the following plasmids were used as shown in Figures 2.6 and 7. They were digested with appropriate restriction enzymes and ligated as shown below.

For example, the deletion mutant Kld! In the production of l, a unique Spe1 site (KN ll were ligated with the unique Nbe 1 site (KCAII) at their normally cohesive ends.

Plasmids and restriction enzymes N del KCI (Nht I) and KNB2 (Nbe I) Kl del KNI (Spt I) and KCAI (lthe I) K2 del KNB2 (Nht 11 and KCA2 (A v+ If) KNI2 del KNI (Spe I) and KCA2 (Awr II) F del Bgl/Spt (Spe I) and and EGAV (Awr If) G del EGAV (Avr I I) and SWS (Spe FG dsl Bgl/Spe pe l) and SWS (Sat l) protease B11/Spe (S pt　]) and SWS　(Nhe　I) duplication Using the Avr II, 1ike I and Spe 1 sites listed and described above With respect to Figure 1, the DNA coding domains overlap and therefore the protein product It is possible to duplicate the same domain. An example of such duplication is mutant S Using WS and SIN, four Kringle domains (overlapping Kringle 1 and 2) ) is the production of an IPA analog containing

The duplicates below were constructed in a similar manner. The amino acid sequences are shown in Tables 4-9 and 12- It is shown in Table 15.

duplicate domain Mutant □ To 2! , [2 Kringle I IKI, 2 to 2 Kling J and 22 to 1.2 to 2 Cli Kringle 1 and 2 SIN Kringle 1 and 22F Finger 2G Growth factor 2FG Finger and Growth Factor 2 Fret ICV Protease domain 2 Prol, 2CV Protease domain In the construction of mutants encoding duplications (1, 2.6 and 7a-1, 7a-2) As shown in Figures , 7b-1 and 7b-2, the following plasmids can be used in appropriate numbers. The enzymes were digested and ligated.

As an example of the generation of a duplication mutant, 2F, the introduced unique Avr It (EGA A plasmid containing V) was introduced at the JI II (115) site (Jl/5pt). ligate with the plasmid containing the inserted Spt1 site via a normal cohesive end. Ta.

Demarium E poem and crab Otsu two two Otsu "Nie! 10 doctors! ? *-2F EGAV (Ayr II) and Bgl/Spe (Spe 1) 2G SWS (Spe l) and EGAV (Awr I 1) 2FG SWS (Spe I) and Bgl/Spe (Spe I) 2K] ]K2 KCAI (Nht I) and KNI (Spc I) 111 2j (2 [CA2 (Awt If) and 1NB2 (Nbe I) 2nd 2g2 KC^2 (Avr If) and KNI (Spe I) S+N SWN (Nhe I) and SWS (Spe I) OMS OMT (Spe I) and SWS (ape l) 2 Pro (ICV) Pen (Awr if) and OMT (apt l) 2 Proj 2CV Pen (Awt II) and SIN (Nbt 1) C) Rearrangement Avr II, Spsl and Nht 1 site individually or in combination plus used within the core to ensure that the final product has the recombinant domain structure. It was possible to rearrange domains or domain blocks.

Examples include heavy and light chains, i.e., light chains and proteins in the amino-terminal half of the protein. The DNA encoding the key molecules in the amino-terminal half of the white matter is replaced.jP A. It was possible to rearrange the cDNA.

The final product was intended for all rearranged mutants.

Other mutants were generated using rearranged domains in a similar manner.

20, the kringle 2 domain is replaced with another kringle 1 domain, Produces a protein with two kringle 1 domains and no kringle 2 An IPAP analog was coded.

2 contains two kringle 2 domains and a kringle 1 domain. There is no IP human body code.

The amino acid sequences of the rearranged variants are shown in Tables 10.11 and 16.

In the construction of mutants encoding rearrangements, the following plasmids are used as plasmids 2.6 and 7. A-1, 7a-2, 7b-1 and 7b-2 are suitably digested with restriction enzymes as shown in Figures, Connected.

As an example, the υLT mutants are Sea urchins were produced.

Mutants Plasmid and restriction enzyme 2KI used KCAI ( Nhe　l), KNI　(Spe　I), niC^2　(Ay+　II) and niN B2 (Spe I) 2 KCAI (Nhe I), Nl (Spe I), KCA2 (person! I ill and KNO2 (Spe I) ULT Bgl/Spc (Spe I), Swn (Nbe I ) and Pen (Ay + I I) Confirmation of mutants It was determined by restriction enzyme analysis, sequencing and/or in-mil+o transcription/translation analysis. The mutation was confirmed. A mutant with sufficient fibrinolytic ability encodes a protein. The wild type IPA was analyzed by enzymogram.

MI3MPI8. From RA, by restriction enzyme analysis and gel electrophoresis, IP A Bj11+old-)1indlll fragment containing the tDNA sequence is isolated separated, Bam) IISHindllll truncated 5P65 vector [Promega Ba Iochic (Promeg! Biolech)]! ::Concatenated.

This orientation (with the 5' end of the cDNA adjacent to the SP6P promoter) Mutant protein is produced by RN human synthesis in IL+O and protein synthesis in MIT+O. This made it possible to analyze the products. 5P65. The 1PA vector also contains the inserted cDNA A, it was a convenient vector for use during deletion generation procedures, for example.

LK444BllS, IPA Regarding Figure 3, the mutated cDNA molecule was inserted into the LK444BIIS vector. Re-cloned. Mutant derivative 5P obtained by restriction enzyme cleavage and gel filtration 55. (B!m old, flindll fragment was isolated from PA. BxmHI fragment, a) 1i force d111 cleavage vector, LK4448 Connected to H3.

This mutant is a CO37 cell line driven by the human β-actin promoter. 5P65.1P^, mutation S! from the derivative by restriction enzyme cleavage and gel filtration. Eleven fragments were isolated.

This fragment was inserted into the Xho I cleavage vector CLH3^XBPV as shown in Figure 5. Connected so that This orientation indicates that the inserted sequence is metallogenic in 0127 cells. It was determined and selected to be under the driving force of the thionine promoter.

CLH3AXSV2DHFR jPA cDNA or mutant was digested with restriction enzyme with Sit I, and the cut Xb was recloned into the vector using the o1 site. IPA cDNA or variant The variant orientation is such that expression is driven by the metallothionein promoter It is. This CLH3AXSV2DHFR vector undergoes metatrexic therapy in CHO cells. Used for stable expression into IPA or modified fP with sate amplification ability. Ru.

Transfection of CO8 cells Expression vector 9- (LK444BIIS) was added to CO5-7 cells (ATCCjcRL) [65]), a transient expression system was used.

Characterize the activity of secreted modified IPAP white matter 2 to 3 days after introduction of exogenous DNA The conditioned medium was analyzed to determine the

3 x 10' cells in IOC plate in DMEM + 10% glutamine The cells were grown for 1 day before transfection.

Add 0 to 20 μg of DNA to 2.0 ml of Tris-buffered saline (pH 7.5). Added. Add 2 mg/ml DEAE-dextran (transfer) to this solution. Immediately before injection, add 25 ml of DEAE-Dextran 50 (prepared by adding) 1 ml was added. Cells were washed in phosphate-buffered saline (PBSI). Washed twice and added transfection solution. Cells were incubated at 37°C for 15-30 min. Incubated for minutes. Then remove the dextran solution and wash the cells twice with PBS. Washed. This solution was mixed with 10ml of DMEM medium (serum-free) and chloroquine (IOml). mM) 100μ! Replaced with.

The cells were incubated at 37°C for 4 hours. Wash the cells twice with PBS , GIT serum-free medium (lQml) was supplied.

Transfection of DHFR-CIO cells Lawrence Chi, Columbia University DUKX Cll0 cells were obtained from Itvrtnce Chasinl. . These cells are deficient in dihydrofolate relactase. This gene is vector It is present in the target CLH3AXSV2DHFR. Before transfecting cells 1008 protein with Alpha Plus Media 10% FBS and 1% Glutamine Medium. Cultured for 24 hours at a density of 7 x 10' cells per plate. 0.5m1) run sfection buffer (its composition is 4 Ntel per 500 ml total volume) g, KCIQ, 185 gSNt2 HPO4 [1,05 gs Dextrose 0.5g and HEPES 2.5gSpit 7.5) in 0.5ml Lasmid IIN^10G to 50μg. 2M CMCI2 309g was added to the above solution and the mixture was allowed to equilibrate for 45 minutes at room temperature. Plate the medium The cells were washed twice with PBS and the DNA solution was added to the cells. So cells were incubated for 20 minutes at room temperature. Next, add 5 ml of medium and was incubated at 37°C for 4 hours. Remove medium and incubate cells at 37°C for 3.5 min. Shocked with 15% glycerol in transfection buffer. 4 After 8 hours, the cells were divided into 1/3 and treated with 0.02 μm of methotrexor. A selection medium was supplied. Cell colonies that survive despite treatment are transfected. They appeared 1 to 14 days after the injection.

The selected colonies are then treated with increasing amounts of methotrexate using known methods [e.g. Biotechnology (Bio/Dingechn) by Shell (Mich*I) et al. oloB), Vol. 3, p. 561 (1985)]. these cells The modified IPA protein produced by Io/Technology, Volume 5, Page 953 (1987) and Ringgold (R ingold) US Pat. No. 4,656,134.

Transfection of C127 cells A DNA preparation of mouse C127 cells was published by a researcher in the assignee's laboratory [ Hsiunl et al., J. Mol.

Appl, Genetics, Vol. 2, p. 497 (1984)] method. Transfection was performed.

Modified IPA proteins were prepared using the previously described method [Bio/Technology by Rowe et al. 5, p. 953 (1987) from a conditioned medium.

Modified IPA assay The m1PA protein in the conditioned medium was quantified using American Diagtustica (^m eic1n Di! gnoslicg) (Greenie, Connecticut, USA) This was done using the Elisa kit sold by (Japan). Coating and detection antibodies are This is anti-human IPAIIG.

Activity was determined using a published spectrophotometric assay for plasminogen activity ratio [Bel. Vrrhcije’n Tbromb by Rani, Hxemos +ts, Vol. 48, p. 266 (1982)]. in assay The measured absorption changes were converted to units with reference to the WHO melanoma IPA standard. Ta.

Determining the specific activity of mjPA protein by dividing it into protein units, The latter is what was measured in the ELISA assay.

The mjPA of the present invention is advantageously combined with a pharmaceutically acceptable carrier material, such as physiological saline. It can be administered orally, intravenously or injected into the artery of the heart lesion. Ru. Administration is generally performed using two commonly used clot lysin enzymes, streptolysin enzymes. This is done as it is done with Nasase and Urokinase.

The 5lPA of the invention may also be used in patients who have had a recent myocardial infarction resulting in blood clots, such as in post-surgical patients. patients requiring treatment for embolism, such as those suffering from deep vein thrombosis and those suffering from deep vein thrombosis. Used therapeutically to dissolve blood clots in human patients.

Example 1 Freeze-dried for urgent treatment of blood clots by balms injection Mix 5 to 10 mg of m1PA with physiological saline and put it into the chamber of the syringe. , mjPA porous was used to inject patients intravenously.

Example 2 For intravenous infusion therapy to rapidly dissolve coronary artery thrombi, lyophilized mfPA was added to approximately The solution was infused intravenously at 10 mm/hour over a period of about 1 hour. After that, about 50■/hour Intravenous infusion was continued for approximately 3 hours.

Example 3 Approximately 1 saline swab before infusion for intravenous treatment for rapid lysis of coronary blood clots The protocol of Example 2 was followed except that 0 μ of porous was intravenously injected.

Example 4 Frozen, dissolved in saline for intravenous treatment of gradual dissolution of deep vein thrombi Intravenous infusion of dry m1PA at approximately 15 mg/hour over approximately 12 to 24 hours. I did it.

Those skilled in the art will appreciate that the amounts set forth above are merely representative and that the individual It will be easily recognized that it varies depending on the characteristics. Moreover, the scope of the present invention Many modifications may be made within the scope of the invention without departing from its scope. However, the +NPA of the present invention is not without limitations, but it is possible to assemble it on your own. It is clear that it can be used for diagnostic purposes, including in-home and in-home video applications. be.

Table 1 aa G T IJ S S T A E S G A A E CT N V @N S S A L -aa N Y C, RN P D RD S K 1” J CY V F K A G -aa FG N G S AA Y RG T HS L T E S G A S C-cTCα; TGACCCCGG ACCCGTrTGTATTMTGAC to GCCTTAG to ACrACCαπA CG to TTC to GGaa OA L G L G K ) I N Y CRN P D G D A K P -＾ CCACGG’TGCACGACTTCTTGGCGTCCGACTGCACC CTCATGACACTACACni GAG (:ACGaa A D I A S) I P IJ Q A A Engineering F A K HRRS P -aa G E RF L CG G X L X S CIJ X L S A A -aa HCF OE RF PP P HHL T V I L G RT Y -aa RV V P G E E E Q K F E V E K -1X V HK -aa E F D D D T Y D N D I A L L Q L K S D S @− aa S RCA Q E S S V V RT V CL P? A D L -aa OL P D 'J T T E CE L S G Y G K) l @ E A L S -aa P F Y S E RL K E A HV RL Y P S S RC’-am T S Q I II L L N RT V T D M L L CA@ G D D - am R5(:(:FOAL-L'1l()ACQGDS(:GP -aa L V CL N D G RM T L V G 5V GLG -aa C to Q K D V P G V Y T K V T N Y L D 　　V　　Z　-Table 2 Propeptide/signal 22 to 108 -29 to -1 finger domain 133 to 246 9 to 46 growth factor domain 268 to 367 54 to 87 kringle 1 liter in 391 to 634 95 to 176 Kringle 2 domain 655 to 900 183 to’264 Table 3 (Characters without a decorative 1 (e.g. 1)) are all related to nucleotide sequences. , and all characters with a decorative character 1 (for example, 11) are related to amino acid sequences. )v Nh 　　　　　　　　　　　　　　　　　　　　　　　■danji Ushiso W Sid Phantom Muni and Servant W Death L H Rainbow U” Group W Plate On a ronsw plate 107168169170171172 +73174175176 wide ・T-N2 5926026126226326426526626726B 269270 271α 扛　　　　　　　　　　・　　・・Heavy chain missing loss F Del (5-51) H4TR52HN4 510G Del (51-92); 50593 ;N4510FG Del (5-92) H4T S93:N4510-3 -:2-1 1 93 94 95 96Protease del(2-226);ITS 267HN451Q EI D To-’N -T661 6754] 2 616t) T, GGG, AAT, GG, to CT AGC, TAC, AGG, GGC----TAC, AGC, TCA , GCTAGG, CTG, ACG, TGG, GAG1851861E17188 189 102103104105 16616716El 169a rg leu thr trp g+u melon -N -u) GAC, CAG, GGC, ACT AGC, T AC, CGT, GGC---CTG, AAG, AAC, CC under ACG, TGC, AGC, ACC, CCT, GCCser ays ser thr pro a la! Table 4 Mutant 2F YDNDIALLQLK5DSSRCAQESSWRTVCLPPADLQLP riWTECELSGYGHεALSPFYSEL'lLK■`HBRLYP Table 5 Mutant 2C IL color Mutant: 2FG ANLHCIACQGDSGGPLVCLNDGR)4TLVGIISWGLG CGQKDVPGVYTKVTNYLOWIRDN MRP17 table Mutant 2 to 11 to 2 MTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNM RP table 8 Mutant lK12 to 2 Table 9 Mutant 2 to 12 to 2 Table 10 GARSYQVI CRD EKTQHI YQQHQSHL I'lPV L R5N Ill VEYCHCN 5GRAQC)l 5VoV)l:SC5E PRCFN GGTCQQA L YFSDFVCQCPEGFAGKCCE I DTRATCYEDOGI 5YRGTWSTAESGAECTNI-IN SSALAOk:PxSGRRPDAI RLG QFRI GGLFADIASHP) IQAAI FAKHRR5PGERFL CGGI LI 5SCHI LSAA) IcFQERFPoHHLTVI L GRTYR WPGE E EQKF EVEKYI VHKEFDDDTYDN D IA LLQLKSDS SRCAOES SWRTVCLPoAD LQLPD WT ECE LS GYG) CHEA LS P FYS ERLKEAHB RLYPS S R CTSQHLLN RTVTDNMLCAGDTR5GGPpAN LHOAC QGDSGGP L VCLNDGRMTLVGI I 514GLGCCQKDVPGVYTKVT NYLDWI RDNMRP Table 11 G to RSYQVICRDE to Ding QMIYQQHQS) ILRPVLR5NRVE YCWCNSGRAOC)ISVPVKSC5EPRCFNfTCQOALY F S DFVCQCP EG FAG) CCCE I DTRATCYEDQ GTSYRGTHS LTES GAS CLPWNSM h LI GKVY TAQN P SAOALGLGKHNYCRNPDGDAKPHCHVLKN PSCSTPACSEGNSDCYFGNGSAYRGTH5LTESGASC LPWNrMILIGK VYTAQNPSAQALGLGKHNYCRN PDGDAKPWCHVLK N RRL Thl EYCDVPS C3TCGLROYSnPQFRI KG GLFA DIAS) IPWQAAI FAKHRRSPGERFLCGGI LI 5S OII　LSAAHCFQERFPPHHLTVr　LGRsYRWPGE　E EQKF EVEKYI Vl(KE FDCIDTYDN D I A LLQLKSD S　S　I’1CAOE　S 5WRTV CLP　PAD　kOLPDI-IT ECεLSGYGKHEALSPFYS ERLKEA)l BRLYPS 5 RCTSQ)l LLN RTVTDNM LCAGDTRS G to PQAN LHDACQfDSGGP LVCLNDGRMT LVGI ItagGLGCG QKDVPG’VYTKVTNYLDHIRDNMI’IP Table 12 (, AR5YQVZ CIIIDEKTQMIYQQHQSWLRPVLR5N RVEYCWCN 5GRAQCH5VPVKS C5Eo IIICFN GGTCQQA LYFSDFVCQCPEG FAGKCCE I DTRA TCY EDQG I 5YRGTH5TA ES GA ECTNWN S S`　LAQKPYSGRRPDA　I　RLGWS ding AESGAECTNWN SSALAQKPYSGRRPDAII’1L(fGN)INYcRNPDRD SKPWCYVFKAGKYrSEFC5TPAC 5EGNSDCYFGNGSAYRII;TH5LTESCASCLPHNSM I LI GKVYTAQN PSAQALGLGKHNYbRNPDGDAK P14C HVLKN RRLT onlyEYCDVPSC5TCGLRQYSQPQF RI KGGLFADI ASHPHQM r FAKHRR5Pf ERF LC G I LI 5 SC) II LSAAHCFQERFP PHHLTVI LGRTYRWPG E　E　EQKF　EVEKYI　VHKE　FDDDTYcN　D　IA　L LQLKS O5S RCAOES 5WRTVCLPPADLQLPD14T ECELSGYGKHEA LS P FYS ERLKEAI (SRLYPS S qCTSQHL LN RTVTD 2 animals 11 tables Mutant OH5 Table 14 Mutant 2 Prot 1CV Table 1S Mutant 2 Prot 2 CV GGLFADIAS)lPIIIQAAIFAK)lRR5PGERFLCG ILmS5o41LSMHCFQERFPPHHLTVrLfRTYRWPGE Table of Contents GARSTSCGLRQYSQPQFRIKGGLFADIASHP AAIF AK) IRR5PGERFLcGIII; ILISSC14PLSAAHCFQ E.R. Table 17 (mIU/ug) MSl 350KN 1“ 485KCI 584KCAI 584KCAI 　　　　　　　　　　　　　　　　517KNB2“ 418KC2745 KCA2”　　　　　　　　　　-617Nd el 1 del cle 1 400KI K2 del ．． IK2” 3201KI, 2 2 502 to 1.2 to 2 v, 1°v, 1. shows very low activity * represents the most preferred molecule Figure 1 Figure 2 Figure 3- Expression vector for COS cells (numbers in parentheses are leotide base position) Figure 4 < Mo Figure 5 0koni overlap area Figure 7a-1 Generation of ULT rearrangement variants Figure 7a-2 Figure 7b-1 Figure 7b-2Figure 7b-2Figure 7b-2 Figure 7b-i KaraF1g”e7b-’force)ra ag N-12cca-1 international search report bu*,,+n+～,1゜yu,. 110. la, PCT/US! ! 91 00465 International Search Report

Claims (1)

[Claims] 1 Biologically active component regarding tissue plasminogen activator (IPA) A method of producing a modified DNA sequence encoding a child, wherein said sequence has a small number of deoxyribonucleate by using at least one endonuclease restriction enzyme. compatible with predictable rearrangements or deletions of acids; a) providing a sequence that encodes a molecule with IPA biological activity and is converted; Beauty b) selected from the group consisting of AvrII, NbeI, SpeI and XbaI A restriction endonuclease that is cleaved by at least one restriction endonuclease converting said sequence with at least two pre-existing restriction sites to create a restriction site; process method including. 2. A DNA sequence produced by the method according to claim 1. 3. Transformed or transfected with a sequence containing the DNA sequence according to claim 2. Specfected parental cells. 4 One of the sites is transformed to produce AvrII, NbeI, SPeI and Xba creating a cleavage site for an enzyme selected from the group consisting of I; cleavage of an enzyme selected from the remaining enzymes of said enzyme group. 2. The method of claim 1, wherein a cleavage site is created. 5 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 6 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 7 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 8 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 9 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 10 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 11 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 12 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 13 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 14 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 15 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 16 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 17 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 18 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 19 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. . 20 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 21 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 22 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 23 [There is an array] a DNA sequence containing and all sequences that hybridize to said sequence under stringent conditions. . 24 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 25 [There is an array] and all sequences that hybridize to said sequence under stringent conditions. Column. 26 further includes substitution by glutamine in place of asparagine at position 451 Claims 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24 or 25. 27 Claims 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24 or 25. A parent cell that has been transformed or transfected with a sequence containing 28 Transformed or transfected with a sequence containing the sequence according to claim 26 the parent cells that were transfected. 29. The parent cell according to claim 27, wherein the parent cell is C127. 30. The parent cell according to claim 28, wherein the parent cell is C127. 31. A molecule produced by the parent cell of claim 27. 32. A molecule produced by the parent cell of claim 28. 33. A molecule produced by the parent cell of claim 29. 34. A molecule produced by the parent cell of claim 30.