A METHOD OF PREPARING AN ACTIVE HUMAN NEUTROPHIL CHEMOTACTIC FACTOR POLYPEPTIDE

Abstract: A simple and efficient method of preparing active recombinant human neutrophil chemotactic factor. The human neutrophil chemotactic factor (NCF) is solubilized from a lysozyme treated transformant cell homogenate using urea or guanidine hydrochloride. The polypeptide is further purified from the solution using conventional protocols such as CM-Sepharose CL-6B, HW-55 column, etc. By using this method, large amounts of active human NCF can be recovered and purified.

Description: A METHOD OF PREPARING AN ACTIVE HUMAN NEUTROPHIL CHEMOTACTIC FACTOR POLYPEPTIDE This invention relates to a method of preparing a human neutrophil chemotactic factor polypeptide from transformant cells harbouring an expression vector for producing the said polypeptide. Human neutrophil chemotactic factor (abbreviated NCF hereinafter) is a physiologically active polypeptide,. which plays an important role in developmental and homeostatic mechanism, such as migration (homing) of T lymphocytes to thymus and lymphonodes, and in the modulation of immuno responses- It has been reported that NCF has biological activities to attract neutrophils and lymphocytes, and to activate these cells (Yoshimura, T. et al., J. Immunol., 139, 788,1987; Walz.A. et al., Biochem. Biophys.Res. Commun. , 149, 755, 1987; Gregory,H. et al., Biochem. Biophys. Res. Commun. , Jj ., 833, 1988). These findings indicate that NCF is a useful immunotherapeutic agent for patients with malignant tumors and immunodeficiency diseases. The present inventors have succeeded in the production of human NCF by recombinant DNA technology using E. col i as a host cell and also in the isolation of the said polypeptide from the soluble fraction of the transformant- homogenate, and a patent application on this invention was filed with the USPTO on May 2, 1988 as Ser. No.189, 164. Furthermore, the inventors investigated the simple and efficient method for preparing the human NCF polypeptide from the transformant-homogenate. It has been unexpectedly found that a large amount of the human NCF polypeptide produced in E. co1i cells harbouring an expression vector encoding the said polypeptide by recombinant DNA technology is mainly distributed in a precipitate fraction collected hy centrifugation from the transformant-homogenate, and that the active human NGF polypeptide can be obtained efficiently from the above precipitate fraction simply by treating with a protein denaturant such as urea and guanidine hydrochloride and removing the said denaturant, for example by dialysis. The object of this invention is to propose the method for preparing a large amount of the active human NCF polypeptide from the transformant-homogenate. The object of this invention is also to propose the simple and efficient method for purification of the active human NGF polypeptide from the precipitate fraction of the transformant-homogenate. Another object will be understood from the following descripton. A typical amino acid sequence of the human NGF polypeptide is represented by the formula [I] shown in Table 1. However, human NCF means not only a polypeptide consisting of said amino acid sequence, but also polypeptides which have similar sequence as represented hy the formula [I] shown in Table 1 and human NGF activity. To describe this invention more in detail, the precipitate fraction of the transformant-homogena e is dissolved with the protein denaturant, for example urea or guanidine hydrochloride preferably at a final concentra ion of more than 4 M, more preferably at 6 to 10 M, followed by removing the said denaturant, such as dialysis, which is carried out preferably against 10-50 mM phosphate or Tris- HC1 buffer (pH 6-8). Then, only the essentially insoluble material containing the modified NCF is reprecipi ated. By removing the resulting precipitate, the solution containing a large amount of the soluble active human NCF polypeptide can be easily obtained. Prior to the separation of the precipitate fraction from the transformant-homogenate it is preferable to carry out the treatment for break-down of nucleic acid in the trans ormant-homegenate by digestion with deoxyribonuclease. The soluble active NCF polypeptide recovered from the above precipitate fraction can be further purified, for example by combination of treatment for salting-out, anion and/or cation exchange chromatography, ultrafi1tration, gel filtration, dialysis, electrophoresis, affinity chromatography using specific antibodies, and so on. The human NCF polypeptide in transformant cells is produced by cultivating the cells transformed with the expression vector constructed with the DNA encoding the human NCF polypeptide. Namely, in the first place, the expression plasmid for producing the human NCF polypeptide can be constructed, for example according to Referential Example 2 with the DNA encoding the said polypeptide, which is isolated, for example according to the method as described in Referential Example 1 or by the total synthesis using conventinal method. As a second step, a transformant cell for producing the human NCF polypeptide can be obtained by introducing the ahove expression plasmid according to the method of Gohen et al. (Proc. atl. Acad. Sci., USA, 6EI, 2110, 1972). As a final step, the human NCF polypeptide can be produced by cultivating the resulting transformant cells under suitable culture conditions. In addition the transformant-homogenate can be prepared by destroying the cells, for example by lysozyme digestion and freeze-thawing, sonication or by using a French press. Chemical and biological properties of the human NGF prepared hy Example was identical with that of the human NGF polypeptide prepared from the soluble fraction of the transformant-homogenate according to the method as described in Referential Example 2, by the peptide mapping analysis performed by high-performance liquid chromato¬ graphy after protease digestion of each human NGF polypeptide, and by neutrophil chεmtactic assay measured in a multiwell hemotaxis Boyden chamber (Neuro Probe, Inc., USA). For simpli ication of the description, the following SUBSTITUTE SHΞET abbreviations are used in the present specification -and claims. A: adenine C: cy osine G; guanine T: thymine I inosine dATP: deoxyadenos ine triphosphate dCTP: deoxycytosine triphosphate dGTP deoxyguanosine triphosphate dTTP: deoxythymidine triphosphate ATP: adenosine triphosphate DNA: deoxyribonuc1eic acid cDNA: complementary DNA kbp: ki 1obase pairs SD sequence Shine-Dalgarno sequence kD: ki 1odaltons SDS: sodium laurylsulf te In the present specification and claims, the nucleotide sequence shown by a single strand is nucleotide sequence of a sense strand, and the left end is a 5 * - terminus and the right end is a 3'-terminus. In the amino acid sequence, the left end is an N-terminus, and the right end is a C-teirminus. The following Example and Referential Examples illustrate this invention more specifically. 8UΞCTΪTUTE SHEET It should be understood however that the invention is " in no way limited to these examples. For a better understanding of the following Referential Examples, Schemata 1 to 3 are attached to the present specifica ion. Schema 1 shows a process of constructing an expression plasmid pHNPlOl (Referential Example 2); Schema 2 shows a process of constructing an expressiαn vector pEP205 (Referential Example 3) ; Schema 3 shows a process of constructing an expression plasmid pHIPH383a (Referential Example 4). Example 1 Production of human NCF polypeptide Transformant cells for producing the human NCF polypeptide, E. coli HBlOl/pHNPlOl obtained in Referential Example 2- (2) was cultivated in the LB broth [composition; 1% tryptone, 0.5% yeast extract, 1% sodium chloride (pH 7.5)], overnight at 37°C . The culture was inoculated in 100-fold volumes of the nutrient medium [composition; 1.5% sodium phosphate, dibasic 12-watεr, 0.3% potassium phosphate, monobasic, 0. IX ammonium chloride, 2 mg/liter vitamin Bi, 0.5% casamino acid, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.4% glycεrol] supplemented with 3-indoleacrylic acid at 20/i. g/ml. The cultivation was done at 35°C for 28 hours. The cells were collected by SUBSTITUTE ishtic ; centrifugation, and suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.02% lysozyme. The cell suspension was allowed to stand in ice water for 30 minutes. Further, freezing in a dry ice/ethanol and thawing 37'C were repeated to disrupt the cells. To the resulting homogenate, magnesium chloride and deoxyribonuc1ease I (Takara Shuzo Co., Ltd., Japan) were added at final concentrations of 1 mM and 2 U/ml, respectively. After incubating at 0 * C for 30 minutes, the insolube pellet was collected by centri fugation and washed twice with 20 mM Tris-HCl buffer (pH 8.0) containing 0.75 M urea and 1% Triton X-100. The washed pellet was dissolved with 6 M guanidrne hydrochloride in 5 mM phosphate buffer (pH 6.5). The clarified extract was obtained by centrifugation and dialyzed against 20 mM phosphate buffer (pH 6.5) (herein¬ after referred to as PB) . After centrifugation to remove the precipitate formed during the dialysis, the dialysate was applied onto a column of CM-Sepharose CL-6B (Pharmacia, Sweden) previously equilibrated with PB. The column was washed with PB, and eluted with a linear gradient of sodium chloride molarity from 0 M to 0.5 M in PB. The fractions containing the human NGF polypeptide were collected and pooled, and concentrated by ultrafi 1tration. Further, the concentrate was subjected to gel filtration on Toyopearl HW-55 column (T0S0H Co., Japan) to obtain the highly purified human NCF polypeptide. By SDS-polyacrylamide gel electrophorεtic analysis, any impurity was not detected in the highly purified human NCF polypeptide preparation. Example 2 Production of human NCF polypeptide The highly purified NCF was obtained by dissolving the washed pellet from the transformant-homogenate as described in Example 1 with 8 M urea in 5 mM phosphate buffer (pH 8.5), followed by using the same procedure as described in the Example L, Referential Example 1 Cloning of cDNA encoding human NCF The cDNA library was constructed by insertion of cDNA which was synthesized with a template polyadenylated mRNA obtained frσm normal human monocyte stimulated with lipopolysaccharide at 10 /-g/ml for 6 hours, into the EcoRI site of the phage vector λ gtlO. One half million individual plaques were screened for hybridization with [ 32 P] -labeled two kinds of chemically synthesized oligonucleαtϊde probes represented by the following formulae, [G] and [D] : A I T A A I T 5'- AA GG TT CT TA GT TTIAT-3' [G] G G G G G C C and • Tϊ ϋ TiTUTΞ SHEET - 9 - A I T I A I T 5' - AA GG TT GA TA GT TT I AT-3' [D] G C G C G G C In the first screening, 13 putative positive clones were obtained. From these positive clones, one clone (termed r-MDNGF 2-1) was selected in the second screening by using another probe. The phage DNA in r-MDNCF 2-1 was subcloned into the pUC19 plasmid. The resulting recombinan plasmid was termed pUG19-1.7-5. The cloned cDNA in a recombinant plasmid pU19-1.7-5 containing a nucleotide sequence encoding human NCF shown in Table 2. Referential Example 2 Producing of human NCF polypeptide Human NCF polypeptide having an amino acid sequence represented by the formula [I] in Table 1, was produced by the following methods. (1) Construction of an expression plasmid pHNPlOl From the recombinant plasmid pUC19-1.7-5 in which human NCF cDNA was inserted as mentioned in Referential Example 1, DNA fragment encoding the polypeptide corresponding to the amino acids from the 18th position- to the 97th position from the N-terminus of human NCF precursor polypeptide (corresponding to the base sequence from the base No.52 to No.291 in Table 2) was isolated by digestion with restriction endonuc1eases Pst I and EcoRI. This DNA fragment was then cloned into a phage vector ^- a » - * "* * ■* " r" * . ' "" M13mpl8 (Takara Shuzo Co., Ltd., Japan) at a region between the restriction εndonuclease cleavage site of Pstl and that of EcoRI in the polylinker sequence. By using the resultant recombinant phage DNA, a specific base sequence being 5' -TTTAAATTATG-3' as inserted between the codon corresponding to Arg at the 27th position from the N-terminus of the human NCF precursor polypeptide and the codon corresponding to Ser at the 28th position, by the technique of site-directed mutagenesis according to the method of unkel et al. (Methods in Enzymol., 154, 367, 1987). The site-directed mutagenesis was carried out using a Muta-Gene in vitro mutagenesis kit according to the instruction manual (Bio-Rad Labs., USA). E. co 1 i JM105 was infected with the recombinant phage DNA, and then it was cultivated to collect the recombinant phage. Then, E. col i CJ236 was infected with recombinant phage obtained as above and cultivated in 2xTY medium [composition; 1.6% tryptone, 1% yeast extract, 0.5% sodium chloride] supplemented with uridine and chloramphenicol at 1 μ. g/ml and 20 μ. g/ml, respectively, at 37 β C for 5 hours. The single-stranded phage DNA containing uracils was isolated from the culture medium. Separately, mutagenic oligodeoxyribonucleotide primer consisting of 33 bases represented by the following formula [E] was chemically synthesized. 5' -GTTTTGCCAAGGTTTAAATTATGAGTGCTAAAG-3' [E] t_riJ •__>■__»■ i, _» t » _-» «__r« . .fa- s. i - l 1 - The 5'-end of the mutagenic primer was previously phosphory1ated. The phosophor lated primer was annealed with the single-stranded phage DNA containing uracils prepared as above in an annealing buffer [20 mM Tris-HCl buffer (pH 7.4) containing 2 mM magnesium chloride and 50 mM sodium chloride] by incubating at 70°C for 10 minutes, followed by cooling down to 30°C at a rate of 1°C per minute. Then, the primer was extended with T4 DNA polymerase in a synthesis buffer [10 mM Tris-HCl buffer (pH 7.4) containing 0.4 mM each deoxynuc1eos idε triphos¬ phate (dGTP, dATP, dCTP, dTTP) , 0.75 mM ATP, 3.75 mM magnesium chloride and 1.5 mM dithi othreito1] to synthesize a complementary strand and the ends were ligated with T4 DNA ligase by sequential incubating on ice for 5 minutes, at 25°C for 5 minutes and at 37 * C for 90 minutes. the reaction was stopped by freezing at -20 * C . The circular double-stranded DNA (heterodup1 ex) was introduced into E.coli JM105 cells, and they were cultivated to isolate the mutagenized double-strandεd replicative form DNA. The nucleotide sequence of the mutagenized DNA was confirmed by sequencing the single-stranded DNA isolated from the culture medium. The resultant mutagenized double-stranded DNA was digested with restriction endonucleases Dral and EcoRI in order to isolate a DNA fragment containing most of the coding region for human NCF polypeptide. The isolated DNA CU__-» ' i -. i - i~ w. »—i- » fragment is, hereinafter, referred to as the NCF " - ; (Dral-EcoRI) -fragment. This NCF (Dral-EcoRI)-fragment was ligated by T4 DNA ligase with a chemically synthesized o1 igodeoxynucleotide adaptor represented by the following formula [F] . 5' -AATTCATGATGAG 3 * -GTAGTACTGAGCT [F] The resultant ligated DNA fragment is referred to as NCF (DraΙ-XhoI)-fragmεnt. Separately, an expression plasmid pEP205 as mentioned in Referential Example 3 was digested with restriction endonucleases Dral and Xho I, and the resulting larger DNA fragment including an ampici11in-resistance gene and a replication origin (hereinafter referred to as the EP205 vεctor-DNA fragnment) was isolated, and this EP205 vector- DNA fragment Tas ligatεd by T4 DNA ligasε with the NCF (Dral-XhoI) -fragment previously prepared in order to construct an expression plasmid pHNPlOl for producing human NCF (see Schema 1). (2) Transformation of Escherichia coli The resulting expression plasmid pHNPlOl was introduced into E. coli HBI01 by the following manner. E. coli HB101 was inoculated in the LB broth [composition; 1% tryptone, 0.5% yeast extract, 1% sodium chloride (pH 7.5)], and cultivated overnight at 30°C . One milliliter of the resulting culture was inoculated in 100 ml of LB broth and further cultivated at 30°C until the turbidity at 800 nm of the culturε reached approximately 0.6. After standing for 30 minutεs in ice watεr, the cells were collected by cεtri fugation. They were resuspended in 50 ml of 50 mM calcium chloride, and allowed to stand for 60 minutes in ice water. Then, the cells were collected by centri ugation and again suspended in 10 ml of 50 mM calcium chloride containing 20% glycerol. To this cell suspension, the expression plasmid pHNPlOl was mixed and incubated sequentially in ice water for 20 minutes and at room temperature for 60 minutes. Then, the LB broth was added to the cell suspension, and it was cultivated under shaking at 37°C for 60 minutes. An aliquot of the rεsulting cell suspension was seeded on the LB agar (1.5% agar) plates containing ampicillin at 25 / n g/ml. After cultivation at 37"C overnight, ampicillin- resistant colonies were sεlεctεd to obtain transformants. Onε of the transformants was named E.coli HB10l/pHNP101 and it was used for production of the human NCF polypeptidε. (3) Production of human NCF polypeptide E. coli HBlOl/pHNPlOl obtained as above was cultivated in the LB broth overnight at 37"C . The culture was inoculated in 100-fold volumes of the nutriεnt mεdium mεntioned in Example supplemented with 3-indo1eacry1 ic acid 20 g/ml. The cultivation was done at 35°C for 28 hours. The cells were collected by centri fugation, and suspended p*.0**m*w~. , _» ,____. in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1% lysozyme ' and 30 mM sodium chloride. The suspension was allowed to stand in ice water for 30.minutes. Further, freezing in a dry ice/ethanol bath and thawing at 37°C were repeated to disrupt the cells. After adding 1/50 volume of 10% eth leneimine polymer, a clarified cεll-extract was obtained by centrifugation. To this cell-extract, ammonium sulfate was added to give a 70% saturation, and the formed precipitate was collected by centrifugation. The precipitate was dissolved in distilled water and it was dialyzεd against 5 mM phosphate buffered saline (pH 6.5). The dialysate was applied onto a column of Sephacryl S-200, and the fractions containing polypeptides having about 6 to 10 kD were collected and pooled. The molecular sizes of polypeptides in each eluate were measurεd by SDS- polyacrylamidε gel elεctrophorεtic analysis. Thε poolεd fraction was dialyzed against PB mentioned in Example. Thεn, the dialysate was applied onto a column of GM- Sepharose GL-6B previously equilibrated with PB. The column was washed with PB, and eluted with a linear gradient of sodium chloride molarity from 0 M to 0.5 M in PB. The fractions containing the human NCF polypeptide were collected and pooled, and concentrated by ultra- filtration. Further, the concentrate was subjected to gel filtration on Toyopearl Hf-55 column to obtain the highly purified human NCF polypeptide. "" Referential Example 3 Construction of an expression vector pEP205 Plasmid pBR322 was digested with rεstriction endonuc1eases Aval and Pvul I, and the resulting larger DNA fragment (about 3.7 kbp in size) was isolated. After filling-in its cohesive ends to blunt-ends with E.coli DNA polymerasε I (Klεnow fragmεnt) in thε presence of dGTP, dATP, dGTP and dTTP, both ends were ligated by T4 DNA ligase to construct a new plasmid vector (designated pBRS6), which was delεtεd a copy numbεr regulatory gene region located near the replication origin of the plasmid pBR322. The plasmid vector pBRS6 was digested with restriction endonucleases EcoRI and * Pstl, and a smaller DNA fragment containing an upstream region of the ampici 1 in-resistance gene (about 0.75 kbp in size) was isolated. The resultant DNA fragment is refεrrεd to as thε Amp (Pst I -EcoRI) - fragment. This Amp (PstI-EcoRI) -fragment was cloned in a phage vector M13mpl8 as mentionεd in Example. By using the rεsultant rεcombinant phage DNA, one basε (T) in the nucleotide sequence of the Amp (PstI-EcoRI) -fragment was changed to another base (C) by the site-directed mutagenesis according to the mεthod as mεntionεd in Rεferential Example 2, in order to eliminatε the specific nucleotidε sequence (AAATTT) recognizable with the restriction endonuc1easεs Dral . "1 ? ■ __> .. :_ • : TC" The single-stranded phage DNA containing uracils was isolated from thε culture medium of E. coli GJ236 infectεd with thε above rεcombinant phagε DNA. As a mutagεnic primer, the oIigodεoxyribonuc1εotidε rεprεsented by the following formula [G] was chemically synthesizεd. 5'-GAGAACTTTGAAAGTGGTC-3' [G] Thε phosphorylatεd primer was annealed with the uraci1-containing DNA templatε. According to the method describεd in Rεfεrεntial Example 2, the desirεd mutagenised double-strandεd DNA was isolatεd. Thε rεsultant mutagenized double-stranded DNA was digested with restriction endonuclεasεs Pstl and EcoRI in order to isolate a DNA fragment corresponding to the Amp (Pstl-EcoRI) -fragment as mentioned above, but not containing the restriction endonucleasε Dral clεavage recognition sequence [hereinafter refεrrεd to as thε mutated Amp (PstI-EcoRI) -fragment] . The mutated Amp (Pstl- EcoRI) -fragment -was ligated with the larger DNA fragment isolated from the vector pBRS6 by digestion with restriction endonucleases EcoRI and Pstl, in order to construct a new vector which wras eliminated the Dral clεavagε recognition sequence in the ampicillin resistance gene of the plasmid vector .pBRS6. This new vector is designated pBRS601. Further, this new vector pBRS601 vas digested with restriction endonucleasε Dral, and thε rεsulting largεr DNA EUBSTΠΌTE SH iE~ ~ fragment was ligated with Smal linker (Takara Shuzo Co. , Ltd. , Japan) by T4 DNA ligase to construct a new plasmid vector. This resulting new plasmid vector is a derivative of plasmid pBRS6 and is not containing any recognition sequεnce for the restriction endonuclεase Dral . This nεw plasmid vector is dεsignated pBRS602. Thε nucleotide sequence of the Smal linker is shown below. 5' -CCCGGG-3' Further, this new vector pBRS802 was digested with restriction endonuclεasε Aat 11 and Sail, and thε resulting largεr DNA fragmεnt was isolated [herεinaftεr rεfεrrεd to as thε pBRS602 (Aat11-Sal I) -fragmεnt] . Sεparatεly, an expression plasmid pHIPH383a for producing human inter leukin-1 a. as mentioned in Referential Example 4, was digεstεd with rεstriction endonucl eases Aat 11 and Sal I , and resulting DNA fragment containing E.coli tryptophan promoter sequεnce and coding region for human inter1 εukin- 1 α was isolated. This resulting DNA fragment is referred to as the trp promoter/IL-1 -DNA fragment. This trp promotεr/IL- 1 a. -DNA fragmεnt was ligatεd with thε pBRS602 (Aat11 -SatI) -fragmεnt by T4 DNA ligase to construct a new expression plasmid (seε Schεma 2). This nεw εxpression plasmid is designated pEP205. r * . »r-n'-:"H rTππ ■ r_r_,_, c._->y_._.r__ Referential Examplε 4 Construction of an expression plasmid pHIPH383a The cloned cDNA encoding human interleukin-1c_ precursor polypeptide was isolated according to the method described in European Patent Publication No-0188920. From the recombinant plasmid pHL4 containing human interlεukin-1 precursor cDNA (Furutani,Y. , et al., Nucleic Acids Rεs., L3, 5869, 1985), thε cDNA insert was isolated hy digestion with restriction endonucleases EcoRI and BstNI, to isolate a DNA fragment (411 bp in size) containing a middlε portion of thε coding region for the mature human interleukin-1 a . Thε isolatεd DNA fragment is corresponding to the nucleotide sequence from base No.398 to No.808 in Table 5 s own in European Patent Publication No.0188920. This DNA fragment was sequentially ligated by T4 DNA ligase with chemically synthesized ol igodeoxyribonuclεotidε adaptors represented by the following formulae [H] and [J] . The resulting DNA fragment is referred to as the SD-IL-1- fragment. The synthetic ol igodeoxyribonucleotide adaptor [Hi was prepared by sequential ligation of the following five kinds of DNA fragments represented by formula [a] — [e] . 5* -AAGTAGTAGGGAAGTTCAG 3'-TTGATCATGCGTTCAAGTGCATT [a] 5'-GTAAAAGGAGGTTTAAA 3' -TTCCTCCAAATTTAATAC [b] 5' -TTATGTCATCACCTTTTAG 3' -AGTAGTGGAAAATCGAAGG [o] 5' -CTTCCTGAGCAATGTGAAATACAACTTTA 3' -AGTCGTTACACTTTATGTTGAAATACTC [d] and 5' -TGAGGATGATCAAATAGG 3'-GTAGTAGTTTATGGTTAA [e] A base sequence of the formula [J] was as follows; 5'-AGGCGTGATGAGTCGA 3'-CCGCACTACTGAGCTCTAG [J] Separately, an expression vector pEP3Q2 (Furutani, Y. , et al., Nucleic Acids Res., J_3 , 5869, 1985) was digested with restriction endonucleases Hpal and BamHI, and the resulting larger DNA fragment containing E.coli tryptophan promoter sequence and an ampicillin resistance gene, was isolated (hereinafter referred to as the EP302 vector-DNA fragment). . The EP302 vector-DNA fragment was ligated by T4 DNA ligase with SD-IL-1-fragment prepared as above to construct an expression plasmid pHIPH383a for producing the mature human inter1eukin-1 a polypeptide (see schema 3). S UBSTITUTE SHEET Tab l ε 1 SerAlaLysGluLeuArgGysGlnCysI leLysThr TyrSerLysProPheHisPro ysPhelleLysGlu LeuArgYallleGluSerGlyProHisCysAlaAsn ThrGluIlellεValLysLεuSerAspGlyArgGlu LeuGysLeuAspProLysGluAsnTrpValGlnArg ValValGluLysPheLeuL sAr AlaGluAsnSer formula [I] STITUTΞ SHEET Tablε 2 Nucleotidε Sεquence of Human NCF Precursor cDNA and Its Deduced Amino Acid Sequence MetThrSerLysLeu AlaValAlaLεuLεu 10 ATGACTTCGAAGCTG GCCGTGGCTCTCTTG 30 AlaAlaPhεLeuI le SerAlaAlaLeuC s 20 GCAGCCTTCCTGATT TCTGCAGCTCTGTGT 60 GluGlyAlaValLεu ProArgSεrAlaLys 30 GAAGGTGCAGTTTTG CCAAGGAGTGCTAAA 90 GluLεuArgCysGln C sI 1εLysThrTyr 40 GAACTTAGATGTCAG TGCATAAAGACATAG 120 SεrLysProPhεHis ProLysPheI 1εL s 50 TCCAAACCTTTGCAC CCCAAATTTATCAAA 150 GluLeuArgVallle GluSεrGlyProHis 60 GAACTGAGAGTGATT GAGAGTGGACGACAC 180 CysAlaAsnThrGlu 11eIleValLysLeu 70 TGCGCCAACACAGAA ATTATTGTAAAGCTT 210 SerAspGlyArgGlu LeuCysLeuAspPro 80 TCTGATGGAAGAGAG CTCTGTCTGGACCCC 240 LysGluAsnTrpVal GlnArgValValGlu 90 AAGGAAAACTGGGTG GAGAGGGTTGTGGAG 270 LysPhεLεuLysArg AlaGluAsnSer 99 AAGTTTTTGAAGAGG GCTGAGAATTCA 297 +c>*:π•*•#n*i•*_♦_.-Tu ά'T t W. t-r s ^r Schema 1 Construction of an expression plasmid pHNPlOl Human NCF cDNA clone: pDG19-l. -5 j— digested with Pstl and EcoRI Pstl-EcoRI-DNA fragment [— cloned into 13m 18 rector j— infected E.coli JM105 Recombinant phage — infected E.coli CJ238 — cultivated in uridine containing medium uracil containing DNA template j— annealed a mutagenic 33-mer primer [E] AAATT T A T T T G GTTTTGCCAAGG AGTGCTAAAG —TCCACGTCAAAACGGTTCC-TCACGATTTC— Uracil containing DNA template extended the primer with T4 DNA polymerase ligated the ends with T4 DNA ligase Heteroduplex j— transformed E.coli J 105 Mutated Double-Stranded DNA j— digested with Dral and EcoRI NCF (Dral-EcoRI)-fragment - to be continued - E HEE" Schema 1 (continued) EcoRI Dral I Xhol MetSerAlaLys AlaGlu AsnSer | AAATTATGAGTGCTAAAG- ■ -GCTGAG AATTCATGATGAC TTTAATACTCACGATTTC- ■ -CGACTCTTAA GTACTACTGAGCT NCF (Dral-EcoRl)-fragment Synthetic DNA [F] { — ligated with T4 DNA ligase NCF (Dral-XhoI)-fragment Expression Yector Expression Plasmid pHNPlOl S SSf«ιu»- -π.*-.*-• Schema 2 Construction of an expression vector pEP205 Plasmid pBR322 j— digested with Aval and Pvull Larger DNA (3.7 kbp)-fragment - filled in the cohesive ends to blunt-ends with DNA polymerase I (Klenow fragment) — ligated with T4 DNA ligase V Pl Amp (Pstl-EcoRI)-fragment pBRSB-larger - cloned into M13mpl8 vector fragment* - infected E.coli JM105 Recombinant phage 1— infected E.coli GJ236* — cultivated in uridine containing medium uracil containing D A template I— annealed a mutagenic oligonucleotide primer [G] CAGAACTTT AAAGTGCTC •GTGTATCGTCTTGAAA TTTCACGAGTAGT- T uracil containing DNA template extended the primer with T4 DNA polymerase ligated the ends with T4 DNA ligase Hetεroduplεx to be continued - S UBSTITUTE SHEET Schema 2 (continued) Hεteroduplex j— transformed E.coli. J 105 Mutated Double-Stranded DNA j— digested with Pstl and EcoRI Plasmid Vector pBRS801 j— digested with Dral Largεr Plasmid Yector pBRS602 I— digested with Aatll and Sail PBRS602 (AatΙI-SalI)-fragment ion Plasmid 3a digested with Aatll and Sal oter/IL-1 -DNA fragment th T4 DNA ligase Expression Yector pEP205 * Schema 3 Construction of an expression Plasmid pHIPH383a Human IL-lα cDNA: pHL4 [— digested with Pstl cDNA insert ij—- ddiiggeesstted with EcoR] * • aanndd BBsstNI Synthetic DNA EcoRI-Bst I-fragment adaptor [H] SD-IL-1-fragment PEP302 Expression Plasmid pHIPH383a

Claims: What is claimed is: 1. A method of preparing an active human neutrophil chemotactic factor polypeptide which comprises cultivat a host harbouring an expression vector encoding the sai polypeptide, and treating the precipitate fraction from transformant-homogenatε hy urεa or guanidinε hydrochlor 2. A mεthod of preparing the said polypeptide accordi to claim 1 wherein the active human neutrophil chemotac factor polypeptide is a polypeptidε consisting of an am acid sequence represεntεd by the formula. SεrAlaLysGluLεuArgGysGlnCysI lεLysThr TyrSerLysProPheHisProLysPhel lεLysGlu LeuArgVallleGluSerGlyProHisGysAlaAsn ThrGluIlelleValLysLeuSerAspGlyArgGlu LεuCysLεuAspProLysGluAsnTrpValGlnArg ValYalGluLysPhεLεuLysArgAlaGluAsnSεr 3. A method of preparing the said polypeptidε accordin claim 1 wherein the host is Escherichia coli.

Inventors: FURUTA RYUJI, MATSUSHIMA KOUJI, YAMADA MASAAKI, YAMAGISHI JUNICHI, FURUTA, RYUJI, MATSUSHIMA, KOUJI, YAMADA, MASAAKI, YAMAGISHI, JUNICHI
Applicants: DAINIPPON PHARMACEUTICAL CO, US GOVERNMENT
Application year: 1989
Main classification: C12N15
IPC codes: C07K1, C07K14, C12N15, C12P21, C12R1