JPH04500602A - Synthetic HIV protease gene and its expression 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] Synthetic HIV protease gene and its expression background field of invention The present invention relates to synthetic genes and their expression products. Specifically, the present invention This invention relates to synthetic protease genes and their expression products.

Description of related technology The presence of protease proteins in purified pyrion preparations is an immunological demonstrated only by method.

HIV protease sequences or fusion proteins with gag and pol sequences are , expressed from bacterial viral DNA. Examples of such disclosures include: 1. Henderso Henderson et al., 1988, “Human retroviruses, cancer and AIDS: Prevention and Treatment ()Iuian Retrovluses, Ca ncer and ＾IDs：＾pproach to Prevention and Therapy)”, Di Bolognesl/(D, Bolognesl ) Supervised by Alan R, Li5s Inc.

) Published by 2nd York, 2nd York State, 135 HIV Protease. The primary sequence was determined by protein analysis and nucleotide sequencing of proviral DNA. It has been determined. Therefore, this protease is 297b of the HIV provirus. It is a 99 amino acid long protein hooded by a p long chain. It has been determined. Traditional experiments on protease genes and their expression by using nucleotide sequences cloned from viral cDNA. It was held. The present inventor's research using synthetic DNA has revealed that the nuclei of proviral DNA The nucleotide sequence and determined amino acid sequence have been proven to be accurate. .

Complete nucleotide pol oven readout of HI V-1 proviral DNA The sequence coding of the protease in the coding frame was determined by previous analysis. has been determined and corresponds to nucleotides 1609-1906. N-terminus and The C-terminal amino acids are proline and phenylalanine, respectively. HIV- This sequence, encoding a 199 amino acid protease, is a 297 bp sequence as follows: It is of length.

260 270’ 280, 290 Infectious human immunodeficiency virus (HI Synthesis encoding a specific enzyme or protease essential for the completion (replication) of V) There are no NA sequences. This DNA sequence may be present in bacterial and/or eukaryotic cells. We expressed this protease by recombinant methods and determined its biochemical and physical properties. By producing enough protease to determine how to design and produce a potent inhibitor of this enzyme, and is desirable in preventing the production of infectious HIV particles.

Summary of the invention The present invention is a gene encoding a human immunodeficiency virus protease.

This gene is related to a protease essential for human immunodeficiency virus infectivity. Consists essentially of synthetic nucleotide sequences.

This protease is preferably essential for HIV-1 or HIV-2 infectivity. These are the proteases of viruses.

A preferred embodiment of the invention is a synthetic gene for the expression of HIV-1 protease. The second coding sequence is represented by the upper row of the nucleotide sequences.

Brief description of the drawing Figure 1 shows expressed HIV proteas analyzed in Western blots. represents ze.

FIG. 2 illustrates a method for the synthesis of the HIV-1 protease gene.

3' overhang is the case below. The complementary strand (not shown) matches the coding strand3 'It had an overhang.

FIG. 3 illustrates the induction of genes at various times.

Figure 4 illustrates the activity of a protease expressed using a synthetic peptide aSa substrate. It is something to do.

Description of preferred embodiments The present invention is a synthetic NA sequence encoding a specific enzyme or protease.

This protease is essential for the infectivity of human immunodeficiency virus (HIV). It is The genes of the present invention can be used recombinantly in bacteria and/or eukaryotic cells. expression of proteases and protein for biochemical and physical characterization. It is desirable for the production of commercially desired amounts of rotease. This characterization is , required for the design and production of potent inhibitors of this enzyme. The present invention Toroviruses, such as HIV-2, human leukemia viruses, such as HTLVI, I I and other human and animal R that cause leukemic sarcomas and other malignancies. NA-containing viruses, including the synthesis and expression of protease genes.

The nucleotide sequences of preferred embodiments of the invention are described by Ratner et al. Obtained from the literature listed above. pol orb encodes the protease of HIV-1 The sequence of the read frame corresponds to nucleotides 1609-1906 .

The N-terminal and C-terminal amino acids are proline and phenylalanine, respectively. It is. This sequence encoding a 99 amino acid protease consists of 29 amino acids as described above. It is 7 bp long. In the present invention, 1 in the above and other genes A desired protease can be expressed by simply substituting the above bases. It is possible to produce a mutant gene.

This sequence was synthesized as five fragments using a DNA synthesizer. this Complementary strands corresponding to the five fragments were also synthesized. 3' over 4 bases -Hang efficiently connects each of these five fragments and connects the protea. Appropriate sequencing was performed to obtain the exact coding sequence of the enzyme gene. genetics Add the nucleotide ATG to the fragment corresponding to the 5' end of the child, and add the nucleotide ATG to the 3' end of the fragment. added TAA.

A prokaryotic expression vector is used to perform cloning and generate a protein encoding the protease. The synthetic sequence was expressed. This expression can be performed in prokaryotes (bacteria) or other suitable expression systems. It can be carried out. Recombinant clones are targeted to span the internal region of the protease gene. colony hybridization using the identification fragment (62 bp). Screened by. Further examine positive colonies for insert size. analyzed. Clones that were positive were expressed and analyzed by Western plot. , protein products were determined using specific antibodies. An example is shown in FIG.

Three of the clones screened so far are illustrated in Figure 1. It expresses a 11,5 kd product that reacts with specific antibodies as shown in FIG. Identified.

The conditions for protease gene induction were investigated using E, coil, and the optimal conditions were obtained. . We have demonstrated that this gene product cleaves both synthetic and natural substrates. This gene product has specific protease activity. It was revealed that This enzyme can be analyzed using special techniques such as affinity chromatography. Purified by ram chromatography method. According to the method of the present invention, protea structure of enzyme, mechanism of its activity, and AIDS caused by virus to obtain therapeutic specific inhibitors of this enzyme for the treatment of diseases such as Sufficient active protease can be produced to study. of the present invention In other cases, other proteins such as the genes listed below for HIV-2 protease A gene that expresses tease can be used.

CCTCAATTCTCTCT! TGG input input G input CCAGT input GTCACA GCA'I'AC in'ITGλGGGTC in GCCAGTAGAAGTCTTG Enter TTAG into CACAGGGGCTGAC and enter ACTCAATAGTAGCAGG. ATAG entered GTTAcGGAACAA'ITATAGCCCAAAAT entered GT AGGGGGλATAGGGGG entered TTC entered T entered ATAC (: entered AGGAAT ATλυWTGTAG entered AATAG entered GTTCTAλATλAAAAGGTA CGGGCCACCATAATGACAGGCGACACCCAATCAACA T’ITTTGGCAGAAATATTCTGACAGCCTTAGGCATG TCATT input τCTAC FIG. 1 shows expression of HIV protease in E. coli. Appropriate expression base Transformed cells with synthetic sequences of HIV protease are induced in vectors to induce bacterial The lysate was subjected to electrophoresis using 5DS-PAGE. Nitrocellulose membrane for proteins After transfer, immunoplots were performed using a specific antibody against HIV protease. I performed the ding method.

Detection of Ag-Ab complexes was performed using 1125 protein A. autoradiolog Rough lane A represents E. coli transformed with the plasmid; B and C are synthetic NAs carrying plasmids encoding HIV protease. Represents E, colt transformed with. On the left, the data is shown in kilodaltons. There are white matter molecular weight markers.

The 11.5 kd band is protease.

The synthetic NA of the present invention does not require any manipulation of (infectious) viral substances. , other methods can eliminate the limit on the amount that can be obtained.

Example Examples were performed using the materials and methods described below.

Plasmids, bacterial chains and chemicals: Plasmid P, a prokaryotic expression vector KK233-2 was purchased from PharIIlacia.

Using P■233-2, 1aq-q host, E, coij JM105 or R Transformation was performed in B791. Cells were grown in M9 minimal medium containing lug/l thiamine. After selection in the ground, these were used for transformation. Used for oligonucleotide synthesis All chemicals were purchased from Applied Biosystems, Inc. Bfosystea 1nc, ). T4 Polinuku Reotide kinase, DNA ligase and E. coli DNA polymerase The Freneau fragment of I is a two-year-old English biolapse (New (England Biolabs). restriction endonuclease, PMSF and PTG are from Boehringer Mannheim (Boehringer Mannheim). MannheI■), Bethesda Research Laboratories (Bethes da Re5earch Laboratories) and Promega (Prom ega), respectively.

DNA synthesis, plasmid construction and screening: DNA fragments are Synthesized using Ibii (ABI) DNA synthesizer (381 human type). All The synthesized fragments were electroporated in a 12% polyacrylamide/8M urea sequencing gel. Purified by electrophoresis. DNA is visualized by ultraviolet shadowing, and the entire length of the DNA is Fragments were eluted from gels known in the art. full length fragmen The purity of the sample was checked using standard techniques.

Appropriate complementary fragments were mixed in equimolar concentrations, annealed, and described elsewhere. Kinase treatment and ligation were carried out. The efficiency of ligation is determined by polyacrylamide gel electrodes. Monitored by pneumophoresis. Linearized plasmid and appropriate concentration of protease enzyme The gene was ligated and used to transform E. coli JM105. recombinant black colonies using an a2bp fragment labeled by kinase treatment. /\Ibridize/! Screened by In. vA using the boiling method Plasmid DNA was isolated on a small scale from the recombinant clones, and the insert size was E. 3′ recess using the Flenow fragment of E.coli DNA polymerase Visualization by autoradiography after labeling the recessed ends. did.

Antibodies against HIV protease.

The polyclonal antibody was tested in rabbits with (1) amino acids 1 to 99 of HIV-1 protease. a complete synthetic sequence of the acid and (1j) preferably a sequence corresponding to the C-terminus of the protease; Generated for Lidecapeptide.

Grown to proliferative phase, induced, and lysed by sonication. whole cell extract were analyzed by NaDodSO4/PAGE and subjected to immunoplot analysis.

Method for analyzing expressed protease activity: Oligopeptides were prepared using a previously reported method (Copeland). and Oroszlan, 1981). It was synthesized using a laser (Applied Blosystems) 430^ model). cleavage The product was transferred to a uBondapack C1g column (Waters As5oci ates) by RP-HPLC. Amino acid composition of peak fractions using a Pico-Tag amino acid analyzer (Waters As5). ociates).

Example 1 This example represents a preferred embodiment.

The nucleotide sequence of the protease gene was determined by the method of Ratner et al. It was decided by. pal open reading frame of protease gene The sequence begins at nucleotide 1609, ends at 1906, and consists of 99 amino acids. code. This sequence and its complement are approximately 60 bases as shown in Figure 2. was synthesized as five separate fragments. 4 bases (shown in the bottom case) The 3' overhang of fragments to selectively ligate the components. Translation start codon ATG and A stop codon, TAA, was added to the appropriate ends of the protease gene.

Add the sequence to the gene so that it has cohesive ends that fit into the Neo I restriction enzyme site. A protrusion was attached to the 5' end. In addition to the 5' overhang at the 3' end of the gene, H ind3 compatible ends were attached. The complementary strand (not shown) is the 3' strand that matches the coding strand. It was equipped with a bahang.

Three clones (P RC, PR-H, and PR-J) to determine optimal induction conditions for the genes. selected.

FIG. 3 shows an example of Western plot analysis of gene products.

Clone PR-C containing the coding sequence for protease was induced and expressed. Ta. Proteins (75 μg of bacterial extract) were electrophoresed in NaDodSo4/PAGE. (i) 1-99 amino acids of HIV-1 protease. The complete synthetic sequence of amino acids and (if) the trinucleotide corresponding to the C-terminus of this protease. A mixture of two protease-specific rabbit polyclonal antibodies against decapeptide. The compound was subjected to immunoplot analysis. FIG. 3A shows 0 at various times, Shows gene induction by 4raM I P TG. Figure 3B shows a 30 minute invitation. Show guidance. With increasing concentration of inducer IPTG, 1 to 5 respectively Concentrations expressed in IIIM, 0.28.0.56.1, 12.2.24 and 4.4 Represents 8. Figure 3C shows cells induced with 1mM IPTG for 60 minutes and treated in various buffers. Analysis after lysing the cells is shown. B1 is 50IDM Tris-HC1, pH 7, 0,150dNaCl, 1mM EDTA, ld PMST, 1mM DTT and and 0.5% NP-40. B2 is NaCl and ED Same as B1 except that there is no TA. B3 is 5hM potassium phosphate, p in H6,0, haMPMSF and 1mM DTT. B4 is p)1 It is the same as B3 except that is 6.5. Position protein molecular weight markers , shown on the left in kilodaltons.

E, colt with plasmid PR-C in Luriapros 0.4 A60 After growing to an optical density of 0 nIg, induction was performed for various times, and Figure 3A. IPTG (isopropyl-β-D-thiogatin) at a concentration of 0.4+aM as shown in Expression was achieved from the tre promoter by adding lactopyranoside).

The cloned gene produces a single unfused protein band of 11.5 kd. It appeared. Expression was maximal after 30 minutes of induction. This level will be reached after 60 minutes. It decreased to about 25%. No expression was detected after 120 minutes of induction and at 0 minutes. won. This form of induction was observed in other clones analyzed (PR-H and PR-H). J) (not shown).

The results of induction for 30 minutes at various inducer concentrations are shown in Figure 3B. It is. I Induction with PTG in the range of 1mM to 4mM showed maximum expression . Similar data were obtained for clones PR-H and PR-J (not shown). figure).

In order to select conditions for efficiently solubilizing the promoter and performing enzyme analysis, we After optimal induction with MIPTG, cells (clone PR -C) was dissolved. 50rBM Tri-HCL pH 7,5,1aMDTT.

1mM PMSF and 0.5% nonidet P-40 buffer When ultrasonication was performed in a liquid system, 50-70% of the protease was contained in the soluble fraction. Release was observed (Figure 3C). This determines the content of the expression product, soluble Calculated by Western plot analysis of extract and insoluble precipitate.

Demonstration of specific proteolytic activity Figure 4 shows the activity of the expressed protease using synthetic peptides as substrates. It is something. Protease analysis was performed on clone PR-C, derivatives (A%B, C) and and underivatized (D) and a control containing only plasmid PKK233-2. The experiments were carried out at 37° C. with 22.5 II g of bacterial lysate obtained from cell lines (not shown).

The nonapeptide was added to the reaction buffer (0.25M potassium phosphate), p)I 7.0 ．． 0.5% (v/v) NP40.5% (v/v) glycerol, 5aM dithio Trait and ZMN were used as substrates in Cl. 25j1 each were collected at 0 hours (A), 1 hour (B), 3 hours CC) and 6 hours later. , analyzed by RP-HPLC. S represents the substrate, Pl and P2 are its represent cleavage products 1 and 2, respectively.

In order to evaluate and evaluate the activity of the cloned HIV-1 protease, p17-p24 cleavage site (Henderson et al., 1988 A synthetic nonapeptide corresponding to 2011) was used as a substrate (4E). in reaction buffer The substrate was mixed with fractions of various cell extracts (see description of Figure 4 above) and Incubated at ℃. Equal volumes of the incubation mixture for various times and analyzed by RP-HPLC. The substrate in the zero time sample is shown in Figure 4A. The peak eluted as a single peak.

Correlates with a significant decrease in the substrate peak after 1 hour incubation. Two newly appearing peaks P1 and P2 are seen, which are standard products. Next By performing amino acid analysis of the collected peaks, product 1 and product 1 were determined. Product 2 is Tyr-Pr, the natural cleavage site determined as shown in Table-1. It was shown to correspond to the expected cleavage product indicating cleavage of the o bond. 3 hours Prolonging the incubation at The peak height increases substantially, indicating that hydrolysis of the Tyr-Pro bond progresses. was.

However, the absorption of the tetrapeptide Pro-118-val-Glu-N)12 The luminous intensity is substantially lower than the absorbance of the pentapeptide with a free C00H-terminal tyrosine. The product 1 peak appeared to be small as expected.

After 3 hours of incubation, products 1 and 2 increase and the substrate peaks are paired. A corresponding decrease was observed.

Uninduced cells, clone PR-C (Figure 4D) and control cells (control reaction using an extract from the sample plasmid PKK233-2 (data not shown). No cleavage products were detected in the reaction. After 6 hours of incubation However, the substrate peak did not decrease, indicating that the nonapeptide was It has been shown that it is resistant to decomposition by This indicates that the amount of urinary chloride in the crude extract to analyze viral protease activity and to facilitate purification and isolation of proteases. makes this substrate particularly useful.

Amino acid composition data for the substrate and its cleavage products are shown in Table-1. observed The amount of amino acids obtained clearly corresponds to the expected amount, indicating that cleavage occurs in the gag precursor. occurs at the predicted cleavage site of the synthetic peptide, which corresponds to the body's p17-p24 site. It shows that

Special Table Sen4-500602 (6) M, W, A B C d ゛□－轡髫一－－１１１・ fragment 1 5’ CCTCAGATCACTCTTTGGCAACGACCCCTCGTC ACAATAAAAGATAGGGGGGCAActaafragment 2 AG　GAAGCTCTATTAGATACAG　GAGCAGATGATAC AGTATTAGAAAGAAATGA(,TTTGCCAGfAa g at fragment 3 GGAAACCAAAAAATGATAGGG G GAATTGGAGGTTT TATCAAAGTAAAGACAGTATGATCAg a@t a fragment 4 CTCATAGAAAT CTGTGGACATAAAAGCTATAGGTAC AGTATTAGTAGGACCTACACCTGT ca≠■ fragment 5 ATAATTGGAAGAAAT CTGTTGACTCAGATT GGTT G CACTTTAAAr port 13'FIG, 4E 9 Yen/￥ (206 nm) Submission of translation of written amendment (Article 184-8 of the Patent Law) 1. Indication of patent application PCT/US 89102996 2. Name of the invention Synthetic HIV protease gene and its expression method 3, patent applicant Address: Springfield, Port, Roya, Virginia, United States le, road, 5285 Name: United States of America 4. Deputy manager (Postal code 100) The scope of the claims 8. The following synthetic nucleotide sequence or a sequence functionally similar to this sequence: A recombinant protease protein produced by a recombinant vector.

CCTCAGATCA CTCTTTGGCA ACGACCCCTCGTCA CAATAA AGATAGGGGGG6゜ GCAACTAAAG GAAGCTCTA TAGATACAGG AGCAGATGAT ACA GTATTAG AAG entered AATGAG’I’rTGCCAGGA 1 piece 0 140 150 160 170AGATGGAAACCA entry A entry T G input T AGGGGG input TT GGAGGTTrTA TCAA input GT input AG TSAT with ACAGT 190 2qO210220230 CAGATACTCA TAGAAATCTG TGGACAT entrance A GCT ATAGGT included C included GT included ↑TAGTAGGACCTACA CCTGTCAACA TAATTGGAA, G input λ input TCTGTTG AC TCAGA’I’rCGTTGCACTTT entry ATTTT 9. (1) The following sequence: CCTCAGATCA CTCTTTGGCA ACGACCCCTCGTCA CAATM AGATAGGGGGC entered ACTAAAG 70 80 90 100 1 yu 0 GAAGCTCTA TAGATACAGG AGCAGATGAT ACA GTAT “ηAG person AGA entry ATGAGTTTGCCAGGA AGATGGAAAACCMAAATGAT AGGGGGAA? r GGAGG T’r’M’A TCAAAGT entered AGAC entered GTATGAT CAGATACTCA TAGAAATCTG TGGACAT entered M GCTA T enter GGTA CAGTAT'I'AGT person GG enter CCTACA Or synthesize a sequence with a structure similar to this sequence, and (2) the recombinant product produced in step (1). inserting the vector into a host cell; (3) express protein; (4) The protein according to claim 8, comprising the step of purifying the protein. Method for producing rotease.

10. Claim 9, wherein the protein is purified by affinity chromatography. The method described in.

1. Contains a synthetic gene with the following sequence or a synthetic sequence structurally similar to this sequence. Become a vector.

CCTCAGATCA CTCT'! 'TGGC input CGACCCCTCGT CACλATAA AGATAGGGGGGCAACT entered AAG Gλ input GCTCTJ% TT input G input T8 CAGG input CCAG input TG input T λCλ GT enters TT enters G enters λG enters ATG enters GTTTGCCAGG enters AAAACC input to AGATG λ input to TGAT GGGGG input ATT GG input G GTTTTA TC input λ input GT input AG person CAGTXTGAT CAGATACTCA TAGAAATCTGTGGACATAAAGCT ATAGGTA CAGTATTAGTAGGACCTACA CCTGTCAACA TAATTGGAAG AAATCTGTTG ACT CAGATTG CACTTrA enters GTT ATTTr 12. Protein produced by the method according to claim 9.

international search report

Claims (7)

[Claims] 1. Synthetic nucleotides of protease essential for human immunodeficiency virus infectivity A human immunodeficiency virus protease-encoding gene consisting essentially of the sequence Denji. 2. Claims in which the protease is essential for retrovirus infectivity The gene described in item 1 below. 3. Retroviruses include HIV-1, HIV-2 and HTLV human leukemia virus The gene according to claim 2, which is selected from the group consisting of: 4. consisting essentially of a synthetic double-stranded nucleotide sequence whose coding sequence is as follows: A gene encoding the human immunodeficiency virus protease. [There is an array] 5. A set containing the synthetic gene for protease, which is essential for retrovirus infectivity. The modified vector is inserted into a host cell, the gene is expressed, and the protease is A method of expressing proteases consisting essentially of isolating them. 6. Retroviruses include HIV-1, HIV-2 and HTLV human leukemia virus 6. The method of claim 5, wherein the method is selected from the group consisting of: 7. The retrovirus is HIV-1, and the gene contains the following nucleotides. 6. The method of claim 5, comprising an array. [There is an array]