KR100557343B1 - Method for preparing a recombinant inhibitor of angiogenesis from transformed insect cells - Google Patents

Abstract

The present invention relates to a method for efficiently preparing an angiogenesis inhibitor using an insect cell transformed with a recombinant vector containing a gene encoding an angiogenesis inhibitor. The transformed insect cells according to the present invention can express angiogenesis inhibitors in a higher yield than other existing expression systems.

Angiogenesis inhibitors, endothelial cell proliferation inhibitors, transformation, insect cells

Description

Method for preparing a recombinant inhibitor of angiogenesis from transformed insect cells             

1 is a schematic diagram of an expression vector pMT / BiP / T-V5-His used to transform Drosophila melanogaster S2 cells to express recombinant tumstatin.

Figure 2 shows the results of Western blot analysis of the expression of toomstatin in untransformed drosophila melanogasster S2 cells and transformed drosophila melanogasster S2 cells. Lane 1 is a cell portion of untransformed cells; Lane 2 shows a media portion of untransformed cells; Lane 3 is a cell portion of transformed S2 cells; Lane 4 is a media portion of transformed S2 cells; Numbers on the left are molecular weight markers (kDa); Arrows indicate recombinant tumstatin.

Figure 3 is the result of performing Western blot of the cell fraction and the medium portion like the non-transformed cell line to confirm the expression of the lithium statin gene in the transformed High Five cell line. Lane 1 shows cell fraction of untransformed cell line; Lane 2 is media of untransformed cell line; Lane 3 shows cell fraction of transformed cell line; Lane 4 is media of transformed cell line; Numbers on the left indicate protein molecular weight; The arrow on the right represents the recombinant lithium statin protein.

FIG. 4 shows the results of investigating His-labeled tumstatin purified from the media by culturing transformed Drosophila melanogasster S2 cells by SDS-PAGE (A) and Western blot analysis (B). M is a molecular weight marker; Lanes 1 and 2 show the results before and after affinity purification of the media portion of transformed S2 cells, respectively; Arrows indicate recombinant tumstatin protein.

Field of invention

The present invention relates to a recombination vector for expressing tumstatin containing a tumstatin gene and a method for producing recombinant tumstatin using insect cells transformed with the vector.

Background of the Invention

Tumstatin is a C-terminal spherical non-collagenic domain of type IV collagen alpha 3 chain that inhibits tumor growth in mouse models (Maeshima et al. , J. Biol. Chem , 275: 21340-8, 2000). Tumstatin also inhibits endothelial cell proliferation extracellularly as a potential inhibitor of angiogenesis with distinct antitumor activity (Maeshima et al. , Science, 295: 140-3, 2002).

Expression of tumstatin has been reported in E. coli, Pichia pastoris and embryonic kidney cells (Maeshima et al. , J. Biol. Chem , 275: 21340-8, 2000; Maeshima et al. , J. Biol. Chem , 276: 15240-8, 2001). However, whether soluble tumstatin is fully available is a requirement for it to be developed as an anticancer agent. To date, expression of tumstatin in insect cells has not been reported due to its expression stability problem.

Thus, the present inventors prepared a recombinant vector pMT / BiP / T-V5-His containing the tumstatin gene in order to stably express the tumstatin in insect cells, transforms insect cells using the same and then adds the tomstatin The present invention was completed by confirming the stable expression.

An object of the present invention is to provide a recombinant vector containing the tumstatin gene and insect cells transformed with the recombinant vector.

Another object of the present invention is to provide a method for producing tumstatin using the transformed insect cells.

In order to achieve the above object, the present invention provides a recombinant vector pMT / BiP / T-V5-His for expressing a tumstatin containing a tumstatin gene and insect cells transformed with the recombinant vector.

In the present invention, the insect cells can be transformed with pMT / BiP / T-V5-His and pCoHygro.

The present invention also provides a method for producing tomstatin, which comprises culturing insect cells transformed with a recombination vector for expressing a tumstatin containing a tumstatin gene. In the present invention, the recombinant tumstatin can be purified by chromatography using Ni-NTA resin.

According to an embodiment of the present invention, a recombinant vector pMT / BiP / T- for expressing tumstatin is expressed in Dipteran insect cells, Drosophila melanogasster S2 cells, and Lepidopteran insect cells, Trichoplusia ni [High Five Cell Line (Invitrogen Co.)]. Provided is a method for producing tumstatin by transforming with V5-His and culturing the transformed insect cell line thus obtained. Such transformation and cultivation may be any method conventional in the field of genetic engineering, so long as it can effectively express the desired tomstatin in high yield. Especially preferably, the transformed insect cell is cultured using a Nunc T-flask.

Drosophila melanogaster Schneider 2 ( Drosophila melanogaster Schneider 2) cells, which are host cells used to express tumstatin in the present invention, are known as one of the cell lines belonging to the genus Drosophila widely used in the field of genetic engineering. It is a cell line.

However, there have been no examples of using the drosophila melanogasster S2 cell line for the expression of tumstatin. The Drosophila melanogasster S2 cell line is already known in the literature ("Drosophila Cells in Culture", Guy Echalier, 1997, Academic Press, U.S.A.) and is readily available from Invitrogen, U.S.A.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Preparation of Host Cells, Vectors and Enzymes Used for Transformation

Drosophila melanogasster S2 cells and Trichoplusia ni [Invitrogen Co.], the host cells used to express human tumstatin , are M3 insect media containing 10% IMS (insect medium supplement, Sigma). (Shields and Sang M3 insect medium, Sigma) was incubated at 27 ℃ using a Nunc (T-25, Denmark) flask.

PMT / BiP / V5-His (3.6kb), which is used for the production of recombinant vectors pMT / BiP / T-V5-His introduced into host cells S2 cells and High Five cells for expression of tumstatin, is expressed in vitro. It is readily available from (Invitrogen Co., CA) and contains a metallothionein promoter, a BiP signal sequence, a V5 epitope tag and a polyhistidine site.

In addition, the selection plasmid pCoHygro (Invitrogen Co.) containing the bacterial hygromycin B phosphotransferase gene under the control of the structural Drosophila Copia 5'-LTR promoter was used to perform transformation. It was.

In the following examples, restriction enzymes were used according to the instructions given by their manufacturer, Promega Co. (U.S.A.) or Takara Co., Japan, respectively.

In the examples below, Western blot analysis was performed on protein samples using SDS-PAGE according to the Lamley method (Laemmli, UK, Nature 227: 680-685, 1970). That is, the proteins electrophoresed on the gel were transferred onto nitrocellulose and reacted with mouse anti-V5 (Invitrogen Co.) multicellular antibody, followed by rabbit anti-mouse IgG alkaline phosphatase conjugate (1: 1000, v / v). Was labeled. After washing, BCIP / NBT solution (Amersco Co., OH) was added and the reaction was stopped with distilled water.

Example 1: Lithium Statin Gene Amplification and Expression Vector Preparation

Total RNA was prepared from thymus tissue to amplify the tumstatin gene (Genbank accession number: AF258351 ), and cDNA was synthesized using reverse transcriptase (Promega Co.) from 10 μg of total RNA. The thus synthesized cDNA was used as a template, and oligonucleotide PCR of the following SEQ ID NOS: 1 and 2 was performed as a primer. At this time, the sense primer was SEQ ID NO: 1, and the antisense primer was SEQ ID NO: 2.

SEQ ID NO: 5'-CCAGGTTTGAAAGGAAAACGTGGAGAC-3 '

SEQ ID NO: 5'-TCAGTGTCTTTTCTTCATGCACACCTG-3 '

The amplified thumstatin gene was subcloned into pGEM-T vector (Promega Co.) using restriction enzyme recognition sites of Bgl II and Xho I to obtain pGEM-TT.

pMT / BiP / V5-His was digested with restriction enzymes Bgl II and Xho I at 37 ° C. for 2 hours. In addition, pGEM-TT obtained above was digested with restriction enzymes Bgl II and Xho I also at 37 ° C. for 2 hours to obtain a Bgl II- Xho I fragment containing the gene of tumstatin . The Bgl II- Xho I fragment of pGEM-TT thus obtained was ligated between the Bgl II and Xho I cleavage sites of pMT / BiP / V5-His at 4 ° C. for 12 hours with T4 DNA ligation. The desired tumstatin expression vector pMT / BiP / T-V5-His was obtained. The structure of pMT / BiP / T-V5-His is shown in FIG. 1.

In the expression vector pMT / Bip / T-V5-His, the gene insertion was in the proper direction and the read frame was confirmed by restriction enzyme mapping and DNA sequencing.

Example 2: Stable Transformation

Lipomfectin was used to calculate the tombstone expression vector pMT / BiP / T-V5-His and the selection plasmid pCoHygro (ratio of 1: 1) prepared in Example 1 in an exponential manner as described below. Drosophila melanogasster S2 cells and Trichoplusia ni [High Five Cell Line (Invitrogen Co.)] were grown.

First, plasmid DNA and lipofectin reagents (Gibco BRL Co., USA) were prepared using shields and Sang M3 insect medium (Sigma Co.) without IMS (insect medium supplement, Sigma), respectively. After diluting to 3 μg of DNA and 15 μg of lipofectin, the mixture was mixed at a ratio of 1: 5 (v / v). The transfection medium was reacted at room temperature for 15 minutes, and DNA, lipofectin, and media mixtures were added to the drosophila melanogasster S2 cells in M3 medium without IMS pre-dispensed in 6-well plates 2 hours ago.

After incubating the plates in a 27 ° C. incubator for 24 hours, the medium in the wells was replaced with M3 medium containing 10% (v / v) IMS but without antibiotics to remove lipofectin, and in this medium 27 Cells were further incubated for 5 days in an incubator.

Cells were then centrifuged at 5,000 rpm for 5 minutes to separate transformed host cells. The isolated host cells were then resuspended in 1.5 ml of selective M3 medium containing 10% (v / v) IMS and 300 g / ml of hygromycin B. The transgenic polyclonal cell populations were selected after 4 weeks of selection using hygromycin B while replacing the selection medium at 5 day intervals. Hygromycin B was routinely maintained in the medium throughout the screening period.

Gene expression was analyzed by Western blot 5 days after induction with 0.5 mM CuSO ₄ to investigate expression of tumstatin in stably transformed S2 cells (FIG. 2). Lane 1 in Figure 2 is the cell portion of the non-transformed cells; Lane 2 shows a media portion of untransformed cells; Lane 3 is a cell portion of transformed S2 cells; Lane 4 is a media portion of transformed S2 cells; Numbers on the left are molecular weight markers (kDa); Arrows indicate recombinant tumstatin.

Western blot using mouse anti-V5 multicellular antibody antibody (Invitrogen) to determine whether the drosophila melanogasster S2 cells transformed with pMT / BiP / T-V5-His stably expressed recombinant tumstatin. The molecular weight was -29 kDa when investigated by the analytical method. This molecular weight was judged to be similar to the predicted molecular weight of the fused tumstatin containing the C-terminal label of V5 and His ₆ . Recombinant tumstatin is present inside and outside (medium) of transformed Drosophila melanogasster S2 cells, and according to densitometric scanning, secreted tumstatin (ie, tumstatin present in the media). This accounted for about 82% of the total tumstatin production.

That is, stably transformed S2 cells secrete the expressed tumstatin effectively extracellularly (in medium). Recombinant tumstatin was not detected in or around untransformed S2 cells. This result suggests that expression of recombinant tumstatin in transformed S2 cells is due to pMT / BiP / T-V5-His containing the tumstatin gene introduced into these cells.

The same experiment was repeated using Trichoplusia ni (High Five Cell Line (Invitrogen Co.)), a Lepidopteran insect cell, instead of Drosophila melanogasster S2 cells. As a result, it was confirmed that tumstatin is expressed in the transformed High Five cell line (FIG. 3). Figure 3 shows the results of Western blot analysis of gene expression 5 days after induction with 0.5 mM CuSO ₄ , lane 1 is the cell fraction of the non-transformed cell line; Lane 2 is media of untransformed cell line; Lane 3 shows cell fraction of transformed cell line; Lane 4 is media of transformed cell line; Numbers on the left indicate protein molecular weight; The arrow on the right represents the recombinant lithium statin protein.

Example 3: Cell Culture and Gene Expression Analysis

The drosophila melanogasster S2 cells transformed in Example 2 above in a non-k (T-25) flask at 27 ° C. in 5 ml of M3 medium containing 10% IMS and 300 μg / ml of hygromycin B Incubated at. Stably transformed S2 cells were incubated in a 27 ° C. incubator for 8 days to analyze cell growth and toomstatin expression in a nonk flask. Expression of tumstatin was induced by addition of 0.5 mM CuSO ₄ after incubation.

Cells were isolated by incubating transformed Drosophila melanogasster S2 cells for 5 days and then centrifuging the culture at 3000 g for 5 minutes. Supernatants were used to identify recombinant proteins extracellularly.

Cell sections were lysis buffer (50 mM Tris / HCl, pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 μg / ml PMSF, 1 μg / ml aprotinin and 1% (v / v) Triton X -100) was shaken for 1 hour, and three freeze-thaw cycles were repeated through a reaction in a -70 ° C freezer with a reaction in a water bath at 37 ° C.

After centrifugation of the cell extract at 10,000 g for 15 minutes to remove foreign substances from the cells, the supernatant was used to confirm the presence of recombinant protein in the cells. In addition to the other formulas indicated, a mixture of extracts in sappo was used to analyze total protein production.

The endothelial cell proliferation assay is a modification of a known method (O'Reilly et al., Cell, 88: 277-85, 1997). Bovine capillary endothelial (BCE) cells were treated with EGM (2% (v / v) bovine fetal serum, 12 μg / ml bovine brain extract, 10 ng / ml human epidermal growth factor, 1 μg / ml hydrocortison ), Containing 50 μg / ml gentamycin and 50 μg / ml: according to user instructions from Clonetics.

About 2 × 10 ⁴ cells in 0.5 ml of EGM were gelatinized in each well of a 24 well plate and incubated at 37 ° C. for 24 hours (5% v / v CO ₂ ). The medium was replaced with 0.25 ml fresh EGM containing antibiotics and different concentrations of recombinant tumstatin were added to each well.

After 30 minutes of incubation, cells were stimulated with 1 ng / ml of basic fibroblast growth factor (bFGF) and 0.5 ml was added by adding EGM containing antibiotics. After 72 hours of incubation, the cells were trypsinized and counted using a hematocytometer.

To confirm that the inhibition observed before counting the cells was not due to the isolation of BCE cells, all wells were examined using an inverted microscope to confirm cell separation.

Endothelial cell proliferation assays were repeated three times. Half concentration of maximal inhibition (ED ₅₀ ) was assessed by probit analysis (Finney DJ, Cambridge, 3, 1997).

The effect of recombinant tumstatin on the inhibition of bFGF-stimulatory endothelial cell proliferation was investigated. Recombinant tumstatin dose-dependently inhibited the proliferation of bovine capillary endothelial cells. The presence of His-labeled and V5 epitope sequences did not affect the inhibition of recombinant tumstatin on bFGF-stimulated endothelial cell proliferation. Half of the maximum inhibitory concentration of recombinant tumstatin (ED ₅₀ ) was about 0.69 μg / ml. The results of the present invention show that a biologically active type of recombinant tumstatin was expressed from S2 cells, which has an anti-proliferative effect on bovine endothelial cells. And this indirectly represents the potential as an angiogenesis inhibitor.

Expression of recombinant tumstatin in T flask cultures of insect cells over time was examined. Initial cell concentration was ⁶ × 10 ⁶ cells / ml. After 7 days of induction with 0.5 mM CuSO ₄ , the accumulation of tumstatin peaks. At this time, the yield of recombinant tumstatin was determined to be about 7.8 mg / l compared to the tumstatin control purified on Western blot analysis. Tumstatin production in each cell was 4.6 μg / 10 ⁷ cells.

The expression levels of these tombstatins from Drosophila melanocytes S2 cells are inadequate data, so other expression systems such as Escherichia coli, Pichia pastoris and 293 times renal cells (Maeshima et al. , J. Biol. Chem , 275: 21340-8, 2000; Maeshima et al. , J. Biol. Chem , 276: 15240-8, 2001).

However, the level of expression than obtained by the present inventors (4.6㎍ / 10 ⁷ cells) is recombinant human tyrosinase cycloalkyl oxy claim 2 expression (1.6㎍ / 10 ⁷ cells) (cyclooxygenase 2) in the switch S2 cells transfected in the previous stable Much superior (Chang et al., Biotechnol. Lett. , 24, 1353-9, 2002).

Example 4: Purification of Recombinant Tumstatin

All treatment steps for purification of tumstatin were performed at 4 ° C. Purification was carried out using a Ni-NTA resin (Qiagen Co., CA) on a chromatography column (Novagen Co., WI) according to the manufacturer's instructions.

Briefly, the resin was equilibrated and bound to the column by binding to a buffer containing 10 mM imidazole. The media portion of the culture was slowly applied to a column containing the resin in equilibrium. Weakly bound proteins were washed out of the resin using a binding buffer, and the resin still attached to the contaminated proteins was removed by raising the imidazole concentration to 100 mM. The recombinant polyhistidine-labeled protein product was finally isolated in buffer containing 1 M imidazole.

The portion containing the tumstatins was isolated by dialysis in phosphate buffered saline (pH 7/4) to remove imidazole. Protein concentrations were determined using a Bradford Protein Assay Kit (BioRad Co., CA) based on bovine serum albumin.

As described above, the purity of the tumstatin which was rapidly purified and homogenized by the one-step Ni ²⁺ chelation affinity purification method was analyzed by SDS-PAGE and silver staining, and the results are shown in FIG. 4A. In addition, Western blot analysis demonstrated that the purified protein was identical to the desired tombstatin (FIG. 4B).

A total of 35 μg of purified polyhistidine-labeled tomstatin (tumstatin-V5-His ₆ ) was obtained in the medium portion of a 70 ml culture. The protein was not particularly contaminated as examined on SDS-PAGE gels stained with silver nitrate.

The present invention provides a recombinant vector (pMT / BiP / T-V5-His) and insect cells transformed with the recombinant vector for producing toomstatin. Recombinant tumstatin produced from a biologically active insect cell purified according to the present invention has anti-proliferative activity against endothelial cells and thus may be developed as an angiogenesis inhibitor. High levels of functional expression have been shown to be a convenient source of recombinant tumstatin for anticancer treatment.

<110> CHUNG, In Sik <120> Method for preparing a recombinant inhibitor of angiogenesis from          transformed insect cells <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 1 ccaggtttga aaggaaaacg tggagac 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 2 tcagtgtctt ttcttcatgc acacctg 27

Claims (6)

A recombinant vector pMT / BiP / T-V5-His for tumstatin expression containing a tumstatin gene, a metallothionein promoter, a BiP signal sequence, a V5 epitope and a poly histidine and having the sequence map of FIG. An insect cell transformed with the recombinant vector of claim 1. 3. The insect cell of claim 2, further transformed with a vector containing a drosophila copia 5'-LTR promoter and a hygromycin B phosphotransferase gene. A method for producing toomstatin, comprising culturing the transformed insect cell of claim 2 or 3. The method of claim 4, wherein the insect cells are Drosophila melanogaster S2 or Trichoplusia ni (High Five cells). The method of claim 4 or 5, further comprising the step of purifying the recombinant tumstatin by chromatography using Ni ²⁺ chelating affinity resin.

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