JPH0227987A - Cdna to code n-acetylglucosamine (beta1-4) galadctosyltransferase - Google Patents

Abstract

PURPOSE:To collect beta1-4GT by synthesizing cDNA to code N-acetylglucosamine (beta1-4)galactosyltransferase (beta1-4GT) containing a specific DNA. CONSTITUTION:A cDNA bank derived from human placenta is screened by using bovine beta1-4GTcDNA as a prove to give 250,000 lambdagt11 recombinant phages, from which cDNA to code the whole length of human beta1-4GT is collected. The cDNA to code the human beta1-4GT is cDNA of the whole length of 1-1194 and a large amount of active standard product of beta1-4GT can be synthesized by recombination with a well-known manifestation vector. In the synthesis, cDNA of the whole length cDNA sequence can be used.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to cDNA encoding N-acetylglucosamine (β1-4) galactosyltransferase.

[Background of the invention]

Most tumor markers have specific structures of sugar chains that are not seen in normal cells, or sugar chain structures that are produced in large quantities in tumors, although the amount is small in normal cases.

The substance that synthesizes this structure is glycosyltransferase, and the appearance of tumor markers due to canceration is thought to be due to abnormal expression of glycosyltransferase. This protein is difficult to purify because it is in such a small amount that analysis of the substance itself has been delayed. To solve these problems, there is an urgent need to isolate enzyme genes and to approach their relationship with canceration from a molecular biological perspective. Furthermore, if the isolated gene is recombined into an expression vector and a large amount of active specimens of the enzyme protein can be obtained, it will be possible to in vitro
This will be the starting point for the development of sugar chain synthesis technology at itro.

[Purpose of the invention]

The object of the present invention is to obtain a cDNA that encodes N-acetylglucosamine (β1-4) galactosyltransferase (hereinafter abbreviated as β1-4GT), which is one of the glycosyltransferases.
The goal is to provide NA.

[Structure of the invention]

The present invention relates to the 376th T of the base sequence shown in FIG.
The base sequence starting from A and ending at the 1197th A and 86
β1-4GT having homology of % or more or containing NA
Concerning cDNA encoding.

Specific examples of the cDNA of the present invention include, but are not limited to, the following.

(i) A cDNA encoding mouse βl-4GT having a nucleotide sequence starting from T at position 376 and ending at 8 at position 1197 among the nucleotide sequences shown in FIG.

(ii) A cDNA encoding mouse β1-4GT having a base sequence starting from the 1st A and ending at the 1197th A among the base sequences shown in FIG.

(iii) A cDNA encoding human β1-4GT having a base sequence starting from C at position 373 and ending at C at position 1194 among the base sequences shown in FIG.

(iv) A cDNA encoding human βl-4GT having a base sequence starting from A at position 1 and ending at C at position 1194 among the base sequences shown in FIG.

The method for producing cDNA of the present invention will be explained below.

(1) cDNA encoding mouse β1-4GT cDNA encoding mouse β1-4GT can be isolated as follows.

Mouse teratocarcinoma F9 cells were grown in vitro (
It is a tumor cell line that can be induced to differentiate in vitro), and it is known that the enzymatic activity of β1-4GT changes depending on the differentiation stage. First, as a first step, β9 cell line and cDNA bank are screened using bovine β1-4GT cDNA as a probe. β9 cD
An NA bank can be created by inducing differentiation of β9 cells with retinoic acid, extracting and purifying mRNA of 3 kb or more, and recombining it with λgtll phage. ORF of bovine β1-4GT
Positive cDNA clones can be obtained by screening 500,000 λgtll phage recombinants using the 5stI-KpnI fragment in the fragment as a probe. The obtained cDNA clone is a full-length cD
If it is not NA, create a Smal fragment as a probe from the 5' end of the cDNA clone with the partially deleted part obtained above, and use the same F 9 c D as a probe.
Full-length cD by rescreening NA banks
NA can be isolated. The entire base sequence of full-length cDNA can be determined by a conventional method (for example, the M13 method using 53%).

(2) cDNA bovine β encoding human β1-4GT
A human placenta-derived cDNA bank was screened using 1-4GT cDNA as a probe, and human β1-4GT was selected from the 250,000 λgtl1 recombinant phages obtained.
cDNA encoding the full length of can be obtained.

Furthermore, Figures 1 and 2 show cDNA encoding mouse β1-4GT and cDNA encoding human β1-4GT.
A is shown respectively. These cDNAs (mouse β1-4
The cDNA encoding GT is a full-length cD from 1 to 1197.
cDNA encoding NA, DNA bank GT is 1-1
By recombining the full-length cDNA of 194) into a known expression system vector, it is possible to synthesize an active preparation of β1-4GT in large quantities. However, for the synthesis of active preparations of β1-4GT by recombination, the above-mentioned full-length c, D
Fragment cDNA of NA can also be used. for example,
In the case of cDNA encoding mouse β1-4GT,
An active preparation of β1-4GT can also be obtained by using cDNA from position 376 T to position 1197 A. Similarly, in the case of cDNA encoding human β1-4GT, by using cDNA from position C 373 to position C 1194, β1-4GT
It is possible to obtain an active preparation of .

In addition, the fragment cDNA was prepared using the primer extension method, Ba131 (
It can be obtained by a method using restriction enzymes).

In addition, as described in the Examples, mouse full-length cDNA and human full-length cDNA have 75% homology in base sequence.
cDNA that has 75% or more homology to DNA in base sequence
An active preparation of β1-4GT can be obtained by recombining A into an expression system vector as described above. Furthermore, the cDNA of mouse 376-11g7 shown in FIG.
There is 86% homology between A and the human cDNA 373-1194 shown in Figure 2, and the mouse cDNA 373-1194 shown in Figure 1 has 86% homology.
Even if a cDNA with more than 80% homology to cDNA 6-1197 is recombined into an expression system vector, β1-4G
An active preparation of T.

The cDNA encoding β1-4GT of the present invention is β1-4
It is extremely useful in providing an active preparation of GT, and β1
By providing an active preparation of -4GT, it becomes possible to synthesize sugar chains in vitro.

The present invention will be further explained below with reference to Examples.

Example 1 Isolation of full-length cDNA encoding human β1-40T and determination of complete base sequence and protein structure. cDNA was synthesized from polyA''m RNA extracted from human placenta by the random primer method, and λgtll The cDNA bank created by phage integration was
Screening was performed using cDNA encoding 1-40T as a probe. The 5stI-KpnI fragment (approximately 0.9 k
b) was labeled with ``P-dCTP'' using the random primer method and used as a probe (Fig. 3).

c DNA bank (λgtll phage bank) 25
0.0 OOPFU was adsorbed onto host bacteria B, Co11 Y1088 at 37°C for 15 minutes, and then transferred to soft agar (0.7% Agar).
) Suspended in LB medium. This is 10CIX14C1
The mixture was evenly spread on 1.0 pieces of LB medium in a square petri dish.

After culturing at 37°C for 8 hours, phage plaques were allowed to appear.

Nitrocellulose filter (hereinafter referred to as NcF) 9X13C soaked in 1M NaCl solution and air-dried
The phage was transferred to NCF by covering 1 liter of the plate on the phage plaque plate and leaving it for 30 seconds. Furthermore, a second sheet of NCF was left on the same plate for 1 minute to transfer the phage. All screenings were performed in duplicate.

NCF was further diluted with 0.5 M NaH, 1,5 M Na
Perform alkaline treatment with C1 solution for 5 minutes and 0.5M T
ris-HC1pfl? , 5, ■, Neutralized with 5M NaCl solution. After air-drying, it was baked at 80°C, and prehybridization was carried out using a buffer composition of 50% formamide, 31% SD, and Denhardt.
) liquid 0.1%, dextran sulfate 5%, poly A
Using 0.05%, the test was carried out at 42°C overnight.

Preparation of cDNA probe: After subcloning into pUc19 vector, it was purified as a plasmid using an ultracentrifuge using a CsC1 density gradient. After cutting with restriction enzyme, 0.7%
Electrophoretic separation was performed using low melting point agarose, and the target band was cut out and purified as probe DNA. Approximately 50nH
The probe DNA was labeled with 32p using the multi-random primer kit method (manufactured by Amerjam).

The labeled probe was diluted with a solution having the same composition as the prehybridization buffer described above, and hybridization was performed at 42° C. overnight. Finally 55℃, 0,2
After washing the NCF with X5SPE stringency, autoradiography was performed and positive clones were picked up. The number of phages was sequentially reduced to 1/10 through secondary, tertiary, and 4th screening, and a single clone was obtained in the 4th screening.

Miniprep method for λgtll phage: Incubate Y1088 host bacteria in YT medium the night before. Phage M, 0, 1. After adding in a range of 0.03 to 0.2 and adsorbing at 37°C for 20 minutes, 15-mL of YT medium was added and cultured with shaking at 37°C for 13 to 14 hours.

After centrifugation (3000 rpm 15 minutes), remove the supernatant and use DE52
Mix in equal amounts on a rotating plate for 30 minutes. Centrifugation (300
0 rpm for 15 minutes), and the supernatant was taken. 5M NaCβ17
8 volumes and 27/40 volumes of cold Improper Tool were added, mixed and left at -20°C for 2 hours. Centrifuge at 4°C (30
After 15 minutes at 0 Grpm, the supernatant was scraped to remove 1 phage from the precipitate.
After suspending in 70% ethanol, transfer to an Eppendorf tube. After centrifugation (6000 rpm 5 minutes),
The supernatant is dried using a hinoki cypress, and the precipitate is dried and suspended in 400 μl of 1TE buffer. After this, phage DNA was purified by ethanol precipitation after phenol treatment twice and phenol/chloroform treatment.

After cutting the phage DNA with ECoRI, it was subjected to electrophoresis and inserted. cDNA results were obtained. Furthermore, S ac
At this point, a rough restriction enzyme map was created by combining restriction enzymes such as I%KpnI and SmaI. This electrophoresis gel was blotted onto a nylon filter, and bovine cDNA was hybridized with each portion to confirm the positional relationship of each of the 18 C-ons. DNA of each clone was prepared by Tendo culture, and E
After cutting with coRi, this EcoRI fragment was transformed into pUC1
All were subcloned into 9 vectors.

Subcloning from λgill phage into pUc19 plasmid;
After cutting with I, it was separated by LMP agarose electrophoresis, cut out and purified. The pUc19 plasmid was cut with EC0RI, the cDNA EcoRI fragment and the pUcL9 DNA amount were combined in terms of moles, and 20μ! The mixture was incubated at 16° C. day and night in the presence of 10 units of 74′ DNA ligase. The host bacteria HBIOI was incubated the night before, and 2 hours before use, it was diluted 20 times with LB medium and cultured with shaking.

Thereafter, the bacteria were removed by centrifugation at 3.00 Orpm for 15 minutes, and the supernatant was replaced with 172 volumes of 50 mM cold CaClx. After further centrifugation, the supernatant was diluted with 1,720 ml of 50 ml J C.
Suspend in aC12. Add 0.3 Wd2 of this bacterial solution to the DNA solution mentioned above, mix and leave on ice for 40 minutes, and incubate at 42°C.
Add heat rapidly for 2 minutes. Pour this bacterial solution onto an LB plate (50μ
g/ml Ampicillin, X-gal 200 D g/
ml containing 100 μg/d of IPTG) using a Conlage stick, and cultured at 37° C. day and night. Among the colonies that appeared, white colonies were picked. To confirm whether or not cDNA has been inserted, labeled cD
Colony hybridization was also performed using NA.

In order to obtain a large amount of cDNA subcloned into pUc19, 11 strains of each plasmid clone were prepared.
The cells were cultured overnight in LB-ampicillin culture solution, and the plasmid was purified by an alkaline method. A restriction enzyme map was created by cutting with various restriction enzymes (Fig. 4). From this result, M
We investigated the protocol for recombining into phage 13 and conducted a search for the base sequence.

Complete base sequence sequencing by M13 phage dideoxy method; M13 phage mp18 and mp19 were used. Each RF DNA was cut at the restriction enzyme site to be recombined. The cDNA incorporated into pUc19 was cut at the desired restriction enzyme site. Both were mixed at the same molar ratio, and a binding reaction was performed at 16° C. day and night using T4 ligase. Transfect host fungus JMIOI as described above, mix with soft agar and indicator fungus JMI 01,
It was spread uniformly on an LB plate (X-gal, containing IPTG) and cultured at 37° C. day and night.

White plaques were collected in YT culture solution 5r111, host strain JMI 01 was added, and cultured with shaking for 8 hours. Centrifugation (3
000 rpm, i5 minutes), the supernatant was taken and heat-treated at 65° C. for 15 minutes. Transfer 5 μl of this onto a filter and
In order to confirm whether the cDNA of interest was inserted into the No. 13 phage, hybridization was performed with labeled cDNA. Only the supernatant of the M13 phage that showed a positive result was used for Sea Fence.

15% polyethylene gelcol 2.5M to supernatant 1rn1
After adding 250 μl of NaCβ solution and mixing, leave on ice for 30 minutes and centrifuge (15,000 rpm, 15 minutes).

Aspirate the supernatant completely with a tapered Pasteur pipette,
Air-dry the phages in the sediment. After phenol treatment, phenol/chloroform treatment, and chloroform treatment, ethanol precipitation is performed twice. After drying, it was dissolved in 10 μl of TE buffer and stored at 4° C. as ssDNA.

ssDN A 4 u 1 , LO4JJ 1 , Bly 7-(0.5 pmol) 1 μl, TM buffer y (0.1M) Lis HCl pH 75.0.1M
Mix 1 μl of MgCl2, heat at 70°C for 10 minutes, and slowly return to room temperature over at least 30 minutes. Centrifuge the water droplets on the wall to the bottom. 2μ
m (20 μCi) of [”S]-d ATP was added, centrifuged after portexing, and placed on ice. During this time, A, G, C,
1', dispense each mixture into 4 μm portions. 1 unit/μl of Klenow fragment in the first sample! j
After adding 1 and centrifuging briefly, add 2.5 to 3.0 μβ to A,
Add to each of the G, C, and T mixtures and mix gently by centrifugation to start the reaction. After being left at room temperature for 20 minutes, 1 μβ of Chase 5 solution was added, briefly centrifuged, and further left at room temperature for 20 minutes. Dry completely using a Spad vac. 5
Add μl of Stop solution and dissolve thoroughly using Vortex.

After heating at 90° C. for 2 to 3 minutes, 2.5 μl is quickly applied to gel for electrophoresis.

Sequence microphoresis: Reagents were prepared as follows. (
1) 40% acrylamide stock solution; add dd LO to 190g acrylamide, 10g bisacrylamide to make 500d. (2) High salt gel solution; 50g urea, 10g sucrose, 15-40% acrylamide stock solution, 25rnl 10xTBE, a little BPB powder, add dd')+20 to the above, 100rnl
After cooling, pass through a filter (0.45 um) and heat at 4°C.
(3) Low temperature gel solution; 250g urea, 75rn
140% acrylamide stock solution, 25mj! 10
Add dd 020 to xTBE to make it 500-, then pass through a filter (0.45 μm) and store at 4°C.

gel 5 size 350 x 425 x thickness 0.4
A gel of mm (equipment manufactured by IBI) was prepared. (
1) Add 120 μβ of 25% ammonium persulfate and 70 μ ITEMED to the 65-mL cold gel solution. (2
) Add 16 μm 25% ammonium persulfate and 20 μ ITEMED to the 15 rd hot gel solution. First 12~
Aspirate (1) of 13FR1 with a 25d pipette, and then aspirate the same amount of ω). Pour gently from the edge of a plate tilted at 45°C. Furthermore, pour the remaining (1) from the center of the plate, let the plate rest, insert a comb, and wait for it to gel.The comb was removed, the above-mentioned sample was placed, and electrophoresis was performed at 74W.After electrophoresis, 5% methanol Urea was removed by soaking in a 75% acetic acid solution for 20 to 30 minutes.The gel was transferred onto 3MM paper, wrapped in plastic wrap, dried at 80°C, and then exposed to Kodak XAR-5 film.

Using the above method, the entire base sequence of human β1-4GT cDNA was determined (Figure 2).

Example 2 Determination of the structure of bovine βl-4GT cDNA clone 5stI-K
The pn I fragment was used as a probe to screen the F9c DNA bank. The method is exactly the same as the screening of human cDNA bank in Example 1.

In the initial screening, 3 out of 5 GT phages were selected.
Positive clones were obtained. A restriction enzyme map was created as described above. Of these, two showed the same map and were almost the same length (named λmGT-10), but one
mG T-2 showed a partially different map. Since it was found that even the longest clone among the former clones did not encode the full length, the 5'-end EcoRI-SmaI fragment was excised and purified, and the same F9 cDNA bank was rescreened. Among the 5 GT phages,
After obtaining four positive clones, a restriction enzyme map was created as usual, and the full-length cDNA human β1-4 GTc D
Hybridization was performed with each part of NA, and the nucleotide sequence of a cDNA clone (λmGT-5) that was thought to encode the full length was determined (Fig. 1).

Mouse full-length βl-4GT [clone (pmG
T-5) The base sequence (1 to 1197) of E is shown in Example 1.
human full-length β1-4GT cDNA (1-1194
), the nucleotide sequence had approximately 75% homology and the amino acid sequence had approximately 83% homology. Furthermore, C-terminal side 20
0 amino acids have extremely high homology (approximately 90%);
The 376-1197 base sequence of mouse β1-4GT and the 373-1194 base sequence of human β1-4CT are approximately 8
There was 0% homology.

Chow-77su-v :/ (Chow-Fasman
) program was used to calculate the protein secondary structure, and the highly homologous regions were rich in α-helices and β-sheets;
On the other hand, the lower part had a so-called random coil structure. Mouse βl-4GT also has 18 at the N-terminus.
The start codon and stop codon were both at the same positions as human cDNA, but there was an insertion of one amino acid in a part with little homology to humans, and all amino acids 400 losses,
The calculated M, W, is 48 KDa. From this, it can be seen that parts of amino acid sequences with low homology between different species are
This site seems to be unrelated to the enzyme's substrate specificity and active center site (Figure 5).

[Brief explanation of the drawing]

Figure 1 shows the nucleotide sequence and amino acid sequence of cONA encoding mouse β1-4GT, Figure 2 shows the nucleotide sequence and amino acid sequence of cDNA encoding human β1-40T, and Figure 3
The figure shows a restriction enzyme map of bovine β1-4GT cDNA, FIG. 4 shows a restriction enzyme map of human β1-4GT cDNA, and FIG. 5 shows the homology between the amino acid sequences of human and mouse β1-4GT.

Claims (2)

[Claims] (1) Of the base sequences shown in Figure 1, 86% of the base sequences start from T at position 376 and end at A at position 1197.
A cDNA encoding N-acetylglucosamine (β1-4) galactosyltransferase containing DNA having the above homology. (2) Among the base sequences shown in Figure 1, N-acetylglucosamine (β1-4) galactosyl contains DNA that has 75% or more homology with the base sequence starting from the 1st A and ending with the 1197th A. cDNA encoding transferase.