KR960010951B1 - New human epidermal growth factor gene - Google Patents

Abstract

The improving method of human epidermal growth factor (I) in E. coli expression consists of culturing E. coli KCCM 10026 having pUE118 plasmid is which DNA sequence of (I) readable by E. coli is substituted for original (I) and has SmaI/ PstI site.

Description

New Human Epidermal Growth Factor Gene

1 shows the basicity of the novel human epidermal growth factor gene of the present invention.

Figure 2 shows the binding state of (a) the synthesized oligonucleotide sequence and (b) the synthesized oligonucleotide sequence for the construction of the human epidermal growth factor gene of the present invention.

3 is a diagram showing the process of inserting the human epidermal growth factor gene of the present invention into the pUC 18 vector.

4 is a photograph confirming the insertion of the human epidermal growth factor gene of the present invention and the presence of a single restriction enzyme site.

The present invention relates to a novel human epidermal growth factor (hEGF) gene. More specifically, the present invention relates to a hEGF gene that is artificially designed and chemically synthesized, capable of mass expression of hEGF in E. coli, and capable of artificial mutagenesis, an expression vector containing the same, and a method of preparing the same.

hEGF consists of 53 amino acids and has three disulfide bonds (Cohen, S. (1962) J. Biol. Chem. 237, 1555-1562; Savage, Ct, Jr. et al. , (1973) J. Biol. Chem. 248, 7669-7672; Savage, CR, Jr. et al., (1972) J. Biol. Hem. 247, 7612-7621). Growth of skin cells (Sporn, MB et al., (1985) Nature (London) 313, 745-747; Sporn MB et al., (1980) N. Engl. J. Med. 303, 878-880) and wounds (Buckley, A. et al., (1985) Proc. Natl. Acad. Sci. USA. 82, 7340-7344) and are known to play a very important role in the regulation at the molecular level.

In addition, the hEGF epithelial cell's ability to inhibit gastric acid secretion in the gastrointestinal tract, in addition to its ability to promote differentiation, has been reported to be very useful for treating diseases such as gastric ulcers (Gregory, H., (1985)). J. Cell Sci.Suppl. 3, 11-17).

On the other hand, since Srarkey et al. Separated and purified hEGF from human urine in 1975 and identified its properties, efforts to continuously obtain hEGF in high yield have been continued (Starkey, RH et al., (1975). ) Scince 189, 800; Cohen. S. et al., (1975) Proc. Natl. Acad. Sci. USA 72, 1317). As one of these efforts, several research groups have reported success in cloning hEGF genes using genetic engineering techniques (Smith, J. et al., (1982) Nucleic Acids Res. 10, 4467-4482; Urdea, MS et). al., (1983) Proc. Natl. Acad. Sci. USA 80, 7461-7465; Oka, T, et al., (1985) Proc. Natl. Acad. Sci. USA 82, 7212-7216). HEGFs prepared using engineering techniques have had the critical problem of not showing sufficient amounts or sufficient activity to be industrially available.

Therefore, in order to solve this problem, many experiments and efforts have been made to determine the most ideal nucleotide sequence capable of expressing hEGF in high yield in a particular host, thereby improving the novel hEGF gene sequence. The need has been very important. In view of this, recent attempts have been actively made to increase the yield and increase the activity of hEGF using protein engineering techniques.

In order to ensure that hEGF is produced in E. coli with high yield, the inventors of the present invention encode the same amino acid as natural hEGF to codon (Grantham et al., (1981) Nucleic Acids Res. 9, 243). -274), replacing the existing hEGF nucleotides, and at the same time selecting a sequence that minimizes the secondary structure of mRNA that can interfere with the expression of hEGF protein in E. coli, the development of a new hEGF gene that satisfies the above purpose This completes the present invention. Therefore, it will be apparent that the hEGF gene of the present invention is a novel gene that is distinct from the known hEGF gene.

Hereinafter, the present invention will be described in more detail.

In the present invention, hEGF gene chemically synthesized to have six novel restriction enzyme sites such as Hpa I, Bpu1102 I, Nsi I, Mlu I, Eco47 III and Afl II, and a Pst I restriction enzyme cleavage site at the 3 ′ end To provide. All of these restriction enzyme sites, namely seven restriction enzyme sites such as Hpa I, Bpu1102 I, Nsi I, Mlu I, Eco47 III, Afl II and Pst I, were seen in the pUC 18 vector as shown in the Examples of the present invention described below. Insertion of the synthetic hEGF gene results in a single restriction enzyme site in the entire vector for all seven restriction enzymes. It is practically important to have so many single restriction enzyme sites in a gene at regular intervals, and in particular, to increase the activity or increase the stability of hEGF through experiments such as causing artificial mutations in specific regions of hEGF. If you want to be very useful. In addition, the Hpa I restriction enzyme site introduced at the 5 'end of the hEGF gene of the present invention, when cleaved with this restriction enzyme, provides a nucleotide sequence that completely matches the native hEGF amino acid sequence, thus avoiding formation of a fusion protein. In this case, the hEGF gene sequence of the present invention may be usefully used.

Therefore, the present invention minimizes the factors that can inhibit the production of E. coli at high yield in E. coli, and enables the production of hEGF in E. coli with high yield and at the same time can be very useful for genetic engineering experiments using the hEGF gene It is a primary object to provide novel hEGF genes that have been artificially designed and chemically synthesized.

As shown in FIG. 1, the hEGF gene of the present invention has a Hpa I restriction enzyme site at the 5 'end and a cleavage site of the Pst I restriction enzyme at the 3' end, and among the internal sequences thereof, Bpu1102 I, Nsi I, Mlu. Artificially designed and chemically synthesized to have I, Eco47 III and Afl II restriction sites.

In particular, the hEGF gene sequence of the present invention is designed to be composed of the codons most frequently used in Escherichia coli without changing the sequence of the final amino acid, designed to increase the expression rate of the gene in Escherichia coli, The restriction enzyme site was designed to maximize its industrial utility. Meanwhile, in order to determine the sequence of the hEGF gene of the present invention, the amino acid sequence of hEGF (Gregory, H., (1975) Nature 257, 325) already known by Gregory was used.

For the assembly of the hEGF gene of the present invention, all 10 strands of complementary oligonucleotides were synthesized (Fig. 2 (a)). The oligonucleotides can be easily synthesized by solid phase phosphite triester synthesis methods commonly used in the art (Narang, SA, Synthesis and Applications of DNA and RNA, (1987), Academic Press), and in the present invention, automated It could be synthesized using a synthetic instrument (Pharmacia LKB Biltechnology, Uppsaoa, Sweden) (see Example 2). Synthesis of the complete hEGF gene using 10 strands of synthetic oligonucleotides used a short-gun ligation method that combines and binds all oligonucleotides at once (see FIG. 2 (b)).

Meanwhile, the hEGF gene of the present invention was inserted into pUC 18 plasmid digested with restriction enzymes Sma I and Pst I. The pUC 18 plasmid containing the hEGF gene of the present invention was named pUE 118 (see FIG. 3) and transformed by the method of Hana, D. into E. coil JM 109 (Hanahan, D.). DNA Cloning Vol.I: A Practical Approach, IRL Press, 1985, pp 109-135). E. coli transformant (Escherichia coil JM 109) containing pUE 118 was named DW / BT0-2040, and was deposited on April 9, 1993 under the KCCM accession number KCCM-10026.

Hereinafter, the present invention will be described in more detail with reference to Examples.

These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1

Design of the hEGF Gene

The hEGF gene sequence of the present invention was designed with the following four considerations in mind: First, it produces a protein having the same amino acid sequence and structure as a natural human hEGF protein; Second, the nucleotide sequence is determined in consideration of the codon most frequently used in E. coli; Third, minimize the formation of secondary structures in mRNA production; Fourth, maximize the utility of the present invention by having a single restriction enzyme site that is experimentally useful within the sequence.

The hEGF gene sequence of the present invention, which satisfies the foregoing objectives, is based on the already known human EGF amino acid sequence and in coliform codons of Grantham et al. (1981) Nucoeic Acid Res. 9, 243-274). The sequence was designed primarily based on the results of the study on the frequency of use. The primary designed sequence is PC-FOLD, a computer software that analyzes the secondary structure of genes for the extent of mRNA secondary structure formation; Version 2.0 (Turner, D. et al., (1987) Cold Spring Harbor Symb. Quant. Biol. 52, 123) program used to investigate the secondary transcript of the transcript without significantly compromising the codon usage of E. coli Sequences were chosen to minimize structure formation. The sequence was designed to have the Hpa I site at the 5 ′ end and to have the cleavage site of the PST I restriction enzyme immediately after the translational stop codon, thereby simplifying the precise insertion, isolation and manipulation of the entire hEGF gene. In addition, the designed gene was designed to have one specific single restriction enzyme site at almost regular intervals. Types and intervals of restriction enzymes are as follows.

Hpa I-22bp-Bpu1102 I-39bp-Nsi I-25bp-Mlu I-35bp-Eco47 III-21bp-Afl II-32bp-Pst I

The sequence of the hEGF gene of the present invention designed as shown in FIG. 1 has a Hpa I restriction enzyme cleavage site at the 5 ′ end, a Pst I restriction enzyme cleavage site at the 3 ′ end, and Bpu1102 I, Nst I , Mlu I, Eco47 III and Afl II restriction enzyme cleavage site can be seen that.

Example 2

Synthesis of Oligonucleotides

The hEGF gene designed in Example 1 was synthesized by dividing all 10 oligonucleotides each having a length between 29 mers (29mers) consisting of 29 bases and 41 mers consisting of 41 bases each (see FIG. 2 (a)). ]. Thus, the entire hEGF gene is complementary to the C1 (35 mer), C2 (34 mer), C3 (29 mer), C4 (39 mer) and C5 (41 mer) oligonucleotides having the same sequence as the transcribed mRNA. N1 (29 mer), N2 (38 mer), N3 (29 mer), N4 (36 mer) and N5 (38 mer) oligonucleotides (see FIG. 2 (b)). Each oligo nucleotide was synthesized using an automated nucleotide synthesizer (Pharmacia LKB Biotechnology, Uppsala, Sweden).

Example 3

Purification and Analysis of Oligonucleotides

Synthesized oligonucleotide was treated with TTD (thiophenol / thioethylamine / dioxane = 1/2/2) solution attached to silica, washed well with methanol and ethanol, and then treated with strong ammonia water, Synthetic oligonucleotides were removed. Strong ammonia water was added to the oligonucleotide solution, and the mixture was kept at 0 ° C. for 12 hours. After the reaction, the solution was concentrated under reduced pressure and concentrated to 0.5 ml after degassing. Concentrated oligonucleotides were first purified with acetonitrile / triethylamine buffer using SEP-PAK cartridge (Watera Inc., MA, USA), followed by polyacrylamide gel (15% acrylamide, pH 8.3 TE-boric acid). Electrophoresis was performed. After electrophoresis, oligonucleotides were identified by short-wavelength ultraviolet light, and only the site was cut. Then, the SEP-PAK cartridge (Waters Inc., MA, USA), which extracted oligonucleotides from the cut gel sections and connected them to a syringe, was used. Purification was carried out by removing the salt with acetonitrile / triethylamine buffer. The purified oligonucleotides of each nucleotide is T ₄ polynucleotide kinase (New England Biolabs, # 201S) by using the then labeled with γ- ^"32 P" -ATP maksam-Gilbert sequencing (Maxam, AM Gilbert, W. ( 1977) , Proc. Natl. Acad. Sci. USA 74, 560-654) were performed to determine the respective oligonucleotide sequences.

Example 4

Ligation of oligonucleotides

100 pmole of each oligo nucleotide with 40 'terminal phosphate group and 40 ul of tris-hydrochloric acid (pH 7.5, 0.1 M) buffer solution were added to the test tube in an Eppendorf test tube, and denatured at 100 ° C. for 3 minutes. The oligonucleotides were recombined while slowly descending to room temperature at. Ligase buffer solution, and T ₄ DNA ligase _{(T 4 DNA ligase, New England} Biolabs, # 202S) was added to 10 units was reacted for 12 hours at 4 ℃. After adding 10 units of the linking buffer solution and T ₄ DNA ligase and reacting at room temperature for 3 hours, 7% polyacrylamide gel electrophoresis was performed and radiographs were made to confirm the assembly state of the gene.

Example 5

Construction and Transformation of the pUE 118 Plasmid

In order to allow the expression of the novel hEGF genes of the present invention, the vectormin pUC 18 plasmids are already known and widely used in general molecular biology experiments (Norrander, J. et al., (1985), Gene 26, 101). The hEGF gene of the invention was inserted. First, the pUC 18 vector was digested with Sma I (New England Biolabs, # 141S) and Pst I (New Englands, # 140S), to which the hEGF gene of the present invention was coupled. The hEGF gene of the present invention has a blunt state having a Hpa I restriction enzyme site at the 5 ′ end and a cleavage site of the Pst I restriction enzyme at the 5 ′ end, thus being double cleaved by Sma I and Pst I without any further manipulation. could be appropriately bound to the pUC 18 vector (Fig. 3).

1% agarose gel electrophoresis of pUC 18 vector containing the hEGF gene of the present invention named pUE 118 and digested with Hpa I, Pst I, Bpu1102 I, Nsi I, Mlu I, Eco47 III and Afl II restriction enzymes, respectively It was confirmed that the hEGF of the present invention was correctly inserted and has a single restriction enzyme site for each restriction enzyme (see FIG. 4). In FIG. 4, the first lane is a 1 kb ladder DNA (BRL, U.S.A.); The second lane comprises pUE 118 cleaved with Hpa I; The third lane comprises pUE 118 cleaved with Pst I; Lane 4 is a poverty cleavage of pUE 118 with Hpa I and Pst I; The fifth lane comprises pUE 118 cleaved with Bpu1102 I; Lane 6 is pUE 118 cleaved with Nsi I; Lane 7 is a pUE 118 cleaved with Mlu I; Lane 8 is pUE 118 digested with Eco7 III; And ninth lanes each indicate the cleavage of pUE 118 with Afl II.

The prepared pUE 118 plasmid was transformed into E. coli JM 109 strain, E. coli JM 109 transformant containing pUE 118 (Escherichia coli JM 109) was deposited on April 9, 1993, Korea Deposit Association (KCCM) Deposited with the number KCCM-10026.

As described and demonstrated in detail above, the gene of the human epidermal growth factor of the present invention is artificially designed to facilitate mass expression and genetic manipulation, and has a Hpa I restriction enzyme site at the 5 'end and Pst I restriction at the 3' end. It has an enzyme cleavage site, and has a Bpu1102 I, Nsi I, Mlu I, Eco47 III and Afl II restriction enzyme sites at regular intervals, thereby maximizing industrial utility.

Claims (3)

Human epidermal growth factor gene having the following nucleotide sequence: A vector pUE comprising the human epidermal growth factor gene of claim 1. Escherichia coli transformed with the vector pUE 118 of claim 2 (Escherichia coli JM 109, KCCM-10026).