KR19990017407A - Expression Vector of Human Growth Hormone and Method of Manufacturing Human Growth Hormone Using the Same - Google Patents

Abstract

The present invention relates to an expression vector of human growth hormone and a method for producing human growth hormone using the same. More specifically, the present invention provides a L-arabinose comprising a fragment of glutathione S-transferase, a DNA fragment encoding a urokinase cleavage site and a human growth hormone gene. ) Inducible expression vectors, recombinant microorganisms transformed therefrom, and electric microorganisms are cultured in a medium to which L-arabinose is added to induce expression, thereby producing a large amount of human growth hormone. The fusion protein prepared from the expression vector of the present invention is produced in high yield through precise L-arabinose induction regulation, and contains an amino acid sequence specifically cleaved by urokinase between the fusion partner and human growth hormone. By treatment with, it is possible to effectively separate the recombinant human growth hormone that retains the activity of the native human growth hormone as it is from the fusion protein.

Description

Expression vector of human growth hormone and a method for producing human growth hormone using the same.

The present invention relates to an expression vector of human growth hormone and a method for producing human growth hormone using the same. More specifically, the present invention provides a L-arabinose comprising a fragment of glutathione S-transferase, a DNA fragment encoding a urokinase cleavage site and a human growth hormone gene. The present invention relates to a method for producing human growth hormone in large quantities by culturing inducible expression vectors, recombinant microorganisms transformed from spheres, and electric microorganisms in a medium containing L-arabinose.

Human growth hormone is the most secreted single-chain protein hormone of the anterior pituitary hormone, a hormone having a molecular weight of about 21,500 Da consisting of 191 amino acid residues. Growth hormone promotes balanced growth by proliferating cells of almost all tissues in the body except the brain and eyes, and has a physiological action that increases bone growth and enlarges skeletal muscle. It is used to treat dwarfism.

Such human growth hormone has been previously extracted and used mainly from human pituitary gland which died from traffic accident or disease, but its amount is greatly limited and only the human pituitary hormone purified from high purity should be used. Not only were they not supplied to all dwarf patients, but they were also expensive. In addition, there are various problems in its use, such as an accident in which a child who receives extracted and purified human growth hormone is infected with a nasty virus and dies, and the US Food and Drug Administration (FDA) died of the human pituitary gland. The use of human growth hormone extracted and purified from mice was prohibited.

In addition to direct extraction from humans, there have been efforts to prepare human growth hormone using chemical synthesis or tissue culture methods, but these have also been disadvantageous in terms of low yield, and thus improvement of production methods has been proposed.

Therefore, the present inventors are conducting research to mass-produce human growth hormone from recombinant microorganisms using genetic engineering techniques, and since the amino acid sequence of the human growth hormone amino terminus is known as an essential factor for biological activity of human growth hormone, Emphasis was placed on the removal of amino-terminal methionine during expression of human growth hormone in Escherichia coli, which is widely used as a host.

E. coli, which is widely used as a host for recombinant protein expression, contains methionine-specific amino peptidase, an enzyme that removes methionine at the start of translation of the amino terminus of an expressed protein.However, when a large amount of foreign protein is expressed in E. coli, Often, this is not properly removed. This phenomenon must be solved when constructing an E. coli expression system of proteins such as human growth hormone, whose amino-terminal amino acid sequence has a great influence on biological activity.

In order to solve the above-mentioned problem, three methods can be mainly used. First, by expressing a secreted signal sequence fused to the amino terminus of the target protein, the target protein is processed to be amino-terminated. It is a method to be secreted in the form of E. coli periplasm (periplasm) or culture medium, this method has the advantage of obtaining a mature protein using the intracellular activity, but has the disadvantage of relatively low expression yield. Secondly, the target protein is expressed alone in E. coli, isolated from E. coli with methionine attached to its amino terminus, and then cleaved with amino peptidase to obtain a mature protein. It is difficult to separate and purify between the removed protein and the protein to which methionine is attached. Third, the target protein is expressed in the form of fusion with other proteins to isolate the fusion protein from Escherichia coli, and then another enzyme (fusion partner) is removed from the fusion protein using enzymes or chemicals to prepare a mature target protein. This method has the advantage of not only providing the target protein from which the amino-terminal methionine has been removed, but also increasing the expression efficiency of the target protein.

Therefore, the present inventors used the third method to mass produce human growth hormone from which amino-terminal methionine was removed from recombinant E. coli. That is, L-Arabinose-inducible expression including a fragment of glutathione S-transferase (hereinafter referred to as 'GST'), a DNA fragment encoding a urokinase cleavage site, and a human growth hormone gene, which is used as a fusion partner. By preparing a vector, transforming E. coli, and then separating the fusion protein and cutting it with urokinase, it was possible to prepare a large amount of mature natural human growth hormone without amino methionine at the amino terminus.

After all, the main object of the present invention is to provide a L- arabinose-inducible expression vector and a recombinant microorganism transformed therefrom comprising a nucleotide sequence of a urokinase cleavage site between a fragment of the fusion partner GST and the gene of human growth hormone, the target protein. It is.

Another object of the present invention is to provide a method for producing human growth hormone by culturing the transformed recombinant microorganism in a medium to which L- arabinose is added to induce expression.

1 shows the cDNA base sequence of human growth hormone cloned from the pituitary gland and the amino acid sequence translated therefrom.

2 is a diagram schematically illustrating the process of constructing the expression vector ΔpG2 of human growth hormone.

3 shows a genetic map of the expression vector ΔpG2 of the present invention.

4 is an electrophoretic image showing the expression pattern of the fusion protein by L-arabinose induction of E. coli transformed with the expression vector ΔpG2.

FIG. 5 is an electrophoretic image showing that human growth hormone is separated from an electrofusion protein by cleaving an inclusion body of GSTt / hGH produced from E. coli transformed with the expression vector ΔpG2 with urokinase.

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

Human growth hormone expression vector of the present invention is a fragment of GST used as a fusion partner, preferably a DNA fragment encoding 69 or 133 amino acids from the GST amino terminus (see the same filed "glutathione S transferase (glutathione) Expression vector of fusion protein using amino-terminal fragment of S-transferase) as fusion partner ”); DNA fragment encoding the urokinase cleavage site -X-Gly-Arg (X is Pro, Thr, Ile, Phe or Leu), most preferably a DNA fragment encoding -Thr-Gly-Arg (see Korean Patent Publication 97-6495); And an L-arabinose-inducible expression vector comprising a human growth hormone gene. Since the fusion protein prepared from this expression vector contains an amino acid sequence specifically cleaved by urokinase between the fusion partner and human growth hormone, human growth hormone can be effectively separated from the fusion protein by treatment with urokinase.

One preferred embodiment of the present invention is a gene fragment encoding 69 amino acids from the GST amino terminus of Schistosoma japonicum, a DNA fragment in which two glycine codons are continuously connected to the 3 ′ end of the fragment, urokinase A DNA fragment encoding a cleavage region Thr-Gly-Arg and a gene of human growth hormone are provided in a sequence of L-arabinose-inducible expression vectors. At this time, the insertion of two glycine codons improves the cleavage efficiency of the expressed fusion protein by urokinase.

By culturing the transformant prepared by introducing the expression vector into E. coli and inducing expression with L-arabinose, it was confirmed that the human growth hormone fused with the GST fragment was expressed in large quantities. That is, the human growth hormone expression vector of the present invention showed higher expression rate and accurate induction regulation than any conventional expression vector.

In addition, it was confirmed that the human growth hormone fused with the GST fragment expressed above was separated into an inclusion body in a transformant, and obtained an inclusion body to treat urokinase, and a fusion protein of GST fragment / human growth hormone. Recombinant human growth hormone was isolated from.

The recombinant human growth hormone thus separated has the same amino acid sequence and molecular weight as the native human growth hormone, and thus will retain the activity of the native human growth hormone.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

(Example 1): cDNA library preparation and human growth hormone gene search

To prepare a human cDNA library, about 2 g of the human pituitary gland was prepared in 4M guanidine isothiocyanate buffer (Chomczynski P. and Sacchi N., Analytical Biochem., 162: 156-159 (1987)). After crushing, the supernatant was collected by centrifugation. To this was added 2M sodium acetate and isopropanol and centrifuged at 15,000 rpm to precipitate total RNA. Precipitated RNA was washed with 75% ethanol and dissolved in sterile distilled water containing 0.1% diethylpyrocarbonate (DEPC), followed by absorbance measurements and formaldehyde gel electrophoresis at 260-280 nm on an ultraviolet spectrophotometer. Sambrook et al., Molecular Cloning 2nd ed., Cold Spring Harbor press, 1989).

Messenger RNA was purified from the extracted total RNA using an oligo (dT) cellulose column (Poly-A Quick ^™ Stratagene, USA). 5 μg of messenger RNA was used as a template, and primary cDNA strands were synthesized using XhoI-oligo (dT) primer and reverse transcriptase (M-MuLV reverse transcriptase; KZAP cDNA synthesis kit, Stratagene, USA). At this time, the dCTP in the dNTP used was methylated primary cDNA by using methylated dCTP (5-methyl dCTP). After the double-stranded cDNA was synthesized from the primary cDNA strand using RNaseH and DNA polymerase, a restriction enzyme EcoRI adapter (adaptor) was connected to both DNA strands with a T4 DNA ligase. The reaction was treated with XhoI restriction enzyme and size fractionated with Sephacryl S 400 column (Stratagene, USA) to collect only high molecular weight cDNA fractions, which were recovered by ethanol precipitation.

Packaging for the above process, the rough reaction in vitro (In vitro packaging; GigaPack II, Stratagene, USA) and E. coli XL1-Bue MRF 'strain {(mcrA183 (mcrCB-hsdSMR- mrr) 173 [F - proAB lacI q Z M15 Tn10 (Tet ^r )] endA1 supE44 thi-1 recA1 gyrA96 relA1 lac} and then amplified.

In order to select a human growth hormone gene from the cDNA library prepared as described above, an oligonucleotide (5'-TTCCCAACCATTCCCTTAT-3 ') corresponding to an amino terminal region from a nucleotide sequence of a known gene and an oligonucleotide (corresponding to a carboxy terminal region) Polymerase chain reaction (PCR) using 5'-CTAGAAGCCACAGCTGCC-3 ') as 5' and 3 'primers, respectively, to remove 191 human growth hormones in which a signal peptide was removed Gene fragments corresponding to amino acids were obtained. At this time, the primer corresponding to the carboxy terminal portion was synthesized to have a nucleotide sequence of the SalI restriction enzyme recognition site in order to facilitate insertion into the expression vector.

The cDNA nucleotide sequence of the human growth hormone gene obtained above and the amino acid sequence translated therefrom are shown in FIG.

Example 2 Preparation of Expression Vector ΔpG2 of Human Growth Hormone

In order to express the human growth hormone gene cloned in Example 1 in Escherichia coli, ΔpCUP-1, which is a fusion protein expression vector for Escherichia coli, was used. ΔpCUP-1 is a DNA fragment encoding 69 amino acids from the GST amino terminus of the Schistosoma japonicum, a DNA fragment encoding the urokinase cleavage site Thr-Gly-Arg, a StuI restriction enzyme recognition site, and L-arabinose-inducible fusion protein expression vector containing human parathyroid hormone gene in a sequence. This expression vector is an E. coli expression vector using an arabinose promoter in which expression is induced by L-arabinose, and has the characteristic of expressing the target protein in the form of a fusion protein using a GST fragment of 69 amino acids as a fusion partner. See also: "Expression vector of fusion protein using amino terminal fragment of glutathione S-transferase as fusion partner".

First, the expression vector was isolated from Escherichia coli MC1061 (KFCC-10979) transformed with ΔpCUP-1, treated with restriction enzymes of StuI and SalI, and then subjected to electrophoresis to obtain vector fragments. Subsequently, the gene of human growth hormone cloned in Example 1 was treated with SalI restriction enzyme and then inserted between the StuI restriction enzyme cleavage plane of ΔpCUP-1 using T4 DNA ligase. The expression vector of the human growth hormone thus prepared was named pCUG.

On the other hand, in order to improve the cleavage efficiency of the expressed fusion protein by urokinase, two glycine codons (GGTGGC) were continuously inserted between the gene of the GST fragment as the fusion partner and the nucleotide sequence corresponding to the urokinase cleavage recognition site. That is, 5 'and 3' oligonucleotides (5'-GGAAACAGTACATATGTCCCCTA-3 ', 5'-AGTACCGGATCCGCCACCCATAGACTG-3') were used to perform PCR using ΔpCUP-1 as a template, and 3 'of the GST fragment as a fusion partner. Gene fragments in which two glycine codons were connected in series were obtained, and then digested with NdeI and BamHI restriction enzymes. On the other hand, the pCUG was treated with BamHI and SalI restriction enzymes to obtain a gene fragment of about 600bp corresponding to the gene for urokinase cleavage recognition and human growth hormone. The gene fragments were linked to vector fragments obtained by cleaving pCUG with NdeI and SalI using T4 DNA ligase, to prepare ΔpG2, the final human growth hormone expression vector (see FIG. 2). 3 shows a genetic map of the expression vector ΔpG2. In Figures 2 and 3, GSTt represents a gene fragment of GST, PTH represents human parathyroid hormone, hGH represents human growth hormone, and UK represents urokinase.

Example 3 Expression of Human Growth Hormone

E. coli MC1061 (E. coli MC1061, F-araD139 Δ (ara-leu) 7696 galE15 galK16Δ (lac) X74rpsL (Str ^r ) hsdR2 (r _k ) with the expression vector ΔpG2 prepared in Example 2 ^- m _k ^+), mcrA mcrB1) the transformant and, him contains the ampicillin an LB (Bacto agar 1.5%, yeast extract 0.5%, NaC1 0.5% and tryptone medium consisting of 1%), a temperature of 37 ℃ in plate medium Cultured under conditions. Selected E. coli colonies were inoculated into TB culture containing ampicillin (media consisting of 1.2% tryptone 1.2%, yeast extract 2.4%, glycerol 0.4%, 0.017MKH ₂ PO ₄ , 0.072MK ₂ HPO ₄ ) and 180 rpm at 37 ° C. Shake culture was carried out. When the concentration of the cultured cells reached an optical density (OD) of 0.5 to 0.6 at 600 nm, l-arabinose was added to the culture to reach a final concentration of 1%, followed by induction of expression from the arabinose promoter. Time was shaken more.

As a result of analyzing the total protein expressed by SDS-PAGE, human growth hormone fused with GST fragment consisting of 69 amino acids was precisely induced by L-arabinose to prepare a high expression rate (see FIG. 4). In FIG. 4, GST / HGH represents a human growth hormone fused with GST 69 amino acids, and lanes 1-7 are each 0, 2, 4, 6, 8, 21, 24 after induction with L-arabinose. The result of time incubation is shown.

The E. coli MC1061 transformed with the expression vector ΔpG2 was named MC1061: ΔpG2, and deposited on July 9, 1997, with the accession number KFFCC-10976 to the microbial preservation center (KCCM), an international depository organization. It was.

Example 4 Isolation of Recombinant Human Growth Hormone Using Eurokinase

In order to separate human growth hormone by treating the fusion protein expressed in Example 3 with urokinase, first, only an inclusion body was isolated from the cell lysate of the cultured recombinant E. coli. It was dissolved in 8M urea and dialyzed in 20 mM Tris-HCl (pH 7.8) to induce refolding of the fusion protein. A uroproteinase of 200: 1 to 1000: 1 relative to the mass was added to the fusion protein sample, and the fusion protein was cleaved under a temperature condition of 25 ° C. in 20 mM Tris-HCl (pH 7.8) containing 0.1 M NaCl.

After the cleavage reaction was started, the reaction samples collected at 0, 2, 5, and 24 hours were analyzed by SDS-PAGE to confirm that human growth hormone was separated as the fusion protein was cleaved by urokinase.

5 is an electrophoretic photograph showing the separation of human growth hormone from fusion proteins by treatment of inclusion bodies with urokinase. In FIG. 5, GST / HGH represents human growth hormone fused with GST 89 amino acids and HGH represents human growth hormone, respectively. In addition, the first lane of Figure 5 is the result of the electrophoresis of the protein of the inclusion body separated from the total protein obtained by L-arabinose induced, it can be seen that the majority of human growth hormone fused to the GST fragment Lane 2 was electrophoresed by cleavage of the protein of the inclusion body with urokinase. As a result, lane S represents purified human growth hormone.

By determining the molecular weight and amino terminal sequence of the human growth hormone separated in this way to obtain the same results as the human growth hormone control, the recombinant human growth hormone expressed in the expression vector of the present invention is the same as the natural human growth hormone It was confirmed.

As described and demonstrated in detail above, the fusion protein of mass-produced GST fragment / human growth hormone by culturing the recombinant microorganism transformed with the expression vector ΔpG2 of the present invention and inducing expression with L-arabinose was treated by urokinase treatment. Recombinant human growth hormone having the same amino acid sequence and molecular weight as the native human growth hormone is isolated. Therefore, the expression vector of the present invention can be produced in high yield through the precise L- arabinose induction of recombinant human growth hormone that maintains the activity of the native human growth hormone as it is.

Claims (7)

An L-arabinose inducible expression vector comprising a fragment of glutathione S-transferase, a DNA fragment encoding a urokinase cleavage site, and a human growth hormone gene, which is used as a fusion partner. The expression vector of claim 1, wherein the fragment of glutathione S-transferase is a DNA fragment encoding 69 or 133 amino acids from the amino terminus. The expression vector according to claim 1, wherein the urokinase cleavage site is a DNA sequence inferred from the following amino acid sequence: -X-Gly-Arg wherein X is P, Thr, Ile, Phe or Leu . A gene fragment encoding 69 amino acids from the amino terminus of glutathione S-transferase, a DNA fragment in which two glycine codons are consecutively linked at the 3 'end thereof, and the kinase cleavage region Thr-Gly-Arg L-Arabinose-inducible expression vector ΔpG2 containing a DNA fragment and a gene of human growth hormone in a sequence. Escherichia coli MC1061: ΔpG2 transformed with the expression vector ΔpG2 of claim 4 (KFCC-10976). (i) L-arabinose inducible expression vector comprising a fragment of glutathione S-transferase, a DNA fragment encoding a urokinase cleavage site, and a human growth hormone gene Transforming Escherichia coli, and culturing the transformant; (Ii) inducing expression of the fusion protein by adding L-arabinose during electric culture, and then culturing again; (Iii) obtaining the cells from the electroculture and crushing the cells to obtain inclusion bodies; And (i) treating urokinase to the inclusion body obtained in the previous period, and obtaining a natural human growth hormone. The method of claim 6, wherein the transformant is MC1061: ΔpG2 (KFCC-10976).