KR20020092089A - Expression vector containing gene of human ferritin, transformant thereof and method for preparing human ferritin using the same - Google Patents

Abstract

PURPOSE: Provided are an expression vector containing gene of human ferritin, an E. coli transformant and a method for preparing human ferritin using the same, thereby mass-producing recombinant human ferritin in high concentration and yield. CONSTITUTION: The expression vector contains human ferritin gene and T7 promotor, wherein the human ferritin gene is characteristically ferritin light chain. E.coli transformant, BL21(DE3)/pT7FerL(KCTC 1016BP) is prepared by transformation with the expression vector. Human ferritin protein is manufactured by the steps of: culturing the E.coli transformant; separating an inclusion body of an insoluble protein; suspending the insoluble protein in an alkali solution; and loading pH buffer solution to the suspension for neutralization.

Description

Expression vector containing a human ferritin gene, E. coli transformant comprising the same and a method for producing a human ferritin protein using the transformant {Expression vector containing gene of human ferritin, transformant etc and method for preparing human ferritin using the same}

The present invention relates to an expression vector of a human ferritin gene, an E. coli transformant comprising the same, and a method for producing a ferritin protein using the transformant.

In order to produce a variety of important proteins that are difficult to obtain in human or animal organs in a microorganism by genetic recombination method, the gene of the target protein is cloned into an expression vector to obtain a recombinant expression vector and transformed into a suitable host cell. Among the various microorganisms, E. coli is known as the most information about its genes and metabolic system compared to other organisms, and is used as a host for producing useful foreign proteins by genetic recombination technology. However, in most cases, the target polypeptide expressed in E. coli is present in the form of insoluble aggregates in cells (Ulman, A.,Gene,1984, 29, 27-31; Nilsson, B. et al.,Embo J.,1985, 4, 1075-1080; DiGuan et al.,Gene,198867, 21-30; La Vallie, E. R. et al.,Bio / Technology,1993, 11, 187-193). In this case, the target recombinant protein can be easily separated from the cell culture in the initial separation process, but during the formation of aggregates, the expressed polypeptide is intermolecular disulfide in the folding intermediate stage. By non-selective binding with other protein impurities (saffron, ribosome, early factors, etc.) of the host cell by bond or hydrophobic interaction, hardly soluble solid aggregates are formed, making it possible to use the desired protein. May be absent (Anna Mituraki et al.Bio / Technology,1989, 7, 690-697). When the protein is produced as insoluble aggregates, in order to purely separate the active protein therefrom, it is generally dissolved using a denaturant such as guanidine hydrochloride or urea, followed by dilution through dilution. You have to go through the folding process. Aggregates produced by disulfide bonds must be treated together with a reducing agent for dissolution. In addition, the refolding process is still a major problem to reduce the production yield due to many problems (Marston, F. A. O.Biochem. J.,1986, 240, 1-12; Mitraki, A. and King,J. Bio / Technology,1989, 7, 690-697).

Iron is an essential nutrient for all forms of organisms, most of which are combined with proteins to participate in many important physiological actions. As a representative example, iron plays an important role in the production of adenosine triphosphate by participating in the role of transporting electrons from the substrate to oxygen in an electron transport system. In relation to this function, below-normal iron production decreases energy production, which leads to rapid fatigue and reduced work efficiency, especially iron deficiency in infancy may damage the brain.

Ferritin, on the other hand, is a protein that stores iron so that it is not toxic to cells and can be used effectively when needed.Most of animal and plant tissue, as well as fungi and bacteria (Ragland, M. et al., J. Biol. Chem ., 1990 , 265, 18339-18344; Grossman, MJ et al., Proc. Natl. Acad. Sci. USA , 1992 , 89 , 2419-242). Ferritin has a significant similarity in sequence to prokaryotic as well as prokaryotic cells, and the structure and function of each are actively studied. One molecule of ferritin consists of 24 subunits consisting of a heavy chain (21 kD; hereinafter referred to as "H-chain") and a light chain (19 kD; hereinafter referred to as "L-chain"). The composition ratio of H-chain and L-chain is not always constant, so the structure and size vary. One molecule of ferritin can hold about 4,500 iron molecules, corresponding to an iron concentration of 0.25 M, an amount capable of producing about 1200 molecules of hemoglobin, and the L-chain has the ability to bind iron compared to the H-chain. This has been reported to be excellent (Levi, S. et al., Biochem. J. , 1992 , 288, 591-596). Until recently, animal ferritin extracts extracted from spleen tissues of animals were mainly used for the prevention and treatment of iron deficiency. As a result of animal virus problems, domestic product license was canceled and manufactured by Korea Food and Drug Administration on March 28, 2000. The suspension of work has been carried out. Therefore, the necessity of developing a process technology capable of mass production of recombinant human ferritin using genetic recombination technology for industrial mass production is required. As a result of studies related to the production of recombinant human ferritin, there have been examples of expression of water-soluble forms in E. coli using the λ _pL promoter for the purpose of physicochemical / biological characterization of human ferritin (Levi, S. et al. , Biochemistry , 1989 , 28, 5179-5184) The expression rate (the ratio of expressed recombinant protein to total E. coli protein,%) is extremely low (1-5%), indicating that the industrial value is very low.

Therefore, the present inventors mass-produced the recombinant human ferritin protein from E. coli in the form of insoluble aggregates using the T7 promoter, and then confirmed the binding properties between the protein molecules in the isolated aggregates, and did not use a denaturing agent or a reducing agent at all. The present invention was completed by revealing that a high concentration of recombinant protein aggregates can be easily converted into a water-soluble form and reactivated to have iron binding ability even by a shift process alone.

The present invention has been made to produce and isolate human ferritin protein of high concentration and high efficiency, the object of the present invention is an expression vector comprising a human ferritin gene, E. coli transformant comprising the same and the transformation It is to provide a method for mass production of ferritin protein using a sieve.

Figure 1 is a schematic diagram showing the manufacturing process of the expression vector pT7-FerL of the present invention,

Figure 2 shows the expression of recombinant human ferritin light chains (hereinafter referred to as "L-chains") in E. coli, followed by whole cell lysates, cell lysates, and cell sedimentation after cell disruption-centrifugation. electrophoresis picture showing the results of reducible SDS-PAGE analysis of pellets and supernatants,

Lane M: size marker

Lane 1: total cell lysate, lane 2: culture supernatant

Lane 3: cell precipitate, lane 4: standard human ferritin (L-chain)

3 is an electrophoretic photograph showing the results of reducing and non-reducing SDS-PAGE analysis of recombinant L-chain human ferritin expressed in the form of insoluble aggregates in E. coli,

Lane M: size marker, lane 1: reducing aggregates

Lane 2: non-reducing aggregate, lane 3: standard human ferritin (L-chain)

R: reducing, NR: non-reducing

FIG. 4 is an electrophoresis photograph of a recombinant L-chain human ferritin expressed in the form of an insoluble aggregate in E. coli, followed by simple dissolution by a pH shift method.

Lane M: size marker,

Lane 1: insoluble aggregate separated before the pH shift process,

Lane 2: the culture supernatant after the pH shift process,

Lane 3: protein precipitate after pH shift process

Figure 5 is an electrophoresis picture showing the iron binding activity of the recombinant L-chain human ferritin protein of the present invention dissolved by the pH shift method.

Lane 1: bovine serum albumin (BSA) (20 ㎍),

Lane 2: recombinant L-chain human ferritin (20 μg),

Lane 3: standard human ferritin (20 µg)

In order to achieve the above object, the present invention provides an expression vector comprising the T7 promoter and human ferritin gene.

In another aspect, the present invention provides an E. coli transformant having the expression vector introduced therein.

In another aspect, the present invention provides a method for mass-producing a ferritin protein using the transformant.

Hereinafter, the present invention will be described in detail.

The present invention provides an expression vector comprising a T7 promoter and a human ferritin gene.

The human ferritin gene can be prepared from human liver cDNA library, and in the present invention, L-chain human ferritin gene was amplified and manufactured using PCR method from human liver cDNA library as a preferred embodiment. The amplified DNA was purified and then digested with restriction enzymes, digested, and inserted into an expression plasmid to prepare an expression vector of the present invention comprising the L-chain human ferritin gene (see FIG. 1 ), which was referred to as "pT7-FerL". Named this.

The present invention also provides a transformant into which the expression vector is introduced.

In the present invention, a transformant prepared by transforming the expression vectors into E. coli. As the host cell, Escherichia coli BL21 (DE3) strain was used, and the transformant transformed by the expression vector pT7-FerL was named "BL21 (DE3) / pT7FerL", and it was Korean life as of May 23, 2001. Deposited to the Engineering Research Institute Gene Bank (Accession Number; KCTC 1016BP).

The present invention also provides a method for mass production of ferritin protein using the transformant.

The method of mass production of the ferritin protein

1) culturing an E. coli transformant transformed with an expression vector or pT7-FerL comprising a T7 promoter and a human ferritin gene;

2) separating insoluble protein aggregates from cells;

3) suspending the insoluble protein of step 2 in an alkaline solution, and

4) dropping the neutral buffer solution to the solution to restore to neutral.

In step 1), BL (DE3) / pT7FerL was used in the embodiment of the present invention as the E. coli transformant.

In step 3), the alkaline solution is preferably a solution having a pH of 10 to 12, more preferably pH 12.

In step 4), the neutral buffer is preferably Tris-HCl buffer of pH 8.

In steps 3) and 4) above, insoluble aggregates comprising recombinant human ferritin protein are readily dissolved in alkaline solutions at high concentrations and reactivated by restoration to neutral pH conditions. This property can be very useful in the purification process. Thereafter, only active recombinant human ferritin can be easily separated by separation through general purification methods such as ultrafiltration, dialysis, and ion exchange chromatography.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Plasmids Expressing L-Chain Human Ferritin Protein

L-chain human ferritin genes were prepared by amplification from human liver cDNA libraries (Clontech, CA, USA) by PCR using primer pairs as set forth in SEQ ID NO: 1 and SEQ ID NO: 2 .

PCR was performed in DNA polymerase reaction buffer (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH 8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100) 100 ng of template DNA, 50 pmol of each primer was added thereto, followed by Tag DNA polymerase. The reaction conditions were 95 ° C./30 sec (denaturation), 52 ° C./30 sec (annealing), and 72 ° C./60 sec (elongation) in 30 cycles in total. Unless otherwise specified in the present invention, all PCR was performed according to the above conditions. After completion of PCR, the amplified DNA was analyzed by electrophoresis on 1% agarose gel. The amplified DNA was purified using a gel extraction kit (Qiagen) and treated with Nde I and Hind III restriction enzymes, respectively, to have Nde I restriction enzyme sites at the 5 'end and Hind III restriction enzyme sites at the 3' end. The expression plasmid pT7-7 was inserted into the NdeI and HindIII sites, and the resulting plasmid was named pT7-FerL ( FIG. 1 ).

Example 2 Preparation of E. Coli Transformant

E. coli BL21 (DE3) (Studier, FA and Moffatt, BA, J. Mol ) by the expression vector pT7-FerL by the method described by Hanahan (Hanahan, D., DNA Cloning , 1985, 1, 109-135, IRSpress). Biol . 1986 , 189, 113-130) strains were then transformed, and colonies resistant to ampicillin were selected.

The transformed strain was deposited with the Korea Biotechnology Research Institute Gene Bank on May 23, 2001 (Accession No .: KCTC 1016BP).

Example 3 Culture of Escherichia Coli Transformant and Production of Recombinant Protein

A portion of the spawn culture cultured overnight was inoculated (1%) in culture medium (LB medium, +100 mg / L ampicillin) and then incubated at 37 ° C. at 200 rpm. When the OD ₆₀₀ of the culture reached 0.4, IPTG (Sigma) was added (0.5 mM) to induce the expression of recombinant genes. After addition of IPTG, the cells were further incubated for 3-4 hours under the same conditions.

As a result, the expressed recombinant human ferritin was confirmed to exist in the form of insoluble aggregates in E. coli cells ( FIG. 2 ).

Example 4 Isolation of Insoluble Aggregates from Cell Culture Fluids

Cell culture was centrifuged at 6000 rpm to recover the cell precipitate. Coliform precipitate was suspended in distilled water and then crushed using an ultrasonic crusher (Branson Sonifier). After centrifugation of the crushed solution at 5000 g for 10 minutes, the supernatant and precipitates were analyzed by SDS-PAGE (14% Tris-Glycine gel) to confirm their recovery. Protein aggregates were washed twice with 0.5% Triton X-100 followed by the required analysis.

As a result, the separated insoluble protein aggregates were analyzed by SDS-PAGE (14% Tris-Glycine gel) and shown in FIG. 3 .

Example 5 Reactivation of Recombinant L-Chain Human Ferritin Contained in Isolated Protein Aggregates

Binding Properties Between Recombinant L-Chain Human Ferritin Protein Molecules Contained in <5-1> Aggregates

In order to determine the binding properties between the constituent proteins in the aggregates (either between the same recombinant protein molecules or between recombinant proteins and other host protein molecules), the following analysis was performed. First, proteins in each aggregate were separated by reducing and non-reducing SDS-PAGE (14% Tris-Glycine gel), and the results of electrophoresis were compared.

As a result, as shown in lanes 1 and 2 of FIG. 3 , it was found that recombinant L-chain human ferritin was the main protein in the aggregate, and protein aggregates generated by direct expression were analyzed under reducing and non-reducing conditions. In comparison, it was found that in both cases almost equal amounts of recombinant L-chain human ferritin were present at the monomer position. The results indicate that L-chain ferritin is mostly bound by hydrophobic interactions between the same molecules in the aggregate, and thus produced by hydrophobic bonds rather than disulfide bonds between the same proteins. Aggregates can be dissociated relatively easily during the purification process and thus have the advantage of being very easy to purify.

<5-2> Reactivation of Recombinant L-Chain Human Ferritin by pH Shift Process: Recombinant Protein Processing Capacity and Recombinant Protein Activity Analysis of Reactivation Process

The insoluble protein obtained from the recombinant E. coli culture was suspended in an alkaline solution of pH 12 and then mixed with Tris-HCl pH buffer (1 M, pH 8) to perform a pH shift process to restore the pH to neutral conditions. Figure 4 shows the results of SDS-PAGE analysis of the protein solution before and after the pH shift process, it can be seen that about 90% of the recombinant human ferritin was dissolved in the pH shift process. In addition, the concentration of human ferritin that can be dissolved in the pH shift process was analyzed in the range of 0.6-1.0 g / l. Recombinant human ferritin, once dissolved by the pH shift process, was found to exist in water-soluble form without reaggregation even after 1.5-fold concentration.

Next, the activity of the water-soluble recombinant human ferritin dissolved in the pH shift process was verified by analyzing iron binding ability. First, a water-soluble recombinant human ferritin solution (50 mg / L) and Hepes buffer (1 M, pH 7) were mixed in a volume ratio of 10: 1, and then in 0.4 mM ferrous ammonium sulfate solution. A process of saturating iron in recombinant human ferritin was performed. After SDS-PAGE, the proteins isolated were subjected to prussian blue staining. Iron-binding proteins are stained but iron-deficient proteins are not stained.

As a result, as shown in FIG. 5 , the BSA (bovine serum albumin) protein (negative control) having no iron binding ability was not stained, but the recombinant human protein dissolved by the pH shift process was clearly stained.

From the above results, it was confirmed that recombinant human ferritin expressed in the form of insoluble aggregates in E. coli was successfully reactivated by a pH shift process.

As described above, the transformant transformed with the expression vector containing the human ferritin gene of the present invention produces a large amount of L-chain protein of recombinant human ferritin, and is produced with recombinant human ferritin protein expression. Aggregated aggregates are due to hydrophobic interactions with weak binding force. Unlike ordinary insoluble aggregates, large aggregates (0.6-1.0 g / l) are easily dissolved in alkaline solutions and reactivated with high efficiency as they return to neutral pH conditions. It can be usefully used for producing large amounts of human ferritin protein.

Claims (10)

Expression vector of human ferritin gene comprising T7 promoter and human ferritin gene. 2. The expression vector of human ferritin gene according to claim 1, wherein the human ferritin gene is a light chain ferritin gene. The expression vector of human ferritin gene according to claim 2, which is pT7-FerL. E. coli transformants transformed with the expression vector of the human ferritin gene of any one of claims 1 to 3. The E. coli transformant (Accession Number: KCTC 1016BP) of Claim 4 which is BL21 (DE3) / pT7FerL. 1) culturing the E. coli transformant of claim 4; 2) separating insoluble protein aggregates from cells; 3) suspending the insoluble protein of step 2 in an alkaline solution, and 4) A method for producing human ferritin protein comprising the step of dropping the pH buffer solution to the solution to restore to neutral. The method of claim 6, wherein the E. coli transformant of step 1) is BL21 (DE3) / pT7FerL. 7. The method of claim 6, wherein the alkaline solution of step 3) is pH 10 to pH 12. 7. The method of claim 6, wherein the pH buffer solution of step 4) is pH 8. 10. The method of claim 9, wherein the pH 8 buffer solution is Tris-HCl buffer.