KR930001388B1 - Method for preparation of expression vector - Google Patents

Abstract

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Description

Method for preparing a new fusion expression vector using DNA encoding a modified polypeptide of the Fc portion of an immunoglobulin

1 shows the sequence and amino acid sequence of a nucleotide encoding a CP polypeptide.

2 shows how to prepare the pTC plasmid.

3 shows a method for preparing a pRHC-10 plasmid.

4 is a diagram illustrating a method for preparing a pRHC-20 plasmid.

5 shows a sequence of 22 oligonucleotides constituting the CP gene.

The present invention relates to a method for stabilizing a material that is difficult to produce due to instability in a microorganism and effectively producing it, that is, a polypeptide modified by immunoglobulin (G) binding to Staphylococcus protein A using protein engineering. The present invention relates to a method for producing an expression vector that can be fused to a DNA encoding an external gene using this DNA and stably expressed in a host cell as a fusion protein, and can easily separate the produced external protein.

When an external gene is expressed in a microorganism, the expression is often not successful. Such inappropriate expression may occur when the stability of messenger mRNA (messenger RNA) is low, or when ribosomal binding sites are inappropriate, or when the resulting protein is unstable and degraded in the host cell. In the latter case, in order to increase the stability of the protein in the cell, the expression of the fusion protein is one way, and the expression of the fusion protein (fusion protein) is also used to facilitate the purification process.

Immunoglobulin G specifically binds to protein A, which is known to occur through interactions with the polypeptides of the Fc region of immunoglobulin G, in particular the C _H 2 -C _H 3 region (Deisenhofer). , J. Biochemistry, 2361, 1981).

The nucleotide sequence encoding the Fe region has been published by Kudo et al. (Gene, 33, 181, 1985). In the present invention, a gene encoding a protein obtained by modifying a C _H 2 -C _H 3 domain (dormain) using protein engineering (see FIG. 1) is synthesized, and the external gene is 3 'end (3'end) of the gene. ) To express an external gene stably in the host cell, and to separate and purify the fusion protein to be produced and to express the foreign protein expressed by fusion to the structural protein.

In order to easily obtain the desired protein from the fusion protein, the modified polypeptide (hereinafter referred to as CP), which can bind to Staphylococcus protein A by changing the structure of the C _H 2 -C _H 3 domain as follows, is referred to as: The coding genes were designed. In other words, the code in the CP gene that transcribes the Asn-Gly sequence was changed to the nucleotide sequence encoding Asn-Ala to remove the Asn-Gly sequence from the CP while minimizing the structure change of the structural protein. By removing the -Pro sequence in the above manner, the Asn-Gly sequence was placed at the linkage of the fusion protein to easily obtain the desired protein, and the Met-X sequence was also removed so that the external protein could be separated by CNBr treatment. (See Figure 1).

In addition, the CP gene does not have a peptide sequence that is specifically degraded by an enzyme, so a linker is encoded that encodes a recognition site for a specific enzyme between the 3 'end of the CP gene and the 5' end of the external gene to be expressed. Thus, cleavage of the fusion protein by enzymes is also possible.

The CP gene was used to cope with the high frequency of codon (codon) used in microorganisms to effectively express, and the restriction enzyme site (resriction enzyme site) is placed so that it is easy to connect the external gene to the end of the CP gene. In order to enhance the stability of the transcribed fusion protein in the host cell, the amino terminal of the structural protein is constructed according to the N-term rule (Bachmair A. et al., Science, 243, 179, 1985). Transcription of the dipeptide, Ser-Thr, which linked amino acids to increase protein stability, was performed.

A plurality of CP genes are linked to each other so that the CP polypeptides are continuously linked as structural proteins to enhance the binding capacity of protein A, and the fusion protein can be purified using a separation purification process of different sizes, for example, gel filtration chromatography. An expression vector was prepared which expresses an external gene so as to separate the external protein from the human body (see FIG. 4).

The present invention is explained in detail by the following examples.

Example 1

22 oligonucleotides (see FIG. 5) that make up the CP gene, consisting of 40-50 bases and cohesive at each end, are synthesized by automated phosphoamidite methods, respectively. Then, these were purified as follows and then assembled by the method described in Example 2 (assembly) to prepare a CP gene (Figure 1).

Deblocking was performed with concentrated ammonia solution at 55 degrees for 8 hours. After ammonia was removed, precipitated with ethanol to purify the oligo DNA, and purified by 8-16% polyacrylamide gel electrophoresis.

Example 2

50-60 picomoles of oligomers (A ₂ -A ₆ and B ₁ -B ₅ ) were each 20 μL of kination buffer (70 mM Tris pH 7.6, 10 mM MgCl ₂ 1 mM ATP, 0.2 mM) Spermidin, 0.5mM DTT) and 2μl of polynucleotide kinase were added and reacted at 37 ° C for 30 minutes and heated at 85 ° C for 10 minutes to stop the reaction. Phosphorylated oligomers (A ₂ -A ₆ and B ₁ -B ₅ ) and non-phosphorylated A ₁ and B ₆ oligomers were mixed with 0.2 p mole each, heated to 90 degrees, and then cooled to room temperature very slowly, annealing. I was.

5 μl of 10-fold concentrated ligation buffer (50 mM Tris pH7.6 10 mM MgCl ₂ , 20 mM DTT, 1 mM ATP) was added, followed by addition of T ₄ DNA ligase and reaction at 15 degrees for 4-10 hours. The reaction mixture was electrophoresed on 1.5-23% agarose gel, and CP DNA fragments were separated and purified by gel elution using a semipermeable membrane.

Example 3

CP DNA fragments extracted and purified with phenol, chloroform and ethanol were digested with pUC 19 plasmid (available from KCTC) with EcoR I and BamH I restriction enzymes and mixed with 0.4 µg of the large pUC 19 plasmid, followed by T ₄ DNA. Ligase was added and reacted at 12 degrees for 8 hours under ligation buffer. E. coli HB101 strain (Boyer et al. J. Mol. Biol. 41, 459, 1969) treated with CaCl ₂ with this reactant was then transformed with ampicillin resistance in a medium containing ampicillin (50 mg / ml). Strains were selected and plasmid DNA was isolated to identify plasmid pUC-C containing CP DNA fragments and plasmids were isolated (see Figure 2).

Example 4

5 μg of the pUC-C plasmid was digested with EcoR I and Pst I, agarose gel electrophoresis, small fragments of plasmid DNA (3866p) were isolated by gel-illution method, extracted with phenol and chloroform, precipitated with ethanol, purified, and nicked. Nick translation buffer (15 mM Tris pH 8.0, 15 mM KCl, 6 mM MgCl₂), And the Klenow fragment and four dNTPs were added and reacted at 20 ° C for 30 minutes to co-determine the DNA fragments in which the cohesive states of the EcoR I and Pst I ends were blunt. Purification was carried out by passing through 50 columns. To insert this DNA fragment immediately after the Tac promoter, ptac 12 (available from KCTC) was digested with pvuII, followed by T₄DNA ligase was treated and ligated in buffer to prepare pTC plasmid. In order to be effective in the transcription of structural genes by the tac promoter, the gene lock I (lac I) encoding the lac operon inhibitor protein (lac) is required. To obtain this gene, the plasmid pIN III (obtained from KCTC) (Masui et al., Biotechnology, 2,1,1984) was digested with Pst I and Sal I, 5.3Kbp DNA fragments were separated on an Ararose gel, and pTC was purified. PTC plasmid fragment and T digested with Pst I and Sal I and isolated by agarose gel electrophoresis₄DNA ligase was ligated. This reactant is CaCl₂Transformed E. coli JM 109 strain, isolate plasmid DNA, correct plasmid by restriction map and DNA sequencing The DNA sequence was identified and named as pRHC-10. The plasmid was designed to effectively express CP genes by the tack promoter and each I gene, and to have BamH I, Xba I, and Sal I restriction enzyme sites for convenient fusion of external genes immediately after the CP gene (Fig. 3). Reference).

Example 5

The pRHC-10 plasmid was partially cleaved with Xba I restriction enzyme, cleaved with Xba I from the pUC-C plasmid to prepare a plasmid so that two genes encoding CP protein were linked in series. A 278 bp DNA fragment and a T ₄ DNA ligase were used to transform the E. coli JM109 strain treated with CaCl ₂ and to analyze the DNA of the transformed strain having the desired plasmid DNA to prepare a pRHC-20 plasmid. . This plasmid was designed such that the gene encoding the structural protein consisting of two consecutive CP polypeptides, as shown in Figure 4, was well reflected by the tack promoter and the Rock I regulatory genes, such as plasmid pRHC-10.

Claims (10)

A polypeptide (CP polypeptide) is prepared by modifying the binding site of immunoglobulin G that binds to Staphylococcus protein A using protein engineering, and fusion of an external gene to the CP gene using DNA encoding the polypeptide. To stably express in a host cell as a fusion protein, and to produce an isolated expression protein. The method of claim 1, wherein the sequence of the synthetic CP DNA and the corresponding amino acid sequence, Method of producing an expression vector characterized in that (Fig. 1). The method of claim 1, wherein the recombinant protein is stabilized by introducing an amino acid combination that stabilizes the protein at the amino acid of the CP protein. The method of claim 1, wherein the CP DNA is a method for producing an expression vector, characterized in that the CP DNA from the code for transcription Asp-Pro. The method of claim 1, wherein the CP DNA is a CP DNA obtained by converting a cord for Asn-Gly transcription to a code for transcription of Asn-Ala. The method of claim 1, wherein at least two CP polypeptides are used as structural proteins. The method of claim 6, wherein the number of CP polypeptides produces recombinant DNA encoding the structural proteins of 2-10. The method of claim 1, wherein the recombinant DNA is a recombinant DNA comprising a portion of a nucleotide sequence encoding a CP polypeptide. The method of claim 1, wherein the foreign gene is expressed as a fusion protein by using a recombinant DNA including CP DNA or a portion thereof. 4. The method of claim 3, wherein the protein stabilizing amino acid is an amino terminal protein stabilization sequence using a combination of 1-20 Ser, Ter, Als or Asp.