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Production of a soluble native form of recombinant protein by the signal sequence and secretional enhancer

A signal sequence and heterologous protein technology, applied in the field of recombinant proteins, can solve the problems of not developing a direct analysis method of signal sequence, impossible and difficult to produce natural form recombinants, etc.

Inactive Publication Date: 2009-02-25
国立水产科学院 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, all signal sequences currently available for expression vectors have only a limited ability to direct the expression of soluble proteins, and use of these vectors resulted in the production of recombinant fusion proteins with signal peptidase cleavage regions, suggesting that it is difficult to produce native forms recombinant
[0006] The reasons why it is difficult to produce recombinant proteins using signal sequences are that 1) the prediction of the production of proteins in soluble form is impossible, so that many researchers have assumed that the expression of recombinant proteins in soluble forms is inherently dependent on the amino acid sequence. physical properties; and 2) there are too many sequences that function as signal sequences, but no direct analytical method has been developed for the function of such signal sequences (Triplett et al., J.Biol.Chem. (Biochemical Journal) 276 :19648-19655, 2001)

Method used

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  • Production of a soluble native form of recombinant protein by the signal sequence and secretional enhancer
  • Production of a soluble native form of recombinant protein by the signal sequence and secretional enhancer
  • Production of a soluble native form of recombinant protein by the signal sequence and secretional enhancer

Examples

Experimental program
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Effect test

Embodiment 1

[0228] Example 1: Cloning Adhesin Gene DNA Polymeric Cassette

[0229] Based on the basic unit of the Mefp1 amino acid sequence represented by SEQ.ID.NO:1 (Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys), by using the basic unit represented by SEQ.ID.NO:2 (5'-TACAAA GCT AAG CCG TCT TAT The forward primer shown in CCG CCA ACC-3') and the reverse primer shown in SEQ.ID.NO:3 (5'-TTT GTA GGT TGG CGG ATA AGA CGG CTTAGC-3') were prepared by the inventors synthesized mefp1 DNA. For the DNA (comprising BamHI / EcoRI / SmaI) synthesized by the left adapter (hereinafter referred to as "La"), the DNA shown by SEQ.ID.NO:4 (5'-GAT CCG AAT TCC CCGGG-3') was used Forward primer and reverse primer shown by SEQ.ID.NO:5 (5'-TTT GTA CCC GGG GAATTC G-3'). For the DNA (comprising Arg / HindIII / SalI / XhoI) synthesized by the right adapter (hereinafter referred to as "Ra"), the DNA prepared from SEQ.ID.NO:6 (5'-TAC AAA CGT AAG CTT GTC GAC C-3 ') and the reverse primer shown by SEQ.ID.NO:7 (5'-TCG AGG TCG ACA...

Embodiment 2

[0250] Example 2: Expression of the adhesion protein mefp1

[0251] In a previous study, Mefp1 was expressed as an insoluble inclusion body when Met-Mefp1 was used as a leader sequence (Kitamura et al., J Polym. Sci. Ser. A 37:729-736, 1999). The inventors introduced the signal sequence OmpASP (OmpA signal peptide) to induce the expression of the protein in soluble form, for its use figure 2 The mefp1 sequence was used as a template for PCR to construct clones carrying ompASP and mefp1 cassettes of different sizes (Table 1).

[0252] In the presence of 50 μg / ml of ampicillin at 30°C, the Escherichia coli BL21 (DE3) transformant produced by using the expression vector containing the signal sequence shown in Table 1 was cultured in LB medium (20 g of tryptone, yeast extract Cultivate in 5.0g, NaCl 0.5g, KCl 1.86mg / l) for 16 hours. The culture solution was diluted 200-fold with LB medium. Incubate the diluted culture solution to OD 600 0.3, and then add IPTG to a final con...

Embodiment 3

[0254] Example 3: Production of Adhesin Mefp1 in Native Form

[0255]To produce Mefp1 with its native N-terminus, based on the results of soluble expression by shortened OmpASP (Table 1), the inventors used pBluescriptIISK(+)-La-7×mefp1-Ra( figure 2 ) as a template and synthesized OmpASP encoding a Factor Xa cleavage site for cleaving the C-terminal end 1-8 -Xa-Mefp1 oligonucleotide was used as forward primer for PCR to construct pET-22b(+)(ompASP 1-8 -Xa-7×mefp1 * )( * : Ra-6×His, Ra derived from right adapter; 6×His derived from His tag) clone. Expression of the constructed vectors was checked by transformation and Western blotting as described in Example 2.

[0256] As a result, this clone produced the soluble protein OmpASP 1-8 -Xa-7×Mefp1 * . Furthermore, 7×Mefp1 with native amino acid termini * Protein removal by OmpASP with Factor Xa protease 1-8 -Xa sequence obtained ( Figure 4 ).

[0257] In order to modify the signal sequence region of the above-mention...

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Abstract

The present invention is drawn to a method for enhancing secretional efficiency of a heterologous protein using a secretional enhancer consisting of a modified signal sequence which comprises the N-region of a signal sequence and / or a hydrophobic fragment of the said signal sequence comprising the said N-region and / or the hydrophilic polypeptide. The method of the present invention can be used not only for production of recombinant heterologous proteins by inhibiting insoluble precipitation and enhancing secretional efficiency of the recombinant protein into the periplasm or the extracellular fluid and but also for transduction of therapeutic proteins by enhancing membrane-permeability of the recombinant protein using a strong secretional enhancer.

Description

technical field [0001] The present invention relates to methods for producing soluble native forms of recombinant proteins by targeting a signal (part of the signal sequence), a secretion enhancer and a protease recognition site. Background technique [0002] One of the most important applications of modern biotechnology is the production of recombinant proteins, especially in soluble native form. Soluble proteins play an important role in the production and recovery of active forms of proteins, their crystallization for functional studies and their industrialization. Recombinant proteins have been expressed in E. coli because E. coli can be easily manipulated, has a fast growth rate, ensures stable expression, is economical, and is easy to scale up. [0003] However, when E. coli is used to express heterologous recombinant proteins, lack of appropriate post-translational chaperones or post-translational processing can lead to misfolding of the expressed protein and aggrega...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63
Inventor 李相俊金英玉南宝惠
Owner 国立水产科学院
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