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Method for improving protein expression efficiency by employing model fitting and gene modification and application thereof

A technology of genetic modification and protein expression, applied in the fields of plant gene improvement, chemical instruments and methods, and botanical equipment and methods, etc., can solve the problems of reducing the persistence and efficiency of expression, and achieve the improvement of expression efficiency, increase expression efficiency, and high efficiency. The effect of versatility and feasibility

Active Publication Date: 2015-09-02
江苏莱森生物科技研究院有限公司
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Problems solved by technology

It can be seen that people can change the position of the "hairpin" structure in some DNA or increase or decrease the corresponding "hairpin" structure according to the situation, so as to improve the transcription efficiency of the target gene and achieve the purpose of improving and increasing the expression efficiency of the recombinant protein ; Finally, at the level of translation, studies have shown that translation is also affected by multiple factors: on the one hand, the translation conditions in the host affect the translation of the target gene to a large extent, such as the abundance of free amino acids, enzyme activity etc., so people can provide a better translation condition by purposefully modifying the host or choosing a better host, thereby improving and increasing the expression efficiency of the recombinant protein; on the other hand, the final product of the translation of the target gene, that is, the target protein (polypeptide chains), their water solubility or whether they can be excreted from the host body in time also affects the translation efficiency to a certain extent, because the two factors of poor water solubility and not being excreted from the host body in time will make the expression of the target gene It quickly reaches a saturated state, thereby inhibiting expression and greatly reducing the persistence and efficiency of expression

Method used

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  • Method for improving protein expression efficiency by employing model fitting and gene modification and application thereof
  • Method for improving protein expression efficiency by employing model fitting and gene modification and application thereof
  • Method for improving protein expression efficiency by employing model fitting and gene modification and application thereof

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Effect test

Embodiment 1

[0030] mRNA secondary structure fitting

[0031] In the M-fold program, input the mRNA sequence of L11. Since the sequence before the start codon, including the SD sequence, etc., can form hydrogen bonds and secondary structures with the sequence after the start codon, and can affect the translation speed of the protein, therefore, enter about 20 nucleotide sequences Perform secondary structure fitting; at the same time, input about 50 nucleotide sequences after the start codon to ensure that the mRNA has enough length to fold after the start codon.

[0032] After fitting with M-fold, it was found that the mRNA of wild-type L11 had a hairpin structure immediately after the start codon ( figure 1 ). Therefore, when the ribosome is translating a protein, it is very likely that reading the mRNA sequence at this position will be blocked, reducing the speed of translating the protein, and ultimately leading to a decrease in the yield of the recombinant protein. At the same time,...

Embodiment 2

[0034] L11 plasmid purification

[0035] A single colony of BL21(DE3)pLysS L11 was inoculated into 20 ml of LB medium containing 10 μg / ml ampicillin (Sigma-Aldrich), and the flask was incubated overnight at 37°C with shaking. The next day, 5ml of saturated BL21(DE3)pLysS L11 cells were transferred to 500ml of terrific broth (Invitrogen) with 10 μg / ml ampicillin, and the flask was incubated overnight at 37°C with shaking. Cells were harvested by centrifugation at 5,500 rpm for 10 minutes with a GS-3 rotor of a Beckman centrifuge. Cells were snap frozen in liquid nitrogen and stored at -80°C.

[0036] Cells were lysed in 20 ml of a solution containing 1 mg / ml lysozyme (Sigma-Aldrich). After the solution was homogeneous, 40 ml of 1% SDS in 0.2N NaOH solution was added to precipitate DNA of chromosome and plasmid at the same time. The solution was kept on ice for 10 minutes and 20 ml of 5M potassium acetate solution (pH 4.8) was added to precipitate the chromosomal DNA while th...

Embodiment 3

[0039] Design of primers for site-directed mutagenesis of L11 plasmid

[0040] The Stratagene QuikChange site-directed mutagenesis kit for vectors was used to construct multiple mutants. Primer design is based on the following principles: 1. The two primers must contain the same mutated sequence on the opposite plasmid strand; 2. The length of the primer should be between 25-45 bases, and the melting temperature (Tm) ≥ 78 °C ; 3. The position of the desired mutation (deletion or insertion) should be in the middle of the primer, and there are 10 to 15 bases with correct sequence on both sides; 4. The minimum cytosine and guanine (GC) content of the primer is 40%, And terminate at one or more C or G bases.

[0041] T m =81.5+0.41(%GC)-675 / N-mismatch % (reaction formula 1)

[0042] T m =81.5+0.41(%GC)-675 / N (for insertion or deletion) (reaction formula 2)

[0043] N is the length of bases in the primer, GC percentage and mismatch percentage are integers.

[0044] The design...

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Abstract

The invention relates to a method for improving protein expression efficiency by employing model fitting and gene modification and an application thereof. The method comprises the following steps: fitting a secondary structure of an mRNA sequence of a target protein ribosomal protein L11 by employing M-fold software, inputting an about early 20-locus nucleotide sequence of an initiation codon and an about back 50-locous nucleotide sequence of the initiation codon and fitting. According to the fitted secondary structure, a hairpin structure behind the initiation codon is removed; targeted mutation is carried out on guanine, cytosine and the like in the hairpin structure; the content of the guanine and the cytosine is lowered; and the guanine and the cytosine are replaced with adenine and thymine; after site-directed mutation is carried out, mutants of the target protein with improved yield are screened by virtue of experiments; the biological functions of the mutants are further verified by virtue of biological function experiments; the mutants mutated by the method do not have a significant effect on the biological function when increasing expression of the ribosomal protein L11; and the method for improving recombinant ribosomal protein expression efficiency based on model fitting, site-directed mutation and experiment screening has relatively high universality and feasibility, and is also applicable to large-scale expression, industrial production and the like of other recombinant proteins.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for improving protein expression efficiency by model fitting and gene modification and an application thereof. Background technique [0002] With the rapid development of modern genetic engineering and bioengineering theory and technology, whether it is in the field of basic research or application fields such as biomedicine, agriculture, environmental protection and food, the use of heterologous expression systems to produce higher levels of target recombinant proteins has become Research hotspots and priorities. Compared with the expression of the original gene in its own host, the use of heterologous hosts to express recombinant proteins has many advantages: first, heterologous expression may significantly increase the expression of the target protein; second, the host usually selected for heterologous expression has relatively thorough The research background, as well as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70G06F19/16C07K14/245C12N15/31
Inventor 屠志刚刘晗青
Owner 江苏莱森生物科技研究院有限公司
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