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Farnesyl transferase variant and preparation method thereof

A farnesyltransferase and variant technology, applied in the biological field, can solve problems such as inability to achieve specific position coupling, and achieve the effect of simple purification and guaranteed activity

Pending Publication Date: 2021-06-25
江苏万邦医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a tool enzyme for directional coupling, FTase solves the shortcomings of traditional coupling technology that the product obtained is a mixture of different numbers of drug molecules and cannot be coupled at a specific position

Method used

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  • Farnesyl transferase variant and preparation method thereof
  • Farnesyl transferase variant and preparation method thereof
  • Farnesyl transferase variant and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: Preparation of farnesyltransferase engineering bacteria

[0063] 1. Construction of farnesyltransferase expression vector and engineering bacteria

[0064] 1. Construction of the nucleotide sequence of farnesyltransferase: the amino acid sequences of EVDDDDK restriction site and α subunit of farnesyltransferase are as follows (Seq ID No.6 and Seq ID No.8, respectively). According to the codon preference of Escherichia coli, the original nucleotides of the amino acid sequence are optimized. This optimization method is a technique known to those skilled in the art. For example, see the Journal of Inner Mongolia University: Comparison of codon usage between Escherichia coli and yeast, 006, VOI.37, NO.1:34-39; get the final nucleotide sequence: Seq ID No.5 and Seq ID No.7. Add 6 His (gene sequence is Seq ID No.3) to the 5' end of the pre-modified farnesyltransferase α subunit to obtain a modified farnesyltransferase α subunit core containing 6 His tags Nucle...

Embodiment 2

[0067] Embodiment 2: High-density fermentation method 1 of engineering bacteria

[0068] 1. Preparation of fermentation medium

[0069] 1.1 Medium

[0070] Seed culture medium: Weigh 27 Typtone, 63g Yeast Extraction, measure 10ml of glycerin into a 5L beaker, stir until completely dissolved, and use a measuring cylinder to set the volume to 2000ml. Divide into 2 250ml shake flasks (50ml TB seed medium per bottle) and 3 2L shake flasks (500ml TB seed medium per bottle).

[0071] 80L basic tank base: Weigh 1080g Typtone, 2520g Yeast Extraction, measure 400ml glycerin into a 10L beaker, stir until completely dissolved, add water to 80L in the fermenter.

[0072] 2. Fermentation process

[0073] 2.1 Primary seed culture

[0074] Add ampicillin to the medium to make the final concentration 100 μg / ml, inoculate into TB seed medium according to 1% inoculum amount, cultivate at 37°C, rotate at 200±20rpm, and cultivate for 14-16 hours.

[0075] 2.2 Secondary seed culture

[0076]...

Embodiment 3

[0086] Embodiment 3: the high-density fermentation method 2 of engineering bacteria

[0087] 1. Preparation of fermentation medium

[0088] 1.1TB culture medium

[0089] Seed culture medium: Weigh 24 Typtone, 48g Yeast Extraction, measure 16g glucose and put it into a 5L beaker, stir until completely dissolved, and use a measuring cylinder to set the volume to 2000ml. Divide into 2 250ml shake flasks (50ml TB seed medium per bottle) and 3 2L shake flasks (500ml TB seed medium per bottle).

[0090] 80L basic tank base: Weigh 960g Typtone, 1920g Yeast Extraction, 184g potassium dihydrogen phosphate, 1312g dipotassium hydrogen phosphate, weigh 640g glucose and put it into a 10L beaker, stir until completely dissolved, add water to 80L in the fermenter.

[0091] 2. Fermentation process

[0092] 2.1 Primary seed culture

[0093] Add ampicillin to the medium to make the final concentration 100 μg / ml, inoculate into TB seed medium according to 1% inoculum amount, cultivate at 37°...

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Abstract

The invention relates to a farnesyl transferase variant and a preparation method thereof, a farnesyl transferase protein sequence is screened out, genes of the farnesyl transferase protein sequence are subjected to high-density fermentation induced expression through an escherichia coli expression system after codon optimization, and the expression quantity is high; The invention further provides a soluble expression method without deformability and renaturation, and the purification step is simple and convenient. The specific enzyme activity detection shows that the prepared farnesyl transferase has very strong enzyme activity, and a tool enzyme capable of being industrially produced is provided for protein coupling.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a farnesyltransferase variant and a preparation method thereof. Background technique [0002] Farnesyl transferase (Farnesyl Transferase, FTase) is composed of two subunits: a 48kDa α subunit and a 46kDa β subunit, and the two subunits are mainly composed of α helices. The α subunit consists of a double layer of parallel stacked pairs of α helices that partially wrap around the β subunit like a blanket. The alpha helix of the beta subunit forms a barrel. The active site is formed by the center of the β subunit flanked by a portion of the α subunit (eg Figure 5 ). [0003] FTase can catalyze the following chemical reactions: [0004] farnesyl diphosphate + protein-cysteine S-farnesyl protein + diphosphate [0005] The enzyme has a hydrophobic binding pocket for farnesyl diphosphate (lipid donor molecule), and mainly adds farnesyl (pentadecarbon isoprenoid) to the carboxyl termi...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70
CPCC12N9/1085C12N15/70C12Y205/01021C12N2800/22
Inventor 文良柱赵珊珊王雪松苗森姜皓容平芳徐晓峰支艳艳李晓稳
Owner 江苏万邦医药科技有限公司
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