Especially-like element hydrazide prepared in cell lysis buffer and synthetic method of specially-like element hydrazide

Abstract

The invention belongs to the field of protein chemical synthesis and modification, and particularly relates to a method for preparing EK hydrazide in a cell lysis buffer and a synthesis method thereof.The synthesis method comprises the following steps that the cell lysis buffer with EK activating enzyme UBA5 overexpression and the cell lysis buffer with EK UFM1 overexpression are obtained respectively, then the two lysis solutions are mixed according to the proportion, and the cell lysis buffer with EK activating enzyme UBA5 overexpression and the cell lysis buffer with EK UFM1 overexpression are obtained; after a hydrazinolysis reagent is added, the activation process of the EK is induced, an in-situ hydrazinolysis reaction is carried out, and finally acid precipitation and high performance liquid chromatography purification are carried out to obtain the EK hydrazide. The method does not need to purify the enzyme UBA5 and the protein substrate UFM1, directly realizes the synthesis of the EK hydrazide through the enzymatic hydrazinolysis reaction in the cell lysis buffer, and has the characteristics of simple operation, high preparation yield, large-scale preparation, low cost and the like.

Description

Technical Field

[0001] The present invention relates to the field of protein chemical synthesis and modification, and in particular to a method for preparing isocyanate hydrazide in cell lysate and a synthetic method thereof. Background Art

[0002] UFM1 is an important ubiquitin-like protein consisting of 83 amino acids. Under the joint action of ubiquitin activating enzyme E1 (UBA5), ubiquitin transferase E2 (UFC1) and ubiquitin ligase E3 (UBL1), ubiquitin undergoes an enzymatic cascade reaction, forming an isopeptide bond between its C-terminus and the lysine side chain of the protein substrate, thereby forming ubiquitin modification and changing the structure and function of the protein. Ubiquitin has been found to exist in many protein substrates, such as tumor suppressor p53, histone H4, autophagy protein p62, etc., and thus plays an important regulatory role in physiological processes such as DNA damage repair, endoplasmic reticulum homeostasis, and protein degradation. Abnormal ubiquitin has also been found to be closely related to human diseases such as cancer, diabetes, and ischemic heart disease. In order to understand the function and mechanism of protein ubiquitin regulation, ubiquitin-modified proteins and their derivative probes are indispensable protein samples or tools, but they are usually difficult to prepare through traditional biotechnology.

[0003] In recent years, a variety of chemical synthesis methods have been developed for the preparation of cytochrome modified proteins and their derivative probes, among which cytochrome hydrazide is a key intermediate in the related synthesis. For example, the prior art 202210101211.9 discloses a cytochrome probe UFM1-Lys-TAMRA and a synthesis method thereof, which uses cytochrome activating enzyme (UBA5) to activate the C-terminus of cytochrome and then perform in situ hydrazinolysis to prepare cytochrome hydrazide; however, in this method, the recombinantly expressed UBA5 and UFM1 need to be purified, such as size exclusion chromatography or affinity chromatography to purify UBA5, which not only increases time and equipment costs, but also causes the loss of protein raw materials. Therefore, it is necessary to develop a new technical solution for the preparation of cytochrome hydrazide. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and to provide a method for preparing isocyanate hydrazide in a cell lysate and a method for synthesizing it. The present invention does not require additional purification of the enzyme UBA5 and the protein substrate UFM1, and synthesizes isocyanate hydrazide (UFM1-NHNH2) by an enzymatic hydrazinolysis reaction in a cell lysate. The method has the characteristics of simple operation, high preparation yield, large-scale preparation, low cost, etc.

[0005] To achieve the above object, the technical solution adopted by the present invention is: a synthetic method for preparing isocyanate hydrazide in a cell lysate, comprising the following steps:

[0006] S1: preparing cell lysates of overexpressed UBA5 and UFM1 cells respectively;

[0007] S2: mixing the two cell lysates, adding a hydrazinolysis reagent to induce the activation process of hydroxyproline and perform an in situ hydrazinolysis reaction;

[0008] S3: Acid precipitation and high performance liquid chromatography purification to obtain the isocyanate hydrazide (UFM1-NHNH2);

[0009] The UBA5 activation and hydrazinolysis reactions are both carried out in cell lysate, and the UBA5 and UFM1 are unpurified proteins in the cell lysate.

[0010] Preferably, in S1, the cell lysate is obtained by lysing a recombinant bacterium; the recombinant bacterium is Escherichia coli BL21 (DE3) transfected with a target gene (UBA5 or UFM1).

[0011] Specifically, the Escherichia coli transfected with the target gene is cultured using a standard method to induce the expression of the target protein, and the cells are resuspended in a lysis buffer and subjected to standard ultrasonic disruption to collect the supernatant to obtain a cell lysate.

[0012] Preferably, the lysis buffer is 30 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, and its pH range is 7.0 to 8.0.

[0013] Preferably, in S2, the volume ratio of the UBA5 overexpressed lysate to the UFM1 overexpressed cell lysate is 2:1.

[0014] Preferably, in S2, the hydrazinolysis reagent is 10 mM MgCl2, 100 mM NH2NH2·HCl, and 2 mM ATP.

[0015] Furthermore, the hydrazine is selected from one or more of hydrazine hydrochloride, hydrazine hydrate and hydrazine acetate.

[0016] Preferably, in S2, the hydrazinolysis reaction is carried out at 37 degrees Celsius for 12 hours.

[0017] Preferably, in S3, trifluoroacetic acid is added to the reaction solution produced in step S2, and after stirring at room temperature for 20 minutes, the precipitate is removed by centrifugation, and the obtained supernatant is purified by high performance liquid chromatography to obtain the isocyanate hydrazide.

[0018] Preferably, the volume concentration of the trifluoroacetic acid is 1%.

[0019] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0020] The present invention simulates the enzymatic process of UBA5 activating UFM1 in cells, and utilizes overexpression to form a large number of thioester intermediates of UFM1-UBA5, thereby realizing the preparation of uvaline hydrazide (UFM1-NHNH2) by in situ hydrazinolysis in cell lysate. Compared with the prior art, the present invention has the following advantages: there is no need to purify the protein raw materials UBA5 and UFM1, avoiding multiple steps such as affinity purification or size exclusion purification, dialysis, etc., and has the characteristics of simple operation, high preparation yield, large-scale preparation, low cost, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The technical solution of the present invention is further described below in conjunction with the accompanying drawings:

[0022] Attached Figure 1 This is the SDS-PAGE analysis of the E. coli cell lysate that overexpresses UBA5 in the present invention;

[0023] Attached Figure 2 is a high performance liquid chromatogram of UFM1 after acid precipitation of cell lysate in the present invention;

[0024] Attached Figure 3 It is the mass spectrum of UFM1 after acid precipitation of cell lysate in the present invention;

[0025] Attached Figure 4 The HPLC chromatogram of the enzymatic reaction in the lysate of the present invention for 12 hours;

[0026] Attached Figure 5 The mass spectrum of the enzymatic reaction in the lysate of the present invention for 12 hours;

[0027] Attached Figure 6 Schematic diagram of the synthesis of isocyanate hydrazide in the present invention. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0029] Example 1

[0030] The gene sequence of UBA5 was cloned into the pET-22b vector by selecting NdeI and XhoI as restriction sites, and the obtained plasmid was transformed into Escherichia coli BL21 (DE3) cells. After cultivation, a single clone colony was picked and amplified in 10 mL of LB medium containing ampicillin resistance (100 μg / mL); after culturing overnight at 37°C and 200 rpm, the obtained bacterial solution was transferred to 1 L of LB medium and continued to be cultured until the absorbance value (OD600) of the bacterial solution reached 0.6-0.8;

[0031] Subsequently, IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.8 mM, and protein expression was induced at 16°C for 16 hours; the bacterial solution was collected by centrifugation at 4°C and 6500 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in 10 mL of lysis buffer (30 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, pH 7.5), and placed in an ice bath to lyse the bacteria with an ultrasonic disruptor; after the ultrasonic disruption was completed, the precipitate was removed by high-speed refrigerated centrifugation (12000 rpm, 4°C) for 30 minutes, and the resulting supernatant could be directly used in the hydrazinolysis reaction of step S2.

[0032] The obtained cell lysate was analyzed by SDS-PAGE. Figure 1 shown.

[0033] Example 2

[0034] The gene sequence of UFM1 was cloned into the pET-22b vector by selecting NdeI and BamHI as restriction sites. The obtained plasmid was transformed into Escherichia coli BL21 (DE3) cells. After cultivation, a single clone colony was picked and amplified in 10 mL of LB medium containing ampicillin resistance (100 μg / mL); after culturing overnight at 37°C and 200 rpm, the obtained bacterial solution was transferred to 1 L of LB medium and continued to be cultured until the absorbance value (OD600) of the bacterial solution reached 0.6-0.8;

[0035] Subsequently, IPTG was added at a final concentration of 0.8 mM to induce protein expression at 37°C for 16 hours; the bacterial solution was collected by centrifugation at 4°C and 6500 rpm for 10 minutes, the supernatant was discarded, the cells were resuspended in 10 mL of lysis buffer (30 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, pH 7.5), and placed in an ice bath to lyse the bacteria with an ultrasonic disruptor; after the ultrasonic disruption was completed, the precipitate was removed by high-speed refrigerated centrifugation (12000 rpm, 4°C) for 30 minutes, and the resulting supernatant was used for the hydrazinolysis reaction in step S2.

[0036] At the same time, a small amount of supernatant was taken for acid precipitation and then analyzed by HPLC and mass spectrometry, respectively. Figure 2 and Figure 3 shown.

[0037] Example 3

[0038] The UBA5 overexpression lysate from 2L cells and the UFM1 overexpression lysate from 1L cells were taken to a final volume of 20mL and 10mL, respectively; the two lysates were directly mixed, and MgCl2, NH2NH2·HCl and ATP were added in sequence to a final concentration of 10mM, 100mM and 2mM, respectively, and then incubated at 37°C;

[0039] After 12 hours, 1% trifluoroacetic acid was added to the mixture and stirred at room temperature for 20 minutes. The precipitate was removed by low-temperature high-speed centrifugation (12000 rpm, 4°C, 10 minutes). The obtained supernatant was verified by high performance liquid chromatography and mass spectrometry, respectively. Figure 4 and Figure 5 After verification, the product was purified by high performance liquid chromatography (C18 or C4 reverse phase chromatography column) (elution gradient: 30-60% acetonitrile, 30 minutes).

[0040] The above are only specific application examples of the present invention and do not constitute any limitation on the protection scope of the present invention. Any technical solution formed by equivalent transformation or equivalent replacement shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a synthetic method of isocyanate hydrazide in a cell lysate, characterized in that: The following steps are involved: S1: preparing cell lysates of overexpressed UBA5 and UFM1 cells respectively; S2: mixing the two cell lysates, adding a hydrazinolysis reagent to induce the activation process of hydroxyproline and perform an in situ hydrazinolysis reaction; S3: Acid precipitation and high performance liquid chromatography purification to obtain the isocyanate hydrazide; The UBA5 activation and hydrazinolysis reactions are both carried out in cell lysate, and the UBA5 and UFM1 are unpurified proteins in the cell lysate.

2. The method for preparing isocyanate hydrazide in cell lysate according to claim 1, characterized in that: In S1, the cell lysate is a lysate of Escherichia coli BL21 (DE3).

3. The method for preparing isocyanate hydrazide in cell lysate according to claim 2, characterized in that: The E. coli transfected with the target gene was cultured using a standard method to induce the expression of the target protein, and the cells were resuspended in a lysis buffer and subjected to standard ultrasonic disruption to collect the supernatant to obtain a cell lysate.

4. The method for preparing isocyanate hydrazide in cell lysate according to claim 3, characterized in that: The lysis buffer contained HEPES, NaCl, DTT and PMSF, and its pH range was 7.0 to 8.

0.

5. The method for preparing isocyanate hydrazide in cell lysate according to claim 1, characterized in that: In S2, the volume ratio of UBA5-overexpressed lysate and UFM1-overexpressed cell lysate was 2:

1.

6. The method for preparing isocyanate hydrazide in cell lysate according to claim 1, characterized in that: In S2, the hydrazinolysis reagent comprises MgCl2, NH2NH2·HCl and ATP.

7. The method for preparing isocyanate hydrazide in cell lysate according to claim 1, characterized in that: In S2, the hydrazinolysis reaction is carried out at 37 degrees Celsius for 12 hours.

8. The method for preparing isocyanate hydrazide in cell lysate according to claim 1, characterized in that: In S3, trifluoroacetic acid is added to the reaction solution produced in step S2, and after stirring at room temperature for 20 minutes, the precipitate is removed by centrifugation, and the obtained supernatant is purified by high performance liquid chromatography to obtain the isocyanate hydrazide.

9. The method for preparing isocyanate hydrazide in cell lysate according to claim 8, characterized in that: The volume concentration of the trifluoroacetic acid is 1%.

10. A hydrazide prepared by the synthesis method according to any one of claims 1 to 9.

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