WO2024055291A1 - Procédé de criblage à haut débit de mutant de sirtuine, mutant de sirtuine et son utilisation - Google Patents

Procédé de criblage à haut débit de mutant de sirtuine, mutant de sirtuine et son utilisation Download PDF

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WO2024055291A1
WO2024055291A1 PCT/CN2022/119322 CN2022119322W WO2024055291A1 WO 2024055291 A1 WO2024055291 A1 WO 2024055291A1 CN 2022119322 W CN2022119322 W CN 2022119322W WO 2024055291 A1 WO2024055291 A1 WO 2024055291A1
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sirtuin
mutant
amc
lysis
activity
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赵维
邹思维
蔡可祝
杨玉蛟
赵国屏
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中国科学院深圳先进技术研究院
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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Definitions

  • the invention relates to the field of biotechnology, and in particular to a method for high-throughput screening of sirtuin mutants, sirtuin mutants and their applications.
  • Sirtuin is a type of protein deacylase that uses NAD+ as a substrate. It is widely present in three domains of life systems: bacteria, archaea, and eukaryotes. It participates in the regulation of gene transcription, chromosome segregation, RNA shearing, cell metabolism, and apoptosis. A series of important biological processes such as death and energy metabolism. In addition to deacetylase activity, sirtuin has also been reported to have other enzymatic activities, such as desuccinylation, demyristoylation, and denonanoylase activities.
  • sirtuin deacylation is closely related to obesity, aging, cancer, neurodegeneration and cardiovascular diseases.
  • crystal structures of sirtuin in different species such as humans, yeast and E. coli, have been solved.
  • the key regulatory sites of sirtuin deacylase activity are currently not fully understood.
  • no drugs targeting sirtuin have been approved for marketing so far.
  • the reason is largely due to the lack of an efficient and accurate detection method for sirtuin enzyme activity.
  • the main methods for detecting sirtuin enzyme activity include isotope labeling, immunoblotting, high-performance liquid chromatography and fluorescence.
  • isotope labeling methods are no longer commonly used due to cumbersome operations and potential safety.
  • Western blotting and high-performance liquid chromatography are only suitable for routine in vitro enzyme activity characterization.
  • coumarin-based fluorescence methods and FRET-based fluorescence methods are It can be used to measure sirtuin enzyme activity on a large scale, but current research still requires in vitro expression and purification to obtain sirtuin protein, which requires a large workload.
  • Direct cleavage of sirtuin after synthesis in vivo often results in inactivation of the enzyme, which limits the use of these methods at high temperatures.
  • sirtuin mutant and its application are also provided.
  • a method for screening sirtuin mutants including the following steps:
  • Each mutant in the sirtuin mutant library is lysed, solid-liquid separation is performed, and the lysis supernatant is collected;
  • the AMC fluorescence method is used to measure the sirtuin deacylation activity of the lysis supernatant in a high-throughput manner, and a sirtuin mutant strain with the required deacylation activity is obtained.
  • sirtuin mutants In the above screening method for sirtuin mutants, error-prone PCR technology is used to obtain a library of sirtuin protein mutants, and then the heterologously expressed sirtuin protein mutants are lysed to obtain a crude enzyme solution, and then a method based on the release of AMC fluorescent groups is used.
  • the in-site detection of deacylation activity of sirtuin mutants mainly includes three steps of library construction, lysis and detection. High-throughput screening of sirtuin protein mutants can be carried out. The steps are simple. Sirtuin synthesized in vivo is directly cleaved through lysis to release the fluorescence of AMC.
  • this method is universal and can screen different sirtuins for enzyme activity, and is not limited to the specific functions of sirtuins in specific environments; more importantly, this method combines error-prone PCR and AMC fluorescence methods.
  • This method combines error-prone PCR and AMC fluorescence methods.
  • high-throughput screening of artificial sirtuin mutants is possible.
  • deacylase sirtuin mutants can be screened in a simple, universal, sensitive and high-throughput manner.
  • an initial sirtuin gene is used to construct the sirtuin mutant library.
  • the initial sirtuin gene includes connected critical region fragments and non-critical region fragments.
  • the steps of constructing the sirtuin mutant library include:
  • an expression vector fragment is obtained.
  • the expression vector is used to express the initial sirtuin.
  • the expression vector fragment is used to express the sirtuin protein.
  • the expression and replication originals are composed of the plasmid carrying the expression vector and the resistance gene of the plasmid carrying the initial sirtuin gene is different;
  • mutant fragment and the expression vector fragment are recombinantly ligated and then transferred into host cells to obtain the sirtuin mutant library.
  • a first primer pair is used to perform error-prone PCR, and the sequences of the first primer pair are as shown in SEQ ID No. 1 to SEQ ID No. 2.
  • a second primer pair is used to perform PCR amplification
  • the third primer pair is used for PCR amplification.
  • the sequences of the two primer pairs are shown in SEQ ID No. 3 ⁇ SEQ ID No. 4.
  • a mutant library with 10-fold coverage can be constructed to screen out the amino acid sites in the critical region of sirtuin, where the mutation The body bank has 2,000-6,000 clones.
  • a cell lysis solution is used for lysis, and the cell lysis solution includes: a non-denaturing detergent and a buffer with a pH of 7 to 8, so The volume ratio of the cell lysis solution to the bacterial solution containing the cells to be tested is 1:20-1:40, the lysis temperature is 20°C-30°C, and the lysis time is 10min-20min.
  • the step of using the AMC fluorescence method to determine the sirtuin deacylation activity of the lysis supernatant includes:
  • the AMC peptide reaction solution contains the AMC peptide with acyl modification, so The final concentration of the AMC peptide is 100 ⁇ M ⁇ 500 ⁇ M;
  • the stop solution includes 2.0mg/mL to 5.0mg/mL trypsin, 2 ⁇ Tris buffer containing 2mM ⁇ 6mM NAM;
  • the fluorescence intensity of the second treatment solution is detected at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and the deacylase activity of sirtuin in the cells to be tested is determined based on the fluorescence intensity of the second treatment solution.
  • the acyl modification in the AMC peptide with acyl modification is acetyl modification, succinyl modification, myristoyl modification, nonanoyl modification or long-chain fatty acyl modification.
  • the AMC peptide is a lysine acylation-modified peptide with a BOC protection group and AMC label.
  • a sirtuin mutant is prepared by the above-mentioned screening method for sirtuin mutants.
  • it is obtained by mutating at least one of the following sites of wild-type sirtuin: alanine at position 76, phenylalanine at position 91, histidine at position 147, histidine at position 155 cysteine at position 185 and proline at position 185.
  • alanine at position 76 undergoes one of the following mutations: A76P, A76V;
  • the phenylalanine at position 91 has one of the following mutations: F91L, F91S;
  • Histidine at position 147 has one of the following mutations: H147L, H147Y;
  • the cysteine at position 155 has one of the following mutations: C155R, C155S, C155Y;
  • the proline at position 185 has one of the following mutations: P185L, P185T.
  • Figure 1 is the schematic diagram of AMC fluorescence detection
  • Figure 2 is a flow chart of mutant library construction
  • Figure 3 shows the gel electrophoresis pattern of the mutant fragments and backbone fragments
  • Figure 4 is a diagram of the sequencing results in Example 1.
  • Figure 5 is a gel electrophoresis diagram of the lysis supernatant
  • Figure 6 shows the fluorescence detection results of the reaction between BL21 cleavage products and peptide fragments
  • Figure 7 shows the fluorescence detection results of the reaction between purified sirtuin protein or sirtuin-containing cell lysate supernatant and substrate;
  • Figure 8 is an example of the fluorescence detection results of the mutant library
  • Figure 9 is a fluorescence detection mutant library enzyme activity and site information diagram
  • Figure 10 is an analysis diagram of the regulatory sites of sirtuin enzyme activity
  • Figure 11 shows the fluorescence detection results of the reaction between cell lysate and AMC-peptide Ac or AMC-peptide Su peptide segments
  • Figure 12 shows the effect of lysate on the enzyme activities of STM1221 and SIRT2.
  • One embodiment of the present application provides a method for screening sirtuin mutants, including the following steps S210 to S230:
  • Error-prone PCR is a method used to construct a library of random point mutants of proteins. It uses low-fidelity DNA polymerase in PCR and adjusts PCR reaction conditions to reduce the accuracy of DNA replication. This enables point mutations in genes. After a mutation is introduced into a gene, the corresponding amino acid changes, thereby affecting the structure and function of the target protein. Error-prone PCR produces a large number of mutation products that are cloned and ligated into expression vectors to generate a library of random point mutants of the target protein. The use of error-prone PCR can expand the size of the mutant library, increase its coverage, and improve screening accuracy.
  • an initial sirtuin gene is used to construct a sirtuin mutant library, and the initial sirtuin gene includes connected critical region fragments and non-critical region fragments.
  • the initial sirtuin is the sirtuin that needs to be mutated.
  • the initial sirtuin is not limited and can be a wild-type sirtuin or other sirtuin that has been mutated and needs to be mutated again.
  • the key region is the region where active sites (i.e., sites that affect enzyme activity) may exist.
  • Active sites i.e., sites that affect enzyme activity
  • Non-critical areas are areas other than critical areas.
  • Figure 2 is a flow chart of mutant library construction
  • the steps of constructing a sirtuin mutant library using error-prone PCR technology include S211 to S213:
  • the mutated fragments are key region fragments with 1 to 4 point mutations. It should be noted that by controlling the template amount of error-prone PCR, DNA fragments in the key region of sirtuin with 1-4 point mutations in each product can be amplified.
  • the plasmid carrying the initial sirtuin gene is the pET28a-sirtuin plasmid with kanamycin (Kanamycin, Kan) resistance.
  • This plasmid is capable of expressing the original sirtuin.
  • the first primer pair is used to perform error-prone PCR.
  • the sequence of the first primer pair is shown as SEQ ID No.1 ⁇ SEQ ID No.2.
  • the sequence shown in SEQ ID No. 1 is ggaaatgatggaaaacccaaga.
  • the sequence shown in SEQ ID No. 2 is ccgacttggcttggctcaag.
  • EP-PCR system formula is shown in Table 2.
  • pET28a-sirtuin plasmid was used as a template
  • random mutation PCR enzyme GeneMorph II Random Mutagenesis Kit, Agilent
  • sirtuin with 1-4 point mutations in each product was amplified by controlling the template amount.
  • DNA fragments in critical regions i.e., mutated fragments).
  • an expression vector fragment is obtained.
  • the expression vector is used to express the initial sirtuin.
  • the expression vector fragment is composed of expression and replication elements for expressing the sirtuin protein.
  • the resistance gene of the plasmid carrying the expression vector is different from that of the plasmid carrying the initial sirtuin gene.
  • the plasmid carrying the expression vector is pTEV5-sirtuin plasmid with ampicillin (Ampiciline, Amp) resistance.
  • This plasmid is capable of expressing the original sirtuin.
  • a second primer pair is used to perform PCR amplification.
  • the sequence of the second primer pair is shown in SEQ ID No. 3 ⁇ SEQ ID No. 4.
  • the sequence shown in SEQ ID No. 3 is cttgagccaagccaagtcgg.
  • the sequence shown in SEQ ID No. 4 is tcttgggttttccatcatttcc.
  • pTEV5-sirtuin was used as a template
  • a high-fidelity enzyme was used to amplify the fragments of sirtuin except the critical region together with the expression vector, and the template was eliminated with DpnI to obtain the vector fragment.
  • S211 and S212 is not limited. S211 can be performed first and then S212, S212 can be performed first and then S211, or S211 and S212 can be performed at the same time.
  • recombinase is used to connect the mutant fragment and the expression vector fragment.
  • the host cell is a competent host cell. Specifically, it can be competent DH5 ⁇ or competent BL21, or other competent host cells.
  • the PCR product fragment is purified, ligated and transformed into a DH5 ⁇ competent state by recombinase, and ampicillin resistance is used to screen out positive clones, that is, target clones. Pick about 10 clones, sequence them, check whether random point mutations occur in key regions and whether the number of mutations meets the requirements, and adjust the initial template concentration based on the results to make the obtained clones reach the desired mutation frequency.
  • this protocol first transforms the recombinant products into DH5 ⁇ , then extracts plasmids from DH5 ⁇ mixed plaques and transforms them into BL21 middle.
  • the BL21 clones that successfully grew on the ampicillin plate were randomly selected and sequenced to check whether the mutation results were as expected. It should be noted that if the fragment ligation product can be directly transferred into BL21, the step of transferring into DH5 ⁇ can be omitted.
  • the enzyme used to perform error-prone PCR mutagenesis of the selected sirtuin gene to establish a sirtuin mutant library is the enzyme in Agilent's GeneMorph II Random Mutagenesis Kit.
  • Each mutant in the sirtuin mutant library is lysed, solid-liquid separation is performed, and the lysis supernatant is collected.
  • cell lysis solution is used for lysis, and the cell lysis solution includes: non-denaturing detergent and buffer solution with pH 7-8.
  • the cell lysate can lyse cells containing sirtuin, and the obtained lysate can be used for AMC fluorescence detection of sirtuin deacylase activity without purification.
  • the cell lysis solution includes a non-denaturing detergent with a mass percentage of 0.5% to 2%, a salt substance of 100mM to 200mM, and a buffer with a pH of 7 to 8.
  • non-denaturing detergents with a mass percentage of 0.5% to 2%, salts from 100mM to 200mM, and buffers with a pH of 7 to 8 can lyse cells containing sirtuin, yielding
  • the cleavage product can be used for AMC fluorescence detection of sirtuin deacylase activity without purification. It is simple to operate and can screen different sirtuins for enzyme activity. It is not limited to the specific functions of sirtuins in specific environments. It has been verified through experiments that the cells were lysed using the cell lysate in this study.
  • the BL21 cell lysates transformed with STM1221 reacted with AMC peptides to produce fluorescence, while the BL21 cell lysates not transformed with STM1221 showed no fluorescence when mixed with AMC peptides.
  • enzyme activity detection has strong specificity.
  • the non-denaturing detergent is at least one of NP-40 (ie, ethylphenyl polyethylene glycol), sodium oxycholate and Triton X-100 (polyethylene glycol octyl phenyl ether).
  • NP-40 ie, ethylphenyl polyethylene glycol
  • Triton X-100 polyethylene glycol octyl phenyl ether
  • the addition of non-denaturing detergent can gently lyse cells and reduce the impact on the activity of released intracellular enzymes.
  • the mass percentage of the non-denaturing detergent is 0.5% to 2%.
  • the mass percentage of the non-denaturing detergent is 0.5% to 1.5%.
  • the buffer is 25mM ⁇ 50mM Tris-HCl buffer. This buffer can both assist in cell lysis and protect the activity of enzymes released by cell lysis.
  • the salt substance is NaCl or KCl.
  • Sodium chloride and potassium chloride can make the solution reach a certain concentration, maintain osmotic pressure, and ensure a certain ionic strength to stabilize proteins.
  • NaCl the same ingredient as physiological saline, is used.
  • the cell lysis solution includes: NP-40 with a mass percentage of 1%, 150mM NaCl, pH7.6, and 25mM Tris-HCl buffer. This cell lysis solution can gently lyse cells and reduce the impact on the activity of released intracellular enzymes.
  • the steps of using cell lysis solution to lyse the sirtuin mutant library include: mixing the cell lysate and the bacterial solution containing the sirtuin mutant library cells in a ratio of 1:20 to 1:40, and lysing at a temperature of 20°C to 30°C. Shake at 300rpm for 10min ⁇ 20min.
  • the solid-liquid separation method is centrifugation.
  • the centrifugation conditions are 4000 rpm and 4°C for 10 minutes. It should be noted that the solid-liquid separation method is not limited to centrifugation, and can also be other solid-liquid separation methods, such as filtration.
  • step S220 includes: picking the BL21 clone into a 96-well plate for culture, and shaking the culture at 37°C and 800 rpm overnight. After that, transfer the saturated bacterial solution 1:20 to a new 96-well plate and continue shaking the bacteria. After about 1.5-2 hours, the OD reaches 0.4. At this time, IPTG with a final concentration of 0.3mM was added to induce sirtuin protein expression at 25°C. After 16 hours, centrifuge the well plate at 4000 rpm and 4°C for 10 minutes to collect bacteria. Then, add 10 ⁇ L of mild cell lysis solution to each well of the 96-well plate.
  • the active ingredient of the lysis solution is 25mM Tris-HCl buffer (pH 7.6) containing non-denaturing detergent. Shake at 300 rpm for 10 min to obtain cell lysate. Finally, centrifuge at 4000 rpm and 4°C for 10 min to obtain the lysis supernatant containing the target sirtuin protein.
  • AMC 7-amino-4-methylcoumarin
  • AMC fluorescence method to measure sirtuin enzyme activity
  • the coumarin amine of AMC is condensed with the carboxyl group of the C-terminal lysine residue of the polypeptide molecule to form an amide bond
  • the AMC-modified polypeptide molecule is synthesized (the detection principle of the AMC fluorescence method is shown in the figure shown in 1).
  • steps of S230 include S231-S233:
  • the AMC peptide reaction solution contains acyl-modified AMC peptides.
  • the final concentration of AMC peptide is 100 ⁇ M ⁇ 500 ⁇ M. Specifically, the final concentration of AMC peptide was 200 ⁇ M.
  • the volume ratio of AMC peptide reaction solution and lysis supernatant is 1:1. This volume ratio facilitates sampling and loading.
  • the AMC peptide is a synthetic lysine acylation-modified peptide with a BOC protection group and AMC label.
  • the acyl modification in the AMC peptide with acyl modification is acetyl modification, succinyl modification, myristoyl modification, nonanoyl modification or long-chain fatty acyl modification.
  • the different deacylase activities of sirtuin can be measured using AMC peptides with different acyl modifications.
  • the acyl modification is not limited to the above-mentioned acyl modifications, and can also be other acyl modifications.
  • AMC-peptide peptides with different acyl modifications can be used according to the deacylase activity measured as needed.
  • the AMC peptide with acyl modification is AMC-peptide Ac (i.e., acetyl-modified AMC-peptide peptide).
  • the specific steps of S231 are: add 10 ⁇ L of reaction solution containing AMC-peptide Ac to each well of the 384-well plate, including 2 ⁇ L of 10 ⁇ Tris buffer (0.5M Tris-HCl, pH8.0, 1.37M NaCl, 27mM KCl, 10mM MgCl 2 ), 1 ⁇ L 20mM NAD + , 0.8 ⁇ L 5mM AMC-peptide Ac , 6.2 ⁇ L ddH 2 O.
  • the volume ratio of the first treatment solution to the stop solution is 1:1. This volume ratio facilitates sampling and loading.
  • the stop solution includes 2.0mg/mL ⁇ 5.0mg/mL trypsin and 2 ⁇ Tris buffer containing 2mM ⁇ 6mM NAM.
  • the tool for detecting fluorescence intensity is a microplate reader. It should be noted that the tool for detecting fluorescence intensity is not limited to a microplate reader, and may also be other tools capable of detecting fluorescence intensity, such as a fluorescence photometer.
  • the BL21 lysate transferred into the blank plasmid is used as a negative control
  • the BL21 lysate transferred into the wild-type pTEV5-sirtuin plasmid is used as the positive control.
  • the fluorescence intensity is directly proportional to the deacetylase activity of sirtuin protein. Therefore, the deacetylation activity of sirtuin can be quantitatively detected based on this proportional relationship.
  • the standard curve method is used for quantitative detection.
  • sirtuin mutant strain with the required deacylation activity
  • a mutant library with 10-fold coverage (screening 2,000-6,000 clones) can be constructed to screen for sirtuin amino acid sites in all key regions, where , the mutant library has 2,000-6,000 clones.
  • the above-mentioned screening method for sirtuin mutants can easily, high-throughput, and reproducibly screen artificial mutants of deacylase sirtuin.
  • Error-prone PCR was used to obtain random point mutation fragments of sirtuin protein coding DNA, and the recombinant plasmid was constructed and transformed into E. coli to obtain a sirtuin protein mutant library.
  • the optimized mild cell lysis solution was used to lyse the heterologously expressed sirtuin protein mutant in a 96-well plate and obtain crude enzyme solution.
  • the deacylation activity of sirtuin mutants was detected in situ using a method based on the release of AMC fluorophores.
  • this method mainly includes three steps: library construction, lysis and detection.
  • High-throughput screening of sirtuin protein mutants can be carried out through 96-well plate culture to induce sirtuin protein and microplate reader fluorescence detection.
  • the main feature of this method is that the steps are simple, and the optimized cell lysis solution allows the sirtuin synthesized in the body to be directly cleaved and released for detection by the AMC fluorescence method; moreover, the method is universal and can screen different sirtuins for enzyme activity without limitation.
  • sirtuin deacylation activity Due to the specific functions of sirtuin in specific environments; more importantly, this method combines error-prone PCR and AMC fluorescence methods to enable high-throughput screening of sirtuin artificial mutations through the seamless connection of bacterial protein expression-lysis-detection. body becomes possible. Subsequent sequencing of sirtuin mutants will provide information on mutation sites, which can analyze the relationship between site mutations and changes in enzyme activity, and predict and verify key sites for sirtuin deacylation activity.
  • AMC fluorescence detection is mostly used to detect the enzyme activity of a single sirtuin, or to screen the substrates and inhibitors corresponding to sirtuin through high-throughput in vitro enzyme activity detection.
  • sirtuin proteins cannot be expressed and purified in vitro, the direct cleavage and release of sirtuin after synthesis in vivo often results in the inactivation of subsequent reaction enzymes, and protein purification and other processes are required before enzyme activity detection can be carried out.
  • This application uses cell lysis solution to gently lyse sirtuin mutants.
  • the obtained cleavage product can be used for AMC fluorescence detection of sirtuin deacylase activity without purification.
  • the operation is simple and can screen different sirtuins for enzyme activity. It is not limited to It is based on the specific functions that sirtuin performs in a specific environment.
  • the present invention organically combines error-prone PCR for library construction and AMC fluorescence method for enzyme activity detection, obtains a random mutant library with controllable frequency through molecular cloning, expresses the protein and obtains enzyme activity information through in-situ detection, thereby constructing a complete set of
  • the sirtuin mutant screening system is simple, reliable and has high throughput. It can screen and discover the deacylation active regulatory site of sirtuin protein in a simple and high-throughput method, providing a basis for subsequent research on the structure and function of sirtuin.
  • This method screens sirtuin enzymatic activity mutants, performs in situ expression and activity measurement in E. coli, minus the tedious protein purification and transfer process, and is very simple; moreover, the use of different acylation modified peptides can be used to test the activity of sirtuin
  • This method can be used to screen different sirtuins for enzyme activity based on different deacylase activities, and is universal.
  • this method combines error-prone PCR and AMC fluorescence methods, and can simultaneously measure multiple proteins in large batches. Test results can be quantified.
  • the method in this study has the advantages of simplicity, universality, and high throughput.
  • One embodiment of the present application also provides a sirtuin mutant, which is prepared by the above-mentioned screening method for sirtuin mutants.
  • This sirtuin mutant can be used to alter deacylase activity.
  • alanine at position 76 undergoes one of the following mutations: A76P, A76V.
  • the phenylalanine at position 91 has one of the following mutations: F91L, F91S.
  • the histidine at position 147 has one of the following mutations: H147L, H147Y.
  • the cysteine at position 155 has one of the following mutations: C155R, C155S, or C155Y.
  • the proline at position 185 has one of the following mutations: P185L, P185T.
  • the reagents and instruments used in the examples are conventional choices in the art. Experimental methods that do not indicate specific conditions in the examples are usually implemented according to conventional conditions, such as conditions described in literature and books or methods recommended by kit manufacturers. The reagents used in the examples are all commercially available.
  • sirtuin protein STM1221 derived from Salmonella was selected as an example, and random point mutations were carried out in its specific site region (V43-N240) to obtain a random point mutant library with 1-3 mutated amino acids.
  • the specific operations are as follows:
  • the sirtuin protein STM1221 has high deacetylase activity.
  • the V43-N240 segment was selected as a possible active site gene fragment. This gene was constructed into the Kan-resistant pET28a-STM1221 plasmid, and amplified by EP-PCR (error-prone PCR) using the first primer pair (sequences shown in SEQ ID No. 1 ⁇ SEQ ID No. 2) The DNA fragment of the key region of STM1221 with 2-4 point mutations in each product (referred to as the mutation fragment) was obtained.
  • pTEV5-STM1221 was used as a template, and the second primer pair (sequences are shown in SEQ ID No.
  • FIG. 4 is a gel electrophoresis diagram of fragment and vector PCR products, in which band 1 represents the mutant fragment, band 2 represents the vector fragment, and band 3 is the 1.1 kb standard.
  • the primers used in PCR are detailed in Table 1.
  • the EP-PCR system formula is detailed in Table 2.
  • step (1) of this example to lyse the induced bacterial liquid of E. coli BL21 transformed with pTEV5-SIRT2 plasmid according to the same method to obtain a lysed supernatant containing SIRT2 protein, and conduct lysis on the lysed supernatant.
  • Gel electrophoresis imaging The gel electrophoresis pattern of the lysis supernatant is shown in Figure 5 for details.
  • Figure 5 takes SIRT2 protein as an example.
  • band 1 is the marker
  • bands 2 and 3 are the protein bands of the supernatant of the lysate induced by 0mM IPTG
  • bands 4 and 5 are the protein bands of the supernatant of the lysate induced by 0.3mM IPTG for 16 hours.
  • the supernatant of the BL21 cell lysate after IPTG induction can clearly show the target band of SIRT2, proving that this method can release the intracellular protein, namely sirtuin, into the supernatant.
  • reaction solution containing AMC-peptide Ac substrate to each well of the 384-well plate, including 2 ⁇ L of 10 ⁇ Tris buffer (0.5M Tris-HCl, pH 8.0, 1.37M NaCl, 27mM KCl, 10mM MgCl 2 ), 1 ⁇ L 20mM NAD + , 0.8 ⁇ L 5mM AMC-peptide Ac , 6.2 ⁇ L ddH 2 O. After that, add 10 ⁇ L of STM1221 or SIRT2 protein solution diluted with ddH 2 O or lysis buffer. The total volume of the reaction system is 20 ⁇ L.
  • the final concentration of Tris is 50mM, the final concentration of NAD + is 1mM, the final concentration of the peptide is 200 ⁇ M, and the final concentration of sirtuin protein is 20 ⁇ L. is 5 ⁇ M.
  • a microplate reader was used to detect the fluorescence intensity at 360nm excitation and 460nm emission wavelengths, and the reaction system without sirtuin was used as a negative control.
  • the measurement results are detailed in Figure 12.
  • Figure 12 shows the effect of lysate on the enzyme activities of STM1221 and SIRT2.
  • Example 2 yielded approximately 80 mutant proteins per 96-well plate. Therefore, by expanding the number of culture colonies, a total of 1,000 STM1221 mutant libraries were obtained in 12 96-well plates.
  • the AMC-ARK Ac peptide used for subsequent detection i.e., the AMC-labeled peptide modified by acetylation was synthesized by Anhui Guoping Pharmaceutical Co., Ltd.
  • BL21 transformed with wild-type STM1221 was first used to conduct lysis and enzyme activity detection experiments.
  • the BL21 bacteria were lysed using the cell lysis solution (i.e., lysis buffer) used in Example 2, and the lysate product was used to detect sirtuin activity.
  • the cleavage products were directly taken for AMC fluorescence detection, but the cleavage products of BL21 in the negative control group also reacted with the peptides to produce fluorescence, and no longer reacted after heat inactivation, proving that there may be other components in the cell fragments of BL21. Enzymes can interfere.
  • sirtuin protein or the protein-containing lysate supernatant from the previous step add 10 ⁇ L of purified sirtuin protein or the protein-containing lysate supernatant from the previous step.
  • 2L E. coli BL21 for cell lysis.
  • the final lysis can obtain at least 1 mL of sirtuin protein with a concentration of 100 ⁇ M. If the culture system is reduced to a 96-well plate, 200 ⁇ L of bacterial solution can obtain at least 10 ⁇ L of protein with a concentration of 1 ⁇ M. Therefore, purified STM1221 protein with an initial concentration of 1 ⁇ M or 5 ⁇ M was selected as a comparison.
  • SIRT2 protein also has strong deacetylation ability
  • purified SIRT2 or BL21-SIRT2 cleavage product was added as a control.
  • the total reaction volume was 20 ⁇ L, and the final peptide concentration was 200 ⁇ M. 37°C, 300rpm shaking reaction for 1h.
  • 20 ⁇ L of 2 ⁇ stop solution 2.0 mg/mL trypsin, 4 mM NAM in Tris assay buffer
  • was added to each well and the reaction was allowed to stand at 25°C for 1.5 h.
  • use a microplate reader to detect the fluorescence intensity at 360nm excitation and 460nm emission wavelengths. The measurement results are shown in Table 4 and Figure 7.
  • Table 4 shows the values of each mutant strain detected by AMC fluorescence, taking a 96-well plate as an example.
  • Figure 7 shows the fluorescence detection results of the reaction between purified sirtuin protein or sirtuin-containing cell lysate supernatant and substrate.
  • the fluorescence intensity of the reaction product is proportional to the deacetylase activity of sirtuin.
  • the BL21 lysate transferred into the blank plasmid is used as a negative control, and the BL21 lysate transferred into the wild-type pTEV5-STM1221 plasmid is used as a positive control.
  • the 12 established above are detected.
  • the deacetylation activity of STM1221 was preliminarily quantified, and 480 cloned strains with large changes in enzyme activity were obtained.
  • An example of the fluorescence detection results of the mutant library is shown in Figure 8. In Figure 8, the shades of gray represent fluorescence intensity, and the last column is the BL21 negative control (4 wells) and W.T.STM1221 positive control (4 wells).
  • STM1221 clones with deacetylase activity reduced by more than 80% were selected for sequencing, about 500 clones, and after excluding clones with more than 5 point mutations in a single clone, 176 mutation sites were obtained.
  • Table 5 shows some mutant sites and fluorescence detection results, which are single-point clones detected by sequencing in the same batch of experiments.
  • two single point mutants STM1221-H147L and STM1221-H147Y, were obtained through random mutation.
  • the enzyme activity fluorescence detection results are shown in Figure 9.
  • the deacetylation ability is reduced. to be equivalent to the negative control.
  • the H147 site in STM1221 has been confirmed to be an absolutely conserved site in the sirtuin protein that determines the deacylation activity. Therefore, the successful mutation of this site and the complete disappearance of the enzyme activity was detected, which proves the feasibility of this method to screen mutants. .
  • FIG. 9 shows the enzyme activity and site information of the fluorescence detection mutant library.
  • picture a is the result of fluorescence detection of the mutant library
  • picture b is the result of repeated fluorescence detection of the mutant library.
  • the point mutation information was obtained by sequencing the clones whose fluorescence was greatly reduced, and these clones were induced to express again in BL21, and the fluorescence was detected by lysing.
  • Figure 9b which is consistent with the first detection. It can be seen that the mutation was detected by fluorescence. The accuracy is higher and the repeatability is better.
  • Figure 10 is an analysis diagram of the regulatory sites of sirtuin enzyme activity.
  • the former site determines the deacetylation activity of STM1221 bigger. Therefore, score information is obtained based on the weight of the mutation site, and the score information of the amino acid site is obtained. The higher the score, the greater its determining role in the active site. It can be speculated from the site information that amino acids such as W67, V75, and H227 in STM1221 may play a decisive role in its deacetylation activity.
  • amino acids such as W67, V75, and H227 in STM1221 may play a decisive role in its deacetylation activity.
  • the corresponding amino acid sites in other typical sirtuins such as human SIRT2, SIRT5, and E. coli cobB were found for mutation and the active site was verified.
  • Sirtuin is a type of enzyme with multiple deacylation activities. It can not only remove the acetyl group on lysine, but also remove succinyl, crotonyl, long-chain fatty acyl, etc. Synthesizing AMC-peptide Acy peptides with different acyl modifications on lysine can be used to detect other deacylation activities of sirtuin proteins and obtain other deacylation active site information. Therefore, the method used in this application is not limited to the detection of deacetylation activity, and can also be applied to the detection of other deacylation activities of sirtuin, which should be included in the protection scope of the present invention.
  • the succinylated AMC-peptide Su peptide segment was synthesized according to the same method as the previous example, and reacted with the cell lysate transformed with the sirtuin protein expression vector to produce fluorescence.
  • the measurement results are detailed in Figure 11.
  • Figure 11 shows the fluorescence detection results of the reaction between cell lysate and AMC-peptide Ac or AMC-peptide Su peptide segments.
  • the BL21 lysate transformed with blank plasmid pUC19 did not react with any acylated peptides.
  • STM1221 which has multiple activities, can react with two peptides to produce fluorescence.
  • the cleavage solution of deacetylase SIRT2 only reacts with AMC-peptide Ac , while the cleavage product of desuccinylase SIRT5 reacts with AMC-peptide Su and produces strong fluorescence. Fluorescence, demonstrating the reaction specificity of these substrates, can be used to detect other enzyme activities, characterize other mutant libraries, and obtain other deacylated active site information.

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Abstract

La présente invention concerne un mutant de sirtuine, un procédé de criblage à haut rendement d'un mutant de sirtuine, et l'utilisation. Le procédé de criblage d'un mutant de sirtuine comprend les étapes suivantes : construction d'une banque de mutants de sirtuine à l'aide d'une technologie PCR à risque d'erreur; lyse de chaque mutant de la banque de mutants de sirtuine, réalisation d'une séparation solide-liquide et collecte d'un surnageant de lysat; et détermination de l'activité de désacylation de la sirtuine dans le surnageant de lysat à l'aide d'un procédé de fluorescence AMC, et obtention d'une souche de mutant de sirtuine présentant l'activité de désacylation requise. En utilisant le procédé de criblage pour un mutant de sirtuine, un mutant de l'enzyme de désacylation sirtuine peut être sélectionné de manière simple, pratique et répétée à l'aide d'un procédé à haut débit.
PCT/CN2022/119322 2022-09-16 2022-09-16 Procédé de criblage à haut débit de mutant de sirtuine, mutant de sirtuine et son utilisation WO2024055291A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006065076A1 (fr) * 2004-12-14 2006-06-22 Cj Corporation Mutant de la 5'-xmp aminase
CN103097545A (zh) * 2010-07-07 2013-05-08 康奈尔大学 Sirt5调节剂及其筛选方法
CN107630011A (zh) * 2006-07-05 2018-01-26 催化剂生物科学公司 蛋白酶筛选方法及由此鉴别的蛋白酶
WO2018064752A1 (fr) * 2016-10-07 2018-04-12 Ranomics Inc. Procédé basé sur la pcr pour produire des banques d'adn mutagènes de saturation multisites
CN108192882A (zh) * 2018-01-18 2018-06-22 天津市湖滨盘古基因科学发展有限公司 一种人的组蛋白去乙酰化酶突变蛋白及其应用
CN113584016A (zh) * 2021-08-17 2021-11-02 安徽金禾实业股份有限公司 一种通过易错pcr技术及高通量筛选提高葡萄糖基转移酶eugt11酶活方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006065076A1 (fr) * 2004-12-14 2006-06-22 Cj Corporation Mutant de la 5'-xmp aminase
CN107630011A (zh) * 2006-07-05 2018-01-26 催化剂生物科学公司 蛋白酶筛选方法及由此鉴别的蛋白酶
CN103097545A (zh) * 2010-07-07 2013-05-08 康奈尔大学 Sirt5调节剂及其筛选方法
WO2018064752A1 (fr) * 2016-10-07 2018-04-12 Ranomics Inc. Procédé basé sur la pcr pour produire des banques d'adn mutagènes de saturation multisites
CN108192882A (zh) * 2018-01-18 2018-06-22 天津市湖滨盘古基因科学发展有限公司 一种人的组蛋白去乙酰化酶突变蛋白及其应用
CN113584016A (zh) * 2021-08-17 2021-11-02 安徽金禾实业股份有限公司 一种通过易错pcr技术及高通量筛选提高葡萄糖基转移酶eugt11酶活方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHI XIAO-YAN, DU LI-MIN: "Sirtuin family and its biological functions", JOURNAL OF INTERNATIONAL PHARMACEUTICAL RESEARCH., vol. 38, no. 5, 1 October 2011 (2011-10-01), pages 349 - 355, XP093146001, DOI: 10.13220/j.cnki.jipr.2011.05.004 *

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