Recombinant cholesterol esterase, polynucleotide thereof, recombinant plasmid, expression system and application thereof

Abstract

The application provides a recombinant cholesteryl esterase, a polynucleotide thereof, a recombinant plasmid, an expression system and application. The application belongs to the technical field of protein engineering, and specifically, the amino acid sequence of the recombinant cholesteryl esterase provided by the application is shown as SEQ ID No. 1, and the nucleotide sequence for encoding the recombinant cholesteryl esterase is shown as SEQ ID No. 2. The sequence of the recombinant cholesteryl esterase provided by the application has a full length of 482 amino acids, is derived from Aspergillus ruber, has the advantage of high thermal stability, and has a thermal stability deviation of less than 10% after thermal storage at 37 DEG C for 11 days and after 30 days of on-board opening, and has a wide application prospect in the field of in-vitro diagnostic reagents.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein engineering, in particular to a recombinant cholesterol esterase, polynucleotide thereof, recombinant plasmid, expression system and application. BACKGROUND

[0002] Cholesterol esterase (EC 3.1.1.13) is an important sterol esterase that can act on fatty acid esters of cholesterol and other sterols in aqueous media, and the reaction catalyzing the hydrolysis of the ester bond generally does not involve a cofactor. Cholesterol esterase widely exists in nature. In the early stage, cholesterol esterase products were mainly derived from animal organs such as liver, pancreas, adrenal gland, gonad, mammary gland, etc. However, the method of extracting internal organs is limited by the source of materials, and the purification process is difficult, which affects the stability of the enzyme preparation. After the 1970s, microbial fermentation production of cholesterol esterase became the main trend. Streptomyces, Pseudomonas, Bacillus, Lactobacillus, Thermofilum, Micrococcus, Aspergillus, Sporosarcina, Saccharomyces, Candida, etc. Many microorganisms have been identified to have the ability to synthesize cholesterol esterase. Studies have shown that cholesterol esterases from different sources have different characteristics in terms of molecular size and structure, substrate specificity, reaction kinetics, etc.

[0003] In 1974, cholesterol esterase was used to measure cholesterol in human serum as a key raw material for diagnostic reagents, which opened up the industrial application of this enzyme. Clinically, the determination of serum cholesterol using kits containing cholesterol esterase has the advantages of simplicity, rapidity, precision, good specificity, suitability for automated instruments, and facilitation of standardization, which has gradually replaced optical, electrochemical, and chemical methods. In medicine, the detection of serum cholesterol plays an extremely important role in the diagnosis and prevention of multiple diseases such as diabetes, cardiovascular and cerebrovascular diseases, hepatobiliary diseases, and kidney diseases.

[0004] At present, the application of cholesterol esterase in in vitro diagnostic reagents has the problem of poor stability. SUMMARY

[0005] The main purpose of the present application is to provide a suitable enzyme source for high-density lipoprotein cholesterol detection kits, which solves the problem of poor stability of existing cholesterol esterase after application in detection reagents.

[0006] To achieve the above purpose, the first aspect of the present application provides a recombinant cholesterol esterase, the amino acid sequence of which is shown in SEQ ID No. 1.

[0007] In order to solve the problem of poor stability of cholesteryl esterase in the prior art, the application provides a recombinant cholesteryl esterase, which is derived from Aspergillus ruber, has a full-length sequence of 482 amino acids after modification, and has 6 histidine tags at the N terminal, and can be used for purification and separation by a nickel column.

[0008] The second aspect of the application provides a polynucleotide, the sequence of which is shown in SEQ ID No. 2, and the polynucleotide is used for efficiently encoding the recombinant cholesteryl esterase of the first aspect.

[0009] Preferably, the sequence of the polynucleotide is shown in SEQ ID No. 2.

[0010] The third aspect of the application provides a recombinant plasmid, which comprises the polynucleotide of the second aspect and can translate and express the recombinant cholesteryl esterase of the first aspect.

[0011] Preferably, the recombinant plasmid further comprises an hp4d hybrid promoter, an Xpr2 terminator and an Xpr2-pre signal peptide.

[0012] Preferably, the empty plasmid of the recombinant plasmid is pINA1317.

[0013] The fourth aspect of the application provides a recombinant cholesteryl esterase expression system, which is a Yarrowia lipolytica Po1h strain capable of stably secreting and expressing the recombinant cholesteryl esterase shown in SEQ ID No. 1.

[0014] Preferably, the recombinant cholesteryl esterase expression system is integrated with a Zeta1-CEN1 expression cassette-Zeta2 fragment, and the nucleotide sequence of the Zeta1-CEN1 expression cassette-Zeta2 fragment is shown in SEQ ID No. 3.

[0015] The fifth aspect of the application provides a preparation method of the recombinant cholesteryl esterase, which comprises the following steps: after high-density culture of the recombinant cholesteryl esterase expression system of the fourth aspect, the fermentation broth is purified by a nickel column to obtain the recombinant cholesteryl esterase.

[0016] More preferably, the preparation method can specifically comprise the following steps:

[0017] Step 1: in vitro synthesis of the nucleotide sequence shown in SEQ ID No. 2, and integration of the sequence into a pINA1317 vector;

[0018] Step two: linearized nucleotide fragments of the recombinant cholesterol esterase are recovered and transformed into the engineered Yarrowia lipolytica, and positive transformants are screened by solid plate of base medium without adding uracil, and the expression level of the cholesterol esterase of the strain is evaluated by the enzyme activity in the supernatant of the fermentation liquor;

[0019] Step three: the strain with high expression of the cholesterol esterase is cultured in large scale;

[0020] Step four: the recombinant cholesterol esterase is purified from the supernatant of the fermentation liquor of the yeast by the method of nickel column affinity chromatography.

[0021] More preferably, the prepared recombinant cholesterol esterase is stored in a protective agent, which is a mixture of 5% mannitol and 2.5% salmon serum protein.

[0022] More preferably, the prepared recombinant cholesterol esterase is stored by freeze-drying.

[0023] The sixth aspect of the present application provides an application of the recombinant cholesterol esterase in the first aspect in the preparation of a high-density lipoprotein cholesterol detection kit.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] 1. The recombinant cholesterol esterase provided by the present application is derived from Aspergillus ruber, has a high expression level in yeast and is easy to extract, and is suitable for being used as an enzyme preparation in a high-density lipoprotein cholesterol detection kit.

[0026] 2. The recombinant cholesterol esterase provided by the present application has extremely excellent thermal stability, and the thermal stability deviation is less than 10% after 11 days of heat storage at 37℃ and 30 days of on-board opening, and has a broad application prospect in the field of in vitro diagnostic reagents. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The plasmid map of the recombinant expression vector pINA1317-CEN1 in the specific embodiment of the present application is shown in the figure;

[0028] Figure 2 The SDS-PAGE electrophoresis result of the purified product in Example 4 of the present application is shown in the figure. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present application, and are not used to limit the parameter range described in the present application, and the reasonable changes derived therefrom are still within the protection scope of the claims of the present application.

[0030] It is to be understood that the endpoints of the ranges of the disclosed ranges are significant as are the values of the ranges for each element. It is to be understood that each individual value, including each endpoint value, in a range is combinable with each of the other values, including each endpoint value, in the same and any other range or ranges, to generate a new range. Each individual value, including each endpoint value, in a range is combinable with each of the other values, including each endpoint value, in the same and any other range or ranges, to generate a new range.

[0031] As described in the background, the enzymatic properties of cholesteryl esterase from different sources are not the same, and cholesteryl esterase as an enzyme preparation of high-density lipoprotein test kit has the problem of poor stability.

[0032] Based on this, the specific embodiment of the present application provides a recombinant cholesteryl esterase, the sequence of which is 482 amino acids long, and the sequence is shown as SEQ ID No. 1. The recombinant cholesteryl esterase is derived from Aspergillus ruber, and a 6-histidine tag is connected to the N-terminus thereof to adapt to nickel column purification.

[0033] More specifically, the nucleotide sequence encoding the recombinant cholesteryl esterase provided in the above embodiment is shown as SEQ ID No. 2.

[0034] Different species will exhibit codon bias when translating the same amino acid sequence due to the presence of synonymous codons and the influence of RNA abundance in the body of the species. Therefore, the polynucleotide sequence capable of encoding the recombinant cholesteryl esterase provided by the present application obtained by the existing codon optimization tool should fall within the protection scope of the present application.

[0035] More specifically, the specific embodiment of the present application further provides a recombinant plasmid containing the nucleotide sequence shown as SEQ ID No. 2 and capable of translating and expressing the recombinant cholesteryl esterase shown as SEQ ID No. 1.

[0036] More specifically, the specific embodiment of the present application further provides a recombinant cholesteryl esterase expression system for secreting and expressing the recombinant cholesteryl esterase shown as SEQ ID No. 1.

[0037] More specifically, the specific embodiment of the present application further provides a method for preparing the recombinant cholesteryl esterase by using the recombinant cholesteryl esterase expression system provided in the foregoing embodiments, specifically comprising the following steps:

[0038] S1: synthesizing the nucleotide sequence shown as SEQ ID No. 2 in vitro;

[0039] S2: The nucleotide sequence shown in SEQ ID No. 2 is introduced into the pINA1317 vector, more specifically, the nucleotide sequence shown in SEQ ID No. 2 is inserted between the Sfil and Kpnl restriction enzyme sites of pINA1317, to obtain a recombinant expression vector pINA1317-CEN1, which has a Zeta1-CEN1 expression cassette-Zeta2 fragment for efficient expression of the recombinant cholesterol esterase, the nucleotide sequence of the Zeta1-CEN1 expression cassette-Zeta2 fragment is shown in SEQ ID No. 3, and the plasmid map of the recombinant expression vector pINA1317-CEN1 is shown in Figure 1

[0040] S3: The expression vector pINA1317-CEN1 is single-enzymatically cut with Notl, and the fragment containing the cholesterol esterase gene is recovered by nucleic acid gel electrophoresis and gel cutting, and the purified fragment is transformed into Yarrowia lipolytica Po1h. Ura + type (uracil auxotrophic marker revertant type) recombinant strains are screened and marked, and the Ura + type recombinant strains are picked and inoculated to prepare seed liquid, and are inoculated into a 20 L fermenter at a 5% inoculation amount, and the recombinant expression strain is cultured at high density;

[0041] S4: The recombinant expression strain is streaked on a YPD plate, and a single colony is picked and inoculated to prepare seed liquid, and is inoculated into a 20 L fermenter at a 5% inoculation amount, and the recombinant expression strain is cultured at high density;

[0042] S5: The fermentation liquid is collected and centrifuged, and the supernatant is subjected to Ni column affinity layer purification, and the recombinant cholesterol esterase is obtained by freeze-drying.

[0043] More specifically, in step S3 of the above embodiment, a linearized fragment with Zeta1 / 2 sites at both ends can be obtained after Notl single enzyme cutting.

[0044] More specifically, in step S4 of the above embodiment, the detection reagent for recombinant cholesterol esterase activity includes reagent R1 and reagent R2, wherein reagent R1 includes: 50 mM PBS buffer, 400 U / L cholesterol oxidase, 2 mL / L triton X-100; and reagent R2 includes: 50 mM PBS buffer, 1000 U / L peroxidase, 1 mmol / L 4-aminoantipyrine, 0.2 mM cholesteryl linoleate.

[0045] ​More specifically, in step S5 of the above embodiment, the protective agent used for freeze-drying of the recombinant cholesterol esterase is a mixture of 5% mannitol and 2.5% salmon serum protein, which can effectively improve the protein structure stability of the recombinant cholesterol esterase during preparation and storage, and reduce the loss rate of enzyme activity.

[0046] The technical solutions of the present application are further described below through specific examples. Unless otherwise defined, all terms, symbols and other scientific and technical terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. In some cases, terms having commonly understood meanings are defined herein for the sake of clarity and convenience in describing the present application. Such definitions herein should not be understood as indicating a significant difference from the commonly understood meanings. The technical methods described or referenced herein are generally well understood by those skilled in the art and can be used by conventional methods. Unless otherwise specified, the use of commercially available kits and reagents, and instruments are carried out according to the protocols and parameters provided by the manufacturers.

[0047] Example 1

[0048] Screening of recombinant expression strain

[0049] The nucleotide sequence shown in SEQ ID No. 2 was designed and synthesized by a commercial company. The nucleotide sequence was introduced into an expression vector pINA1317, and the nucleotide sequence shown in SEQ ID No. 2 was located between the Sfil and Kpnl restriction enzyme sites in the multiple cloning site of the vector. The final recombinant plasmid was named pINA1317-CEN1.

[0050] The glycerol bacteria containing the aforementioned pINA1317-CEN1 expression vector were streaked on LB plates containing 50 μg / mL of kanamycin resistance and incubated in a 37°C incubator overnight. Single colonies on the plates were picked and cultured in LB liquid medium containing 50 μg / mL of kanamycin resistance at 37°C and 220 rpm for 14-18 hours. The target expression vector was extracted using a plasmid extraction kit. NotI fast enzyme and the corresponding buffer were added and incubated at 37°C for 1.5 hours. The NotI enzyme cutting system of the pINA1317-CEN1 recombinant expression vector is shown in Table 1.

[0051] Table 1

[0052]

[0053] The target fragment and the mixed fragment containing the recombinant cholesterol esterase gene were separated by nucleic acid gel electrophoresis after enzyme cutting, and the linearized fragment was extracted using a gel recovery kit.

[0054] 10 μL Yarrowia lipolytica liquid from the strain preservation tube was taken into 5 mL YPD liquid medium, and cultured at 30°C with 200 rpm shaking for 24 hours. The liquid was taken into a 1.5 mL centrifuge tube, and centrifuged at 12000 rpm for 1 min. The volume of the collected bacteria was adjusted to 100 μL, and washed twice with sterile water. Then, 0.6 mL of 0.1 M lithium acetate was added, and the mixture was incubated at 30°C for 1 hour. The mixture was centrifuged at 4000 rpm for 1 min, and the supernatant was discarded. Then, 40 μL of 0.1 M lithium acetate was added to obtain the competent cells.

[0055] 1 μg of the linearized fragment and 50 μg of single-stranded salmon sperm DNA were added to 40 μL of the prepared competent cells, and mixed by blowing. The mixture was incubated at 30°C for 15 min. Then, 350 μL of 40% PEG4000+0.1 M lithium acetate buffer and 16 μL of 1 M dithiothreitol were sequentially added, and the mixture was further incubated at 30°C. After 1 hour, the centrifuge tube was taken out and placed in a 39°C metal bath for heat shock for 10 min. Then, 0.6 mL of 0.1 M lithium acetate was added, and the mixture was incubated at 30°C for 30-60 min. 100-200 μL of the bacterial liquid was taken and spread on solid basic medium without leucine (20 g / L glucose, 10% YNB: 1.7 g of amino acid-free yeast nitrogen source+5 g of ammonium sulfate), and placed in a 30°C incubator for 4-5 days. Then, the plate was taken out, and 20 single colonies were picked and inoculated in 5 mL of YPD liquid medium (10 g of yeast powder, 20 g of peptone, and 50 g of glucose per liter of pure water), and cultured at 30°C with 200 rpm shaking for 120 hours. 200 μL of the fermentation liquid was taken and centrifuged at 12000 rpm. The supernatant was reserved for qualitative activity detection. The single colony with the highest activity was re-inoculated and cultured. When the yeast grew to the exponential phase, 50% glycerol was added, and the mixture was frozen and preserved at -80°C.

[0056] Example 2

[0057] Detection of recombinant cholesterol esterase activity.

[0058] The enzyme activity detection reagent for recombinant cholesterol esterase was prepared according to the formula shown in Table 2.

[0059] Table 2

[0060]

[0061] The reaction parameters were set on the Miyakawa 880 biochemical instrument as follows: the detection wavelength was set to 546 nm, the sample volume was set to 10 μL, the reagent R1 volume was set to 150 μL, the reagent R2 volume was set to 150 μL, the reaction mode was rate method, and the reading points were 35-45. Water and 100 U / L cholesterol esterase were used as the calibrants.

[0062] The detection results of the 20 monoclonal small-scale cultures after 120 hours are shown in Table 3.

[0063] Table 3

[0064]

[0065] Example 3

[0066] High-density culture.

[0067] The positive single colony numbered 3 in Table 3 was streaked onto a YPD solid plate, and cultured at 30°C for 3 days. A single colony was inoculated into 5 mL of YPD liquid medium, and cultured at 30°C, 200 rpm for 24 hours. The culture was scaled up to 250 mL of YPD medium at a 2% inoculation amount, and cultured at 30°C, 200 rpm for 24 hours. The culture was transferred into a fermenter with a volume of 20 L at a 5% inoculation amount. The parameters of the fermenter were set as follows: the liquid volume of the fermenter was 15 L, the medium used was YPD liquid medium, the stirring speed was 500-1000 rpm, the temperature was set to 28-30°C, the pH was adjusted to about 7.0 by adding ammonia water, and the DO was controlled at 20-50%. The wet weight of the bacteria and the enzyme activity of the recombinant cholesterol esterase were determined every 24 hours, and glucose was added at the same time. After 120 hours of fermentation, the fermentation broth was centrifuged, and the supernatant had a volume of about 9 L, the wet weight of the bacteria was 48 g / 100 mL, and the enzyme activity of the recombinant cholesterol esterase in the supernatant was 37500 U / L.

[0068] Example 4

[0069] Product purification.

[0070] The fermentation broth harvested in Example 3 was centrifuged at 4°C, 4000 rpm for 30 min. The supernatant was concentrated and the pigment was removed using a tangential flow ultrafiltration system. The concentration ratio was 4-5 times, and the replacement buffer was 20 mM Tris-HCl+300 mM Nacl (pH 7.8).

[0071] The nickel column was equilibrated with 5-10 times the volume of the filler with 20 mM Tris-HCl+300 mM Nacl (pH 7.8) buffer. The ultrafiltration concentrated liquid was loaded onto the nickel column. The nickel column was equilibrated with 20 mM Tris-HCl+300 mM Nacl (pH 7.8) buffer until the ultraviolet detection was stable. The target protein was eluted with 20 mM Tris-HCl+300 mM Nacl+20 mM imidazole, 20 mM Tris-HCl+300 mM Nacl+50 mM imidazole, and 20 mM Tris-HCl+300 mM Nacl+400 mM imidazole, and the elution peak was collected. The total amount of the eluate was 700 mL, the enzyme activity was 140 KU / L, and the SDS-PAGE electrophoresis result of the purified product is shown in Figure 2As shown, the target protein band is clear, mainly concentrated in the 400 mM imidazole eluate, and substantially free of impurities.

[0072] The 400 mM imidazole eluate containing the recombinant cholesteryl esterase was loaded into a dialysis bag, and dialyzed 3-4 times against 50 mM phosphate buffer (pH 7.8) to obtain the pure recombinant cholesteryl esterase.

[0073] Example 5

[0074] Effect of protective agents on the stability of recombinant cholesteryl esterase protein

[0075] According to Table 4, protective agent formulations 1-6 were prepared, and 10 mL of the pure recombinant cholesteryl esterase obtained in Example 4 was mixed with each of the protective agent formulations 1-6.

[0076] Table 4

[0077]

[0078] The recombinant cholesteryl esterase solution with the protective agent added was aliquoted into cryovials at a volume of 1 mL, and freeze-dried using a freeze dryer to obtain a lyophilized powder. 1 mL of sterile water was used to reconstitute the lyophilized powder, and the relative deviation in enzyme activity of the cholesteryl esterase before and after lyophilization was recorded. The reconstituted solution was placed at 4°C and 42°C, and the relative deviation in enzyme activity was detected on day 0 and day 14, with the results shown in Table 5.

[0079] Table 5

[0080]

[0081] As can be seen from Table 5, both formulation 4 and formulation 6 with added salmon serum protein can effectively improve the stability of the recombinant cholesteryl esterase, and the use of 5% mannitol and 2.5% salmon serum protein as the lyophilization protective agent results in the lowest loss of enzyme activity during lyophilization and the best long-term storage stability at 4°C and 42°C.

[0082] Example 6

[0083] Application of recombinant cholesteryl esterase in high-density lipoprotein cholesterol detection reagent

[0084] High-density lipoprotein cholesterol detection reagent RA was prepared, and the components of reagent RA are shown in Table 6.

[0085] Table 6

[0086]

[0087] High-density lipoprotein cholesterol detection reagent RB was prepared, and the components of reagent RB are shown in Table 7.

[0088] Table 7

[0089]

[0090] The detection reagent RA / RB was used to detect high-density lipoprotein cholesterol quality control (level 1 range: 0.89-1.33 mmol / L, level 1 target value: 1.11 mmol / L; level 2 range: 0.89-1.33 mmol / L, level 2 target value: 1.93 mmol / L) and 20 serum samples before and after 11 days of heat storage in a 37℃ water bath and before and after 30 days of on-board opening, and the specific data are shown in Tables 8 and 9.

[0091] Table 8

[0092]

[0093] Table 9

[0094]

[0095] As can be seen from Tables 8 and 9, the values of the serum samples and the quality control are basically consistent, proving that the recombinant cholesterol esterase provided by the application as the raw material of the high-density lipoprotein cholesterol detection kit has high stability and can meet the basic performance of detection.

[0096] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited to this. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present disclosure.

Claims

1. A high-density lipoprotein cholesterol detection kit, characterized by, The reagent RA comprises the following components: 800 U / L of recombinant cholesteryl esterase, 50 mM of piperazine-1,4-bis(2-ethanesulfonic acid) buffer, 400 U / L of cholesteryl oxidase, 20 KU / L of catalase, 2 mL / L of triton X-100; The reagent RB comprises the following components: 50 mM of piperazine-1,4-bis(2-ethanesulfonic acid) buffer, 1000 U / L of peroxidase, 1 mM of 4-aminoantipyrine, 0.5 mM of N,N-bis(4-sulfobutyl)-3-methyl aniline, 1 g / L of sodium azide, The recombinant cholesteryl esterase is obtained by encoding the nucleotide sequence shown in SEQ ID No.

2. The N-terminal of the recombinant cholesteryl esterase is connected with 6 histidine tags.

2. The high-density lipoprotein cholesterol test kit of claim 1, wherein, The recombinant plasmid comprises:

3. A recombinant plasmid, characterized in that, The polynucleotide of claim 1; hp4d hybrid promoter, Xpr2 terminator and Xpr2-pre signal peptide; pINA1317 empty plasmid. The recombinant cholesteryl esterase expression system is a Yarrowia lipolytica Po1h strain capable of stably secreting and expressing the recombinant cholesteryl esterase shown in SEQ ID No.

1.

4. A recombinant cholesteryl esterase expression system, characterized by, The Zeta1-CEN1 expression cassette-Zeta2 fragment is integrated into the recombinant cholesteryl esterase expression system, and the nucleotide sequence of the Zeta1-CEN1 expression cassette-Zeta2 fragment is shown in SEQ ID No.

3.

5. The recombinant cholesteryl esterase expression system of claim 4, wherein, The preparation method is to purify the fermentation broth by using a nickel column after high-density culture of the recombinant cholesteryl esterase expression system of claim 4 or 5, so as to obtain the recombinant cholesteryl esterase.

6. A method for producing a recombinant cholesterol esterase, characterized by, The method comprises the following steps:

7. The production method according to claim 6, wherein Step one: in vitro synthesis of the nucleotide sequence shown in SEQ ID No. 2, and integration of the sequence into an empty plasmid; Step two: recovery of the linearized nucleotide fragment of the recombinant cholesteryl esterase, and transformation of the fragment into Yarrowia lipolytica engineering bacteria, solid plate screening of positive transformants by using a basic medium without adding uracil, and evaluation of the expression level of cholesteryl esterase of the strain in the supernatant of the fermentation broth; Step three: scale-up culture of the cholesteryl esterase high-expression strain; Step four: purification of the recombinant cholesteryl esterase from the fermentation supernatant of the yeast by the method of nickel column affinity chromatography. ​

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