阅读说明：本技术 重组人胰岛素前体的酶切转换方法 (Enzyme digestion conversion method of recombinant human insulin precursor ) 是由 聂洪霞 刘海峰 黄菁 许波 吕晓林 张金迪 解福生 郝向慧 于 2020-12-29 设计创作，主要内容包括：本发明公开了重组人胰岛素前体的酶切转换方法,包括：(1)将初步纯化的重组人胰岛素前体溶液加入缓冲液进行浓度和pH值的调整得到酶切反应底物混合液；(2)向酶切反应底物混合液中加入重组赖氨酰肽内切酶进行重组人胰岛素前体的酶切转换反应。本发明选择重组赖氨酰肽链内切酶作为工具酶,与常用的重组胰蛋白酶相比,单位前体蛋白所需的酶更少,酶切位点单一,酶切过程更高效,使人胰岛素前体的酶切转换效率达到95％以上；在放大酶切时,没有过酶切的顾虑且酶切后获得的目的蛋白纯度更高；重组赖氨酰肽链内切酶酶切最适pH范围离酶切前后蛋白的pI更远,有利于目的蛋白的稳定且对酶切环境中的盐浓度耐受范围更大。(The invention discloses an enzyme digestion conversion method of a recombinant human insulin precursor, which comprises the following steps: (1) adding the preliminarily purified recombinant human insulin precursor solution into a buffer solution for adjusting the concentration and the pH value to obtain a mixed solution of enzyme digestion reaction substrates; (2) and adding recombinant lysyl endopeptidase into the enzyme digestion reaction substrate mixed solution to perform enzyme digestion conversion reaction of the recombinant human insulin precursor. The invention selects recombinant lysyl endopeptidase as tool enzyme, compared with common recombinant trypsin, the enzyme required by unit precursor protein is less, the enzyme cutting site is single, the enzyme cutting process is more efficient, and the enzyme cutting conversion efficiency of the human insulin precursor reaches more than 95%; when the enzyme digestion is amplified, the concern of enzyme digestion is avoided, and the purity of the target protein obtained after enzyme digestion is higher; the optimal pH range of the recombinant lysyl endopeptidase during enzyme digestion is farther from the pI of the protein before and after enzyme digestion, which is beneficial to the stability of the target protein and has larger tolerance range to the salt concentration in the enzyme digestion environment.)

1. An enzyme digestion conversion method of a recombinant human insulin precursor is characterized by comprising the following steps: (1) adding the preliminarily purified recombinant human insulin precursor solution into a buffer solution for adjusting the concentration and the pH value to obtain a mixed solution of enzyme digestion reaction substrates; (2) and adding recombinant lysyl endopeptidase into the enzyme digestion reaction substrate mixed solution to perform enzyme digestion conversion reaction of the recombinant human insulin precursor.

2. The enzyme digestion conversion method according to claim 1, characterized in that in step (1), the concentration of the precursor protein in the precursor solution of recombinant human insulin obtained by primary purification is controlled to be 8-25 mg/ml; preferably, the concentration of the precursor protein is controlled to be 15-20 mg/ml.

3. The enzyme digestion conversion method according to claim 1, wherein the buffer solution in step (1) is any one of sodium carbonate-sodium bicarbonate buffer solution, borax-sodium hydroxide buffer solution or Tris-hydrochloric acid buffer solution.

4. The enzymatic conversion process of claim 3, wherein said buffer is borax-sodium hydroxide buffer.

5. The enzymatic conversion process according to claim 3 or 4, characterized in that the concentration of said buffer is 0.01-0.05 mol/L; preferably, the concentration of the buffer solution is 0.02-0.035 mol/L; most preferably, the concentration of the buffer is 0.025 mol/L.

6. The enzyme digestion conversion method according to claim 1, characterized in that in step (1), the pH value of the enzyme digestion reaction substrate mixture is controlled to be 8-10.5; preferably, the pH of the mixture of substrates for the enzymatic digestion is controlled to 9 to 10.

7. The enzyme digestion conversion method according to claim 1, characterized in that in step (1), the final concentration of the recombinant human insulin precursor protein in the enzyme digestion reaction substrate mixture is controlled to be 0.02-0.03 mol/L.

8. The enzyme conversion method according to claim 1, wherein the recombinant lysyl endopeptidase is controlled in terms of w/w: the proportion of the recombinant human insulin precursor is 1:15000-1: 500; preferably, the recombinant lysyl endopeptidase is controlled: the proportion of the recombinant human insulin precursor is 1:15000-1: 1000; most preferably, the recombinant lysyl endopeptidase is controlled: the proportion of the recombinant human insulin precursor is 1:15000-1: 2000.

9. The enzymatic conversion process according to claim 1, wherein the temperature of the enzymatic conversion reaction in step (3) is 15-30 ℃, preferably 20-25 ℃.

10. The enzyme digestion conversion method according to claim 1, characterized in that the enzyme digestion reaction time of the enzyme digestion conversion reaction in step (3) is 12-48 h; preferably, the enzyme digestion reaction time is 18-24 h.

Technical Field

The invention relates to an enzyme digestion conversion method of an insulin precursor, in particular to an enzyme digestion conversion method of a recombinant human insulin precursor, belonging to the field of enzyme digestion conversion of the recombinant human insulin precursor.

Background

Diabetes has become a chronic non-infectious disease that threatens human banking, in addition to cardiovascular and cerebrovascular diseases and malignancies. According to the global diabetes overview assessment of the International Diabetes Federation (IDF)2017, about 4.25 hundred million adults aged 20-79 suffer from diabetes globally, wherein the number of diabetic patients in china is as high as 1.144 million, and china has become the country with the largest number of diabetic patients worldwide.

At present, insulin mass spectrum is one of the most effective diabetes treatment medicines, and the emergence of insulin provides a better treatment means for vast diabetics and plays an important role in improving the life quality of the diabetics. With the development of science and technology, the industrial production of insulin has undergone a great change from the extraction of animal insulin, the organic synthesis of insulin to the present recombinant human insulin and its analogues. The production of recombinant human insulin and its analogs is generally based on obtaining precursor protein of recombinant human insulin and its analogs, so that the preliminary purification and enzyme digestion of the precursor protein of recombinant human insulin and its analogs become necessary purification steps. As mentioned in chinese patent CN101253196A, the cleavage site of trypsin is the carboxyl-terminal residues of lysine and arginine when the amino acid sequence of the protein contains arginine, so it is likely to generate hybrid protein with more arginine carboxyl-terminal residues, which results in increased impurities in the subsequent samples, increased difficulty in sample purification and inevitably reduced sample yield.

Therefore, the development of a specific and efficient enzyme digestion conversion method for recombinant human insulin and analogues thereof can improve the purity of intermediate samples, reduce the difficulty of impurity production and subsequent purification processes, and have important market value and application prospect.

Disclosure of Invention

The invention mainly aims to provide a specific and efficient enzyme digestion conversion method for a recombinant human insulin precursor.

The above object of the present invention is achieved by the following technical solutions:

the invention discloses an enzyme digestion conversion method of a recombinant human insulin precursor, which comprises the following steps of (1) adding a primarily purified recombinant human insulin precursor solution into a buffer solution to adjust the concentration and the pH value to obtain an enzyme digestion reaction substrate mixed solution; (2) and adding recombinant lysyl endopeptidase into the enzyme digestion reaction substrate mixed solution to perform enzyme digestion conversion reaction of the recombinant human insulin precursor.

As a preferred embodiment of the invention, in the step (1), the concentration of the precursor protein in the solution of the recombinant human insulin precursor obtained by primary purification is controlled to be 8-25 mg/ml; more preferably, the concentration of the precursor protein is controlled to be 15-20 mg/ml.

As a preferred embodiment of the present invention, the buffer solution in step (1) may be any one of a sodium carbonate-sodium bicarbonate buffer solution, a borax-sodium hydroxide buffer solution or a Tris-hydrochloric acid buffer solution, and is preferably a borax-sodium hydroxide buffer solution; wherein, the concentration of the buffer solution can be 0.01-0.05 mol/L; preferably, the concentration of the buffer solution is 0.02-0.035 mol/L; most preferably, the concentration of the buffer is 0.025 mol/L.

As a preferred embodiment of the present invention, the solution of the recombinant human insulin precursor obtained by the primary purification in step (1) is buffered in a buffer

As a preferable embodiment of the invention, in the step (1), the pH value of the enzyme digestion reaction substrate mixed solution is controlled to be 8-10.5; preferably, the pH of the mixture of substrates for the enzymatic digestion is controlled to 9 to 10.

As a preferred embodiment of the invention, in the step (1), the final concentration of the recombinant human insulin precursor protein in the substrate mixture solution of the enzyme digestion reaction is controlled to be 0.02-0.03 mol/L.

The inventor finds through experiments that the proportion of the recombinant lysyl endopeptidase to the recombinant human insulin precursor protein has obvious influence on the enzyme digestion conversion efficiency in the enzyme digestion conversion reaction, and finds through experiments that the enzyme digestion conversion reaction is carried out on the recombinant lysyl endopeptidase and the recombinant human insulin precursor protein according to the following proportion, so that the enzyme digestion conversion efficiency is favorably improved:

preferably, the recombinant lysyl endopeptidase: recombinant human insulin precursor ═ 1:15000-1:500 (w/w); more preferably, the recombinant lysyl endopeptidase: recombinant human insulin precursor ═ 1:15000-1:1000 (w/w); most preferably, the recombinant lysyl endopeptidase: the recombinant human insulin precursor ═ 1:15000-1:2000 (w/w).

The temperature of the enzyme digestion reaction of the enzyme digestion conversion reaction in the step (3) can be 15-30 ℃, and preferably, the temperature of the enzyme digestion reaction is 20-25 ℃; the enzyme digestion reaction time can be 12-48 h; preferably, the enzyme digestion reaction time is 18-24 h.

The finishing time of the enzyme digestion conversion of the recombinant human insulin precursor obtained by the primary purification is a variable value within a certain range under the combined action of the enzyme addition amount and the enzyme digestion temperature. The invention finds that the enzyme digestion conversion efficiency can be effectively improved when the enzyme digestion conversion reaction is carried out by combining the following reaction conditions: namely, recombinant lysyl endopeptidase: the recombinant human insulin precursor is 1:15000-1:500(w/w), the temperature of enzyme digestion reaction is 15-30 ℃, and the time of enzyme digestion reaction is 12-48 h; further preferably, the recombinant lysyl endopeptidase: the recombinant human insulin precursor is 1:15000-1:2000(w/w), the temperature of the enzyme digestion reaction is 20-25 ℃, and the time of the enzyme digestion reaction is 18-24 h.

The method can ensure that the enzyme digestion conversion efficiency of the human insulin precursor reaches more than 95 percent under the action of the specified single enzyme digestion site enzyme, and compared with the production process using the conventional enzyme, the method improves the enzyme digestion conversion efficiency and the purity of subsequent products and reduces the difficulty of the subsequent purification process. Meanwhile, the production process is simplified, and a good production process is provided for large-scale production.

Compared with the prior mainstream technology, the technical scheme of the invention has the following beneficial effects:

(1) the invention selects the recombinant lysyl endopeptidase as the tool enzyme, and compared with the common recombinant trypsin, the recombinant lysyl endopeptidase uses less enzyme required by unit precursor protein, has single enzyme cutting site and more efficient enzyme cutting process.

(2) The recombinant lysyl endopeptidase is used as a tool enzyme, compared with the commonly used recombinant trypsin, the recombinant lysyl endopeptidase is used, enzyme digestion is avoided during amplified enzyme digestion, and the purity of the target protein obtained after enzyme digestion is higher, so that the subsequent purification steps are more facilitated.

(3) The recombinant lysyl endopeptidase is selected as a tool enzyme, and compared with the common recombinant trypsin, the optimal pH range of the recombinant lysyl endopeptidase during enzyme digestion is farther from the pI of the protein before and after enzyme digestion, so that the stability of the target protein is facilitated, and the tolerance range of the recombinant lysyl endopeptidase to the salt concentration in the enzyme digestion environment is wider.

Drawings

FIG. 1 comparison of liquid phase maps of recombinant human insulin protein after enzymatic cleavage for example 1 and comparative example 1.

FIG. 2 example 2 UPLC profile of recombinant human insulin after enzyme cleavage.

FIG. 3 Mass map of recombinant human insulin after enzyme cleavage in example 3.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

1. Test material and detection method

1.1 test materials and instruments

Buffer substances such as tris (hydroxymethyl) aminomethane and phosphoric acid were purchased from Sigma, conventional reagents such as sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Chemicals, Inc., national drug group, and anhydrous sodium sulfate (Na)₂SO₄) Purchased from Sigma, acetonitrile (CH)₃CN, HPLC grade), the analytical high performance liquid instrument is Agilent 1260, the analytical chromatographic column is Kromasil-C18-5 μm-(4.6 x 250 mm). The LC-MS is Q-active-Plus LCMS from Saimearfei, and the UPLC analytical column is Hypersil GOLD (100 × 2.1-3 um).

1.2 detection method

1.2.1 liquid phase detection method

Detecting samples before and after enzyme digestion by adopting a reverse-phase chromatography method, wherein an apparatus is an Agilent 1260 analysis type high-performance liquid phase, and an analysis chromatographic column: Kromasil-C18-5 μm-(4.6 x 250 mm). Mobile phase a was 0.2mol/L anhydrous sodium sulfate (ph 2.3): acetonitrile 90:10, mobile phase B water: acetonitrile 50: 50. The flow rate was 1.0ml/min, the column temperature was 40 ℃ and the detection wavelength was 280nm, and gradient elution was carried out according to the parameters in Table 1.

TABLE 1 analytical HPLC gradient elution procedure

1.2.2 liquid quality detection method

In order to confirm the digestion effect and ensure the correct digestion result, the product after digestion was subjected to a mass spectrometric detection method for substance confirmation.

The UPLC detection method of the recombinant human insulin precursor enzyme digestion sample comprises the following steps: column: hypersil GOLD (100 x 2.1-3um), mobile phase A: 0.1% formic acid, mobile phase B: acetonitrile, flow rate: 0.3ml/min, column temperature: the gradient elution was carried out at 35 ℃ and a detection wavelength of 280nm according to the parameters in Table 2.

TABLE 2 gradient elution procedure for UPLC detection method of samples

1.3 conversion efficiency of enzyme digestion

The enzyme digestion conversion efficiency refers to the ratio of the molar weight of the protein obtained by the reagent after enzyme digestion to the molar weight of the protein which should be obtained theoretically after enzyme digestion, wherein the theoretical molar weight of the protein after enzyme digestion is the molar weight of the recombinant human insulin precursor before enzyme digestion, the masses of the recombinant human insulin precursor and the protein after enzyme digestion are respectively obtained by calculation according to respective standard curves, correspondingly, the molar weight of the recombinant human insulin precursor is 7088, the molar weight of the protein after enzyme digestion is 5721, and the specific calculation formula is as follows

EXAMPLE 1 enzymatic conversion of recombinant human insulin precursor

Taking a proper amount of the recombinant human insulin precursor solution obtained by primary purification, detecting the concentration of a sample by a high performance liquid chromatography, calculating the volume V1 diluted to the concentration of 15g/L, adding a proper amount of tris (hydroxymethyl) aminomethane according to the volume, uniformly stirring to ensure that the final concentration is 0.03mol/L (calculated according to the volume after dilution), measuring the pH value, adding a proper amount of purified water to adjust the volume of the sample solution to be close to V1, adjusting the pH to 9.0 by hydrochloric acid, and finally adjusting the volume of the solution to be V1;

calculating the content of the recombinant human insulin precursor protein, and then calculating the content of the recombinant human insulin precursor protein according to the content of the recombinant lysyl endopeptidase: accurately weighing an appropriate amount of recombinant lysyl endopeptidase according to the proportion of 1:1000(w/w), adding the tool enzyme while stirring the solution, completely dissolving, and then stirring and carrying out enzyme digestion at room temperature, wherein the room temperature is 23 ℃, and the room temperature is controlled to be 23 +/-1 ℃ within 24 hours.

The concentration of the primary purified recombinant human insulin precursor is about 25g/L, 40ml is measured, 243.4mg of tris (hydroxymethyl) aminomethane is added with stirring, a proper amount of purified water is added to adjust the volume to 60ml, the pH is adjusted to 9.03, and the volume is finally adjusted to 66.7 ml.

Weighing 1.00mg of recombinant lysyl endopeptidase, adding the solution, stirring and dissolving, timing, carrying out sampling detection after carrying out enzyme digestion for 16 hours, wherein the protein purity after enzyme digestion is 91.9%, and the enzyme digestion conversion rate is calculated to be about 87.2%. This shows that according to the digestion conversion method of this example, the purity of the recombinant human insulin after digestion is up to 90% or more, and the digestion conversion rate is about 87.2%.

Example 2 enzymatic conversion of recombinant human insulin precursor

Taking a proper amount of the recombinant human insulin precursor solution obtained by primary purification, detecting the concentration of a sample by a high performance liquid chromatography, calculating the volume V2 diluted to the concentration of 10g/L, adding a proper amount of borax according to the volume, uniformly stirring to ensure that the final concentration is 0.02mol/L (calculated according to the volume after dilution), measuring the pH value, adding a proper amount of purified water to adjust the volume of the sample solution to be close to V2, adjusting the pH value to 9.4 by using sodium hydroxide, and finally adjusting the volume of the solution to be V2;

calculating the content of the recombinant human insulin precursor protein, and then calculating the content of the recombinant human insulin precursor protein according to the content of the recombinant lysyl endopeptidase: accurately weighing a proper amount of recombinant lysyl endopeptidase according to the proportion of 1:10000(w/w), adding tool enzyme while stirring the solution, completely dissolving, and then stirring and carrying out enzyme digestion at room temperature, wherein the room temperature is 20 ℃, and the room temperature is controlled to be kept within the range of 20 +/-1 ℃ within 24 hours.

The concentration of the primary purified recombinant human insulin precursor is about 20g/L, 50ml is measured, 123.7mg of borax is stirred and added, a proper amount of purified water is added to adjust the volume to 90ml, the pH value is adjusted to 9.39, and the volume is finally adjusted to 100 ml.

Weighing 0.1mg of recombinant lysyl endopeptidase, adding the solution into the solution, stirring and dissolving, timing, performing sampling detection after enzyme digestion for 22 hours, wherein the protein purity after enzyme digestion is 93.1%, and the enzyme digestion conversion rate is calculated to be about 88.4%. This shows that according to the method of this example, the purity of the target protein after enzyme digestion is up to 90% or more, and the conversion rate of enzyme digestion is about 88.4%.

EXAMPLE 3 enzymatic conversion of recombinant human insulin precursor

Taking a proper amount of the recombinant human insulin precursor solution obtained by primary purification, detecting the concentration of a sample by a high performance liquid chromatography, calculating the volume V3 diluted to the concentration of 20g/L, adding a proper amount of tris (hydroxymethyl) aminomethane according to the volume, uniformly stirring to ensure that the final concentration is 0.02mol/L (calculated according to the volume after dilution), measuring the pH value, adding a proper amount of purified water to adjust the volume of the sample solution to be close to V3, adjusting the pH value to 9.5 by using hydrochloric acid or sodium hydroxide, and finally adjusting the volume of the solution to V3;

calculating the content of the recombinant human insulin precursor protein, and then calculating the content of the recombinant human insulin precursor protein according to the content of the recombinant lysyl endopeptidase: accurately weighing a proper amount of recombinant lysyl endopeptidase according to the proportion of 1:2000(w/w), adding tool enzyme while stirring the solution, completely dissolving, and then stirring and carrying out enzyme digestion at room temperature, wherein the room temperature is 25 ℃, and the room temperature is controlled to be kept within the range of 25 +/-1 ℃ within 24 hours.

The concentration of the primary purified recombinant human insulin precursor is about 25g/L, 40ml is measured, 121.13mg of tris (hydroxymethyl) aminomethane is added with stirring, a proper amount of purified water is added to adjust the volume to 45ml, the pH is adjusted to 9.52, and the volume is finally adjusted to 50 ml.

Weighing 0.5mg of recombinant lysyl endopeptidase, adding the solution into the solution, stirring and dissolving, timing, performing sampling detection after performing enzyme digestion for 20 hours, wherein the protein purity after the enzyme digestion is 92.8%, and the enzyme digestion conversion rate is calculated to be about 87.8%. This shows that according to the method of this example, the purity of the target protein after enzyme digestion is up to 90% or more, and the conversion rate of enzyme digestion is about 87.8%.

Comparative example 1 enzymatic conversion of recombinant human insulin precursor

The endonuclease used in this comparative example was recombinant trypsin, and other conditions were the same as those in example 1.

Through detection, the protein purity after enzyme digestion is 57.2%, and the enzyme digestion conversion rate is calculated to be about 53.7%.

Comparative example 2 two-step purification of recombinant human insulin precursor and cleavage after precipitation

Centrifuging the pichia pastoris fermentation liquor, and performing copper ion chelation chromatography and SP cation exchange chromatography on the supernatant to obtain a purified human proinsulin precursor; the precursors of the aspart islets were precipitated by addition of 20mM Zn ions, centrifuged and the pellet was dissolved in 100ml of 50mM Tris. Detecting the concentration of the sample by high performance liquid chromatography, calculating the protein content to be 1g, adjusting the pH value to be 8.8 by hydrochloric acid, adding 0.2mg of lysyl endopeptidase (Lys-C) according to the mass ratio of the enzyme to the protein of 1:5000, and stirring and carrying out enzyme digestion at 30 ℃. After 16 hours, a sample is taken for RP-HPLC analysis, the purity of the target protein after enzyme digestion is 90.3 percent, and the enzyme digestion conversion rate is about 87.1 percent.

Comparative example 3 two-step purification of recombinant human insulin precursor and cleavage after precipitation

Centrifuging the pichia pastoris fermentation liquor, and performing copper ion chelation chromatography and SP cation exchange chromatography on the supernatant to obtain a purified human proinsulin precursor; and adding 20mM Zn ions to precipitate the precursors of the insulin aspart, centrifuging, and dissolving the precipitate by using 100ml of 25mM borax. Detecting the concentration of the sample by high performance liquid chromatography, calculating the protein content to be 1g, adjusting the pH value to be 8.8 by sodium hydroxide, adding 0.2mg of lysyl endopeptidase (Lys-C) according to the mass ratio of the enzyme to the protein of 1:5000, and stirring and carrying out enzyme digestion at 30 ℃. After 16 hours, a sample is taken for RP-HPLC analysis, the purity of the target protein after enzyme digestion is 95.6 percent, and the enzyme digestion conversion rate is about 92.1 percent.

Comparative example 4 two-step purification of recombinant human insulin precursor and cleavage after precipitation

Centrifuging the pichia pastoris fermentation liquor, and performing copper ion chelation chromatography and SP cation exchange chromatography on the supernatant to obtain a purified human proinsulin precursor; and adding 20mM Zn ions to precipitate the precursors of the insulin aspart, centrifuging, and dissolving the precipitate by using 100ml of 25mM borax. Detecting the concentration of the sample by high performance liquid chromatography, calculating the protein content to be 1g, adjusting the pH value to be 9.5 by sodium hydroxide, adding 0.2mg of lysyl endopeptidase (Lys-C) according to the mass ratio of the enzyme to the protein of 1:5000, and stirring and carrying out enzyme digestion at 25 ℃. Sampling for 18h, and performing RP-HPLC analysis, wherein the purity of the target protein after enzyme digestion is 98.4%, and the enzyme digestion conversion rate is about 95.3%.

Comparative example 5 two-step purification of recombinant human insulin precursor and cleavage after precipitation

Centrifuging the pichia pastoris fermentation liquor, and performing copper ion chelation chromatography and SP cation exchange chromatography on the supernatant to obtain a purified human proinsulin precursor; and adding 20mM Zn ions to precipitate the precursors of the insulin aspart, centrifuging, and dissolving the precipitate by using 100ml of 25mM borax. Detecting the concentration of the sample by high performance liquid chromatography, calculating the protein content to be 1g, adjusting the pH value to be 9.5 by sodium hydroxide, adding 0.2mg of lysyl endopeptidase (Lys-C) according to the mass ratio of the enzyme to the protein of 1:5000, and stirring and carrying out enzyme digestion at 20 ℃. And sampling for 24h, and performing RP-HPLC analysis, wherein the purity of the target protein after enzyme digestion is 98.1%, and the enzyme digestion conversion rate is about 94.7%.