A method for efficiently expressing ovine growth hormone

Abstract

The application discloses a method for efficiently expressing sheep growth hormone. The application provides a method for preparing sheep growth hormone, which comprises the following steps: 1) preparing a recombinant vector for expressing sheep growth hormone; the recombinant vector contains a Trx label, a thrombin cleavage site and sheep growth hormone; 2) co-transferring the recombinant vector and a vector for expressing a molecular chaperone into E. coli to obtain a recombinant bacterium for expressing a sheep growth hormone fusion protein; 3) inducing the recombinant bacterium to express the sheep growth hormone to obtain sheep growth hormone with a Trx label; and 4) cleaving the sheep growth hormone with the Trx label by thrombin to obtain sheep growth hormone; the method of the application significantly improves the soluble expression of growth hormone, and the soluble expression of sheep growth hormone is greatly improved by changing expression conditions and co-expressing with a molecular chaperone.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for efficiently expressing sheep growth hormone. Background Technology

[0002] Growth hormone (GH) is a protein hormone synthesized, stored, and secreted by the acidophilic cells of the anterior pituitary gland in animals. Its structure consists of a peptide chain of approximately 186-191 amino acids, with a molecular weight of approximately 21-22 kDa. Growth hormone not only increases body weight and height but also participates in the regulation of carbohydrate, protein, and lipid metabolism. In animals (e.g., cattle), growth hormone is mainly used to increase milk production and lean meat percentage, and improve feed conversion ratio.

[0003] There are two main ways to obtain growth hormone: the first is direct extraction from the pituitary gland of animals or humans; the second is through genetic engineering. The first method was used a long time ago when technology was relatively limited. At that time, human growth hormone content only accounted for 4%-8% of the pituitary gland's dry weight, and typically one pituitary gland could yield 3-5 mg of growth hormone. In the 1970s, with the development of technology, especially genetic engineering, the second method was commonly used to produce large quantities of recombinant growth hormone to replace natural growth hormone. In 1985, the US FDA approved recombinant human growth hormone produced through genetic engineering.

[0004] From the late 20th century to the early 21st century, scientists used various methods to express and purify sheep growth hormone, primarily using prokaryotic expression. However, low expression levels and inclusion bodies remained unresolved, and the activity of these inclusion bodies after renaturation could not be guaranteed. The presence of inclusion bodies was mainly due to the strain expressing the hormone at too high an intensity, making it impossible to ensure the correct folding of the synthesized peptide chains, thus forming inactive inclusion bodies.

[0005] Therefore, how to efficiently prepare sheep growth hormone has become a problem worth exploring. Summary of the Invention

[0006] One object of the present invention is to provide a method for improving the expression of soluble prokaryotic growth hormone in sheep.

[0007] The method provided by this invention includes the following steps:

[0008] 1) The recombinant vector and the vector expressing the molecular chaperone were co-transformed into Escherichia coli to obtain recombinant bacteria;

[0009] The recombinant vector contains a Trx tag and sheep growth hormone;

[0010] 2) Inducing the expression of the recombinant bacteria to enhance the soluble expression of sheep growth hormone;

[0011] The sheep growth hormone mentioned, specifically taking sheep growth hormone as an example, is any one of the following:

[0012] a) Proteins including amino acid sequences from positions 133 to 322 of sequence 2;

[0013] b) Proteins with tags at the ends of the protein sequences shown in a);

[0014] c) The protein shown at positions 125-322 of sequence 2;

[0015] d) and any of a)-c) have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology and have the same function.

[0016] The inducing agent used in the above induction was IPTG.

[0017] The aforementioned improvement in the soluble prokaryotic expression of sheep growth hormone is reflected in the fact that the recombinant bacteria prepared with vectors expressing molecular chaperones have a higher level of soluble sheep growth hormone expression after induction than the recombinant bacteria prepared with vectors without expressing molecular chaperones.

[0018] Another object of the present invention is to provide a method for preparing sheep growth hormone.

[0019] The method provided by this invention includes the following steps:

[0020] 1) Preparation of a recombinant vector expressing sheep growth hormone;

[0021] This recombinant vector contains a Trx tag and sheep growth hormone;

[0022] 2) The recombinant vector and the vector expressing the molecular chaperone were co-transformed into Escherichia coli to obtain recombinant bacteria; then the recombinant bacteria were induced to express to obtain sheep growth hormone with the Trx tag;

[0023] 3) Remove the Trx tag from the sheep growth hormone with the Trx tag to obtain sheep growth hormone;

[0024] The sheep growth hormone specifically refers to sheep growth hormone, specifically any of the following:

[0025] a) Proteins including amino acid sequences from positions 133 to 322 of sequence 2;

[0026] b) Proteins with tags at the ends of the protein sequences shown in a);

[0027] c) The protein shown at positions 125-322 of sequence 2;

[0028] d) and any of a)-c) have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology and have the same function.

[0029] In the above method, the recombinant vector is obtained by inserting a nucleic acid molecule containing the sheep growth hormone encoding gene into an expression vector with a Trx tag;

[0030] The nucleic acid molecule containing the sheep growth hormone encoding gene further includes a restriction enzyme recognition site, a thrombin cleavage site, a purification tag, the sheep growth hormone encoding gene, and another restriction enzyme recognition site.

[0031] Furthermore, the restriction enzyme recognition site and the other restriction enzyme recognition site are MScI and BamHI, respectively;

[0032] Alternatively, the purification label in an embodiment of the present invention may be a His label;

[0033] Alternatively, the nucleic acid molecule containing the sheep growth hormone encoding gene is sequence 1.

[0034] In the above method, the expression vector with the Trx tag is derived from pET-32a(+).

[0035] Alternatively, the expression vector with the Trx tag is a linear vector backbone obtained by double digestion of the pET-32a(+) vector with MSc1 and BamH1.

[0036] In the above method, the vector for expressing the molecular chaperone is pGro7.

[0037] In the above method, the conditions for inducing expression are: 0.025mM-1mM IPTG, induction culture at 20℃-37℃ for 20 hours.

[0038] In the above method, the removal of the Trx tag from the sheep growth hormone with the Trx tag is achieved by thrombin enzymatic digestion of the sheep growth hormone with the Trx tag.

[0039] Alternatively, the enzyme digestion conditions are 0-4℃ for 8 minutes.

[0040] The above method further includes the following steps:

[0041] Before or after thrombin digestion, the pre-digestion product or the post-digestion product is purified using the purification tag.

[0042] Alternatively, the application of the above-mentioned expression vectors with the Trx tag and the above-mentioned expression molecular chaperone vectors in improving the soluble prokaryotic expression of sheep growth hormone is also within the scope of protection of this invention.

[0043] Alternatively, the application of the above-mentioned expression vectors with Trx tags, expression vectors for molecular chaperones, and thrombin in the preparation of sheep growth hormone or in increasing sheep growth hormone production is also within the scope of protection of this invention.

[0044] Alternatively, the application of the above-mentioned sheep growth hormone, expression vector with Trx tag, expression molecular chaperone vector and thrombin in the preparation of sheep growth hormone or to increase the yield of sheep growth hormone is also within the scope of protection of this invention.

[0045] Alternatively, the application of the above-mentioned sheep growth hormone, expression vector, and expression molecular chaperone vector in the preparation of recombinant bacteria expressing sheep growth hormone is also within the scope of protection of this invention.

[0046] Alternatively, the application of pET-32a(+) vector and pGro7 vector after double digestion with MSc1 and BamH1 in improving the soluble expression of sheep growth hormone is also within the scope of protection of this invention;

[0047] Alternatively, the application of the pET-32a(+) vector, pGro7 vector, and thrombin after double digestion with MSc1 and BamH1 in the preparation of sheep growth hormone or to increase sheep growth hormone production is also within the scope of protection of this invention.

[0048] Alternatively, the aforementioned recombinant vectors or recombinant bacteria are also within the scope of protection of this invention;

[0049] Alternatively, the application of the recombinant vector or recombinant bacteria in the preparation of sheep growth hormone or in increasing the yield of sheep growth hormone is also within the scope of protection of this invention.

[0050] The main technical solution of this invention utilizes TransB(DE3) for expression. By modifying the pET-32a(+) vector, it retains the Trx-Tag and Thrombin restriction sites. The Trx-Tag facilitates the formation of correctly folded proteins containing disulfide bonds, increasing protein solubility. The thrombin restriction site is removed after fusion expression. Furthermore, another expression chaperone vector, pGro7, is simultaneously introduced during transformation. The GroEL / GroES expressed in this vector forms a hydrophobic structure, allowing for correct folding of the peptide chain after synthesis. In summary, soluble expression of sheep growth hormone can be achieved.

[0051] The modified pET-32a(+) vector was created by removing redundant sequences on pET-32a(+) using restriction endonucleases MSCI and BamHI. The protease cleavage sites and His tag sites were rearranged using PCR for later purification. The original T7 promoter of the vector was used to initiate the expression of the target gene. In order to prevent the fusion tag from damaging the biological function of the recombinant protein and interfering with its natural structure or immunogenicity, the Trx tag was removed using inexpensive thrombin. The conventional vector achieved high-efficiency expression of sheep growth hormone in Escherichia coli, and increased the expression level of soluble sheep growth hormone.

[0052] Experiments have shown that this invention has the following advantages:

[0053] (1) Sequence modification: Based on codon preference, the recombinant sheep growth hormone nucleotide sequence was redesigned to make oGH easier to express.

[0054] (2) Vector modification: The sequence of the fusion protein Trx-hisogh was designed to facilitate the removal of the Trx tag in the later stage, and unnecessary redundant sequences on pET-32a(+) were removed. Moreover, the original T7 promoter of the vector was used to start the expression of the target gene. Through all the above modifications, the high-efficiency expression of sheep growth hormone in Escherichia coli TransB(DE3) was successfully achieved using the conventional vector pET-32a(+).

[0055] (3) Significantly improve the soluble expression of growth hormone. By changing the expression conditions and co-expressing with molecular chaperones, the soluble expression of sheep growth hormone was greatly improved.

[0056] (4) The initial vector used in this invention is common, the bacterial strains are common, and the induction expression parameters are simple to control, so the cost is low, it is easy to implement, the method is simple, it is easy for technicians to master, and it is easy to promote. Attached Figure Description

[0057] Figure 1 The engineered bacteria BAC ID NO:1 was induced to express.

[0058] Figure 2 The solubility of the protein after induction expression of engineered bacteria BAC ID NO:1 was detected.

[0059] Figure 3 To optimize the IPTG concentration for induction of BAC ID NO:1 expression in engineered bacteria.

[0060] Figure 4 Optimization of the induction temperature for BAC ID NO:1 expression in engineered bacteria.

[0061] Figure 5Detection of protein solubility after optimization of expression conditions for engineered bacteria BAC ID NO:1.

[0062] Figure 6 Detection of the solubility of the protein induced by engineered bacteria BAC ID NO:2.

[0063] Figure 7 Optimization of enzyme digestion conditions for the fusion protein Trx-hisogh.

[0064] Figure 8 The fusion protein Trx-hisogh was further purified after enzymatic digestion to obtain the final crude protein hisogh.

[0065] Figure 9 The relative molecular weight of the final crude protein was determined by Western blot detection and LC-MS.

[0066] Figure 10 The structures of the fusion protein Trx-hisogh and the enzyme-digested hisogh are shown. Detailed Implementation

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0068] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0069] The following examples use sheep growth hormone as an example.

[0070] The specific parameters of LC-MS in the following embodiments are as follows:

[0071] Experimental instruments and materials

[0072] Exactive Plus EMR (Thermo Fisher Scientific, US), HPLC L-3120 (RIGOL, CN), ACN (Fisher), FA (Fisher), deionized water

[0073] Exactive Plus EMR Mass Spectrometry

[0074] Acquisition mode: Positive ion. Acquisition range: m / z 1000~m / z 9000; HCD: 35.0 eV; Resolution: 17500; Microscan: 10; Mass lock: Off; Maximum ion implantation time: 200 ms; Sheath gas flow rate: 30; Auxiliary gas flow rate: 10; Purge flow rate: 0; Spray voltage: 3.5 kV; Capillary temperature: 350℃; Auxiliary gas heating temperature: 310℃; S-prism RF setting: 200. Data processing software: ProteinDeconvolution (v4)

[0075] HPLC (RIGOL, L-3120)

[0076] Chromatographic column: Agilent C3 column (3.5 μm, 150 mm × 4.6 mm). Column temperature: 40℃. Mobile phase A: H2O (containing 2% ACN, 0.1% FA); Mobile phase B: ACN (containing 10% H2O, 0.1% FA). Flow rate: 0.25 ml / min. Sample loading volume: 50 μl. Detection wavelength: 280 nm. Gradients are shown in Table 1 below:

[0077] Table 1

[0078]

[0079] Experimental method: Take a certain amount of sample, add 6 μl of 5% FA and mix well, centrifuge at 10000g for 5 min, and take the supernatant for LC-MS detection.

[0080] Example 1: A method for efficient expression of sheep growth hormone using Escherichia coli.

[0081] I. Preparation of recombinant bacteria expressing sheep growth hormone

[0082] 1. Obtaining the target gene sheep growth hormone

[0083] Based on the known natural amino acid sequence of sheep growth hormone oGH (NCBI: NM_001009315.3, derived from sheep), and following the codon preference of E. coli, the coding sequence of oGH (sequence 1, positions 59-631) was designed without altering the amino acid sequence. Relevant sequences were then added to both ends of this coding sequence to obtain the following target gene, sheep growth hormone: restriction endonuclease site 1 (MscI) - thrombin cleavage site sequence - His tag sequence - coding sequence of oGH - restriction endonuclease site 2 (BamHI), named the target gene sheep growth hormone.

[0084] The nucleotide sequence of the target gene, sheep growth hormone, is shown in Sequence 1 of the sequence listing. In Sequence 1, positions 1-6 are the MScI restriction site, positions 7-22 are the linker, positions 23-40 are the thrombin restriction site, positions 41-58 are the His tag sequence, positions 59-628 are the coding sequence for oGH, positions 629-631 are the stop codon, and positions 632-637 are the BamHI restriction site.

[0085] 2. Modification of pET-32a(+) vector

[0086] pET-32a(+)(Novagen, 69015-3) was digested with both MSc1 and BamH1 enzymes, and the 5747bp fragment was collected, which is the linearized pET-32a-Trx.

[0087] The linearized pET-32a-Trx removed the S-Tag tag, the original Thrombin, and the redundant sequence of the enterokinase site on pET-32a(+), while retaining the Trx-Tag. The Trx-Tag facilitates the formation of correctly folded proteins containing disulfide bonds, increasing protein solubility. The thrombin cleavage site was rearranged and added in sequence 1 to facilitate the later cleavage of the Trx-tag and protein purification.

[0088] 3. Preparation of recombinant expression vector pET-32a-Trx-his-oGH

[0089] The target gene sheep growth hormone prepared in step 1 above was ligated with the linearized pET-32a-Trx prepared in step 2 above to obtain the recombinant expression vector pET-32a-Trx-his-oGH.

[0090] This recombinant expression vector expresses the recombinant sheep growth hormone Trx-hisogh (structure shown in Figure 1) with the Trx tag. Figure 10 (See the image above). The amino acid sequence of the recombinant sheep growth hormone Trx-hisogh with the Trx tag is sequence 2. In sequence 2, positions 1-109 are the Trx tag, positions 110-126 are the linking sequence and thrombin recognition sequence, positions 127-132 are the his tag, and positions 133-322 are the oGH protein.

[0091] The recombinant expression vector pET-32a-Trx-his-oGH is obtained by replacing the fragment between the MSc1 and BamH1 restriction sites in the linearized pET-32a-Trx with the target gene sheep growth hormone shown in sequence 1.

[0092] 4. Preparation of recombinant bacteria expressing recombinant proteins

[0093] 1) Preparation of recombinant strain BAC ID NO: 1

[0094] The recombinant expression vector pET-32a-Trx-his-oGH prepared in step 3 above was transformed into Escherichia coli TransB(DE3). Positive clones were selected on LB plates containing ampicillin (100ug / mL) and identified by double digestion of plasmid MSCI and BamHI (631bp was identified as positive). Sequencing confirmed that the clone was successful and the positive recombinant bacterium BACID NO:1 was obtained.

[0095] The positive recombinant bacterium BAC ID NO:1 is a TransB(DE3) engineered bacterium containing pET-32a-Trx-his-oGH.

[0096] 2) Preparation of recombinant strain BAC ID NO: 2

[0097] The recombinant strain BAC ID NO:1 obtained in 1) above was prepared as competent cells. The molecular chaperone expression vector pGro7 (takara, 3340) was transformed into the engineered strain BAC ID NO:1. Positive clones were selected on LB plates containing ampicillin (100ug / mL) and chloramphenicol (20ug / mL) to obtain the engineered strain BAC ID NO:2.

[0098] II. Induced expression of recombinant sheep growth hormone with a Trx tag derived from engineered bacteria BAC ID NO:1

[0099] 1. Induced expression of recombinant sheep growth hormone Trx-hisogh

[0100] The engineered bacteria BAC ID NO:1 prepared above was cultured in LB medium for 10 hours to obtain a bacterial suspension. The bacterial suspension was then added to LB medium at a ratio of 1:100 and cultured at 37 degrees Celsius for 3 hours. A portion of the bacterial suspension was taken out to collect the bacterial cells (referred to as the whole bacterial sample of BAC ID NO:1 before induction). IPTG (final IPTG concentration of 1 mM) was added to the remaining bacterial suspension, and the culture was induced at 37 degrees Celsius for 4 hours. The bacterial cells were collected by centrifugation at 5000g for 10 minutes to obtain the whole bacterial sample of BAC ID NO:1 after induction. The sample was then detected by SDS-PAGE gel electrophoresis.

[0101] The results are as follows Figure 1 As shown, 1 is the whole bacterial sample of BAC ID NO:1 before induction, and 2 is the whole bacterial sample of BAC ID NO:1 after induction. The engineered strain BAC ID NO:1 expresses approximately 34KD of recombinant sheep growth hormone Trx-hisogh (sequence 2) with a Trx tag.

[0102] 2. Protein detection in recombinant sheep growth hormone cells

[0103] The whole bacterial sample obtained from BAC ID NO:1 after induction was resuspended in lysis buffer (i.e., solution A below) and then sonicated (200W ice bath, sonication for 2 seconds, pause for 4 seconds). The sample was then centrifuged at 17,000×g for 30 minutes using a low-temperature ultracentrifuge. The supernatant and precipitate were collected and the supernatant (potential target protein) was purified and enriched using a nickel column.

[0104] Prepare solution A (pH 8.0): 50 mM Tris, 500 mM NaCl, 20 mM imidazole, 10% glycerol, and the remainder is water.

[0105] SDS-PAGE gel electrophoresis was performed, and the results are as follows: Figure 2 As shown, 1 is the whole bacterial sample before BAC ID NO:1 induction, 2 is the whole bacterial sample after BAC ID NO:1 induction, and 3 is the eluted sample of the ultrasonic supernatant after BAC ID NO:1 induction enriched by nickel column. It can be seen that there is a small amount of recombinant sheep growth hormone Trx-hisogh (34KD) with Trx tag in the supernatant of engineered bacteria BAC ID NO:1, indicating that the soluble protein of this target protein is very small.

[0106] 3. Exploring the concentration of inducer in induced expression

[0107] Based on the results in section 2, the expression conditions for engineered bacteria BAC ID NO: 1 were explored:

[0108] The engineered bacteria BAC ID NO:1 prepared above was cultured in LB medium for 10 h to obtain a bacterial suspension. The bacterial suspension was then added to LB medium at an inoculation ratio of 1:100 and cultured at 37 degrees Celsius for 3 h. IPTG was then added, with final IPTG concentrations of 0.025 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM, respectively. The culture was induced at 37 degrees Celsius for 4 h, and the bacterial cells were collected by centrifugation at 5000g for 10 minutes and detected by SDS-PAGE gel electrophoresis.

[0109] The results are as follows Figure 3 As shown, 1-6 are whole bacterial samples of engineered strain BAC ID NO:1 before induction, and samples of engineered strain BAC ID NO:1 after induction with 0.025mM, 0.05mM, 0.1mM, 0.5mM, and 1mM, respectively. It can be seen that the IPTG concentration does not affect the expression of recombinant sheep growth hormone with the Trx tag. Therefore, the minimum IPTG concentration of 0.025mM was selected in the later stage.

[0110] 4. Exploring the temperature in induced expression

[0111] Based on the results in section 3, the expression conditions for engineered bacteria BAC ID NO: 1 were explored:

[0112] The engineered bacteria BAC ID NO:1 prepared above was cultured in LB medium for 10 h to obtain a bacterial suspension. The bacterial suspension was then added to LB medium at a ratio of 1:100 and cultured at 37°C for 3 h. IPTG was added to a final concentration of 1 mM, and the culture temperature was adjusted to 20°C, 25°C, 30°C, and 37°C for 20 h, 15 h, 10 h, and 4 h, respectively. The bacterial cells were collected by centrifugation at 5000g for 10 minutes, resuspended in lysis buffer, and then sonicated (ice bath 200W, sonication for 2 s, pause for 4 s). The cells were then centrifuged at 17,000×g for 30 minutes using a low-temperature ultracentrifuge, collected, and detected by SDS-PAGE gel electrophoresis.

[0113] The results are as follows Figure 4 As shown, 1-5 are whole bacterial samples of engineered strain BAC ID NO:1 before induction and whole bacterial samples of engineered strain BAC ID NO:1 after induction at 20℃, 25℃, 30℃ and 37℃ respectively. It can be seen that temperature does not affect the expression of recombinant sheep growth hormone with Trx tag. Therefore, the minimum temperature of 20℃ was selected in the later stage.

[0114] 5. Induced expression of recombinant bacterial BAC ID NO:1

[0115] Based on the results of 3 and 4, the experimental results of 3 and 4 were combined. The engineered bacteria BAC ID NO:1 prepared above was cultured in LB medium for 10 h to obtain a bacterial suspension. The bacterial suspension was then added to LB medium at a ratio of 1:100 and cultured at 37 degrees Celsius for 3 h. IPTG was added to a final concentration of 0.025 mM and cultured at 20 degrees Celsius for 20 h. The bacterial cells were collected by centrifugation at 5000g for 10 min, resuspended in lysis buffer, and then sonicated (ice bath 200W, sonication for 2 s, pause for 4 s). The cells were then centrifuged at 17,000×g for 30 min using a low-temperature ultracentrifuge. The supernatant and precipitate were collected, and the supernatant was purified and enriched using a nickel column for SDS-PAGE gel electrophoresis detection.

[0116] The results are as follows Figure 5 As shown, 1-6 represent: 1. Whole bacterial sample before BAC ID NO: 1 induction; 2. Whole bacterial sample after BAC ID NO: 1 induction; 3. Ultrasonic precipitation sample after BAC ID NO: 1 induction; 4. Ultrasonic supernatant sample after BAC ID NO: 1 induction; 5. Nickel column eluted sample after BAC ID NO: 1 induction; 6. Nickel column eluted sample after BAC ID NO: 1 induction. It can be seen that... Figure 2Compared with the results of nickel-permeable column chromatography, the content of recombinant sheep growth hormone Trx-hisogh with the Trx tag in the supernatant of engineered bacteria BAC ID NO:1 did not change, indicating that the optimized induction conditions did not change the content of soluble Trx-hisogh protein, which still exists in the form of inclusion bodies in the precipitate or bacterial cells.

[0117] III. Inducible expression of recombinant sheep growth hormone with a Trx tag derived from engineered bacteria BAC ID NO:2

[0118] Since BAC ID NO:2 originates from BAC ID NO:1, and the induction expression conditions for BAC ID NO:1 are applicable to BAC ID NO:2, the induction expression conditions in section 5 of the above-mentioned second part are used to induce the culture of BAC ID NO:2, as follows:

[0119] 1. Induction culture

[0120] The engineered bacteria BAC ID NO:2 was cultured in LB medium for 10 hours to obtain a bacterial suspension. The bacterial suspension was then added to LB medium at a ratio of 1:100 and cultured at 37 degrees Celsius for 3 hours. IPTG was added to the medium at a final concentration of 0.025 mM and cultured at 20 degrees Celsius for 20 hours. The bacterial cells were then collected by centrifugation at 5000g for 10 minutes.

[0121] 2. Broken bacterial cells

[0122] The bacterial precipitate from step 1 above was added to solution A at a volume ratio of 1:5 for resuspending. The mixture was then sonicated in an ice bath (200W, 2s sonication, 4s intermittent). It was then centrifuged at 17,000×g for 30 minutes using a low-temperature ultracentrifuge, and the supernatant and precipitate were collected.

[0123] 3. Affinity chromatography

[0124] The supernatant obtained in step 2 above was loaded onto a nickel column and fully bound to the nickel column at 4°C. The column was then washed with 2 times the volume of solution A and eluted with 5 times the volume of solution B. The eluent (containing recombinant sheep growth hormone Trx-hisogh with the Trx tag) was collected.

[0125] Prepare solution A (pH 8.0): 50mM Tris, 500mM NaCl, 20mM imidazole, 10% (volume percentage) glycerol, balance water;

[0126] Prepare solution B (pH 9.0): 25mM Tris, 250mM NaCl, 200mM imidazole, 5% (v / v) glycerol, balance water;

[0127] SDS-PAGE gel electrophoresis was performed, and the results are as follows: Figure 6As shown, 1-4 are whole bacterial samples of BAC ID NO:2 before induction, whole bacterial samples of BAC ID NO:2 after induction, ultrasonic precipitation samples of BAC ID NO:2 after induction, and ultrasonic supernatant samples of BAC ID NO:2 after induction, respectively; 5-7 are ultrasonic supernatant samples of BAC ID NO:2 after induction eluted by nickel column, respectively; 8-9 are ultrasonic supernatant samples of BAC ID NO:1 after induction under the same conditions eluted by nickel column, respectively. It can be seen that compared with BAC ID NO:1, the content of recombinant sheep growth hormone Trx-hisogh with the Trx tag in the supernatant of engineered bacteria BAC ID NO:2 is significantly increased, indicating that the soluble protein content of recombinant sheep growth hormone Trx-hisogh with the Trx tag in engineered bacteria BAC ID NO:2 is high.

[0128] According to Bradford assay, the Trx-hisogh protein content in the elution of recombinant BAC ID NO:2 after expression and purification was approximately 7.5 mg / mL, totaling 38 mL; while the Trx-hisogh protein content in the elution of recombinant BAC ID NO:1 after expression and purification was approximately 760 μg / mL.

[0129] Therefore, the engineered strain BAC ID NO:2 has a higher content of the target protein Trx-hisogh than the engineered strain BAC ID NO:1.

[0130] IV. Obtaining Recombinant Sheep Growth Hormone via Enzymatic Digestion

[0131] To prevent the fusion tag from impairing the biological function of the recombinant protein, interfering with its natural structure, or causing immunogenicity, the Trx tag was removed using inexpensive thrombin. Experiments showed that 0.005 IU was used to cut 1 μg of protein.

[0132] The specific method is as follows:

[0133] 1. Affinity chromatography

[0134] The eluents of the three engineered bacteria BAC ID NO: 2 (containing recombinant sheep growth hormone Trx-hisogh with the Trx tag) were loaded onto a nickel column and fully bound to the nickel column at 4°C. The column was washed with 2 times the volume of solution A and eluted with 5 times the volume of solution B. The eluent (containing recombinant sheep growth hormone hisogh) was collected.

[0135] 2. Ultrafiltration

[0136] Using a Millipore ultrafilter with a molecular weight cutoff of 10 kDa, the function is to remove small molecule impurities and salts and concentrate proteins.

[0137] Take the eluent obtained in step 1 above, dilute it with 10 times the volume of solution C, perform ultrafiltration, repeat 4 times, and obtain a purified recombinant sheep growth hormone Trx-hisogh solution with the Trx tag.

[0138] 3. Enzyme digestion

[0139] Collect 30 mL of purified Trx-tagged recombinant sheep growth hormone Trx-hisogh solution (protein content 7.5 mg / mL), add thrombin (Anhui Jingke Biotechnology Co., Ltd., JK-034B, specific activity 2000 U / mg) at a rate of 0.005 IU / ug protein, and terminate enzyme digestion with PMSF at 4°C.

[0140] Sample collection (of the enzyme digestion product) begins 2 minutes after cutting (starting from the time thrombin is added), and samples are collected every 2 minutes.

[0141] SDS-PAGE gel electrophoresis was used to analyze samples at different cutting times, and the results are as follows: Figure 7 As shown: 1 is the original sample before enzyme digestion (recombinant sheep growth hormone with the Trx tag without the addition of thrombin), and 2-12 are the samples after enzyme digestion for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 minutes, respectively. It can be seen that 8 minutes of enzyme digestion can obtain satisfactory results and 22KD recombinant sheep growth hormone hisogh. Therefore, an enzyme digestion time of 8 minutes was selected.

[0142] The amino acid sequence of recombinant sheep growth hormone hisogh is sequence 2, positions 125-322.

[0143] V. Purification of recombinant sheep growth hormone Hisogh

[0144] Prepare solution C (pH 9.5): 20 mM Tris, balance water;

[0145] 1. Affinity chromatography

[0146] The enzyme digestion products obtained from the above four steps were loaded onto a nickel column after 8 hours and fully bound to the nickel column at 4°C. The column was then washed with 2 times the volume of solution A and eluted with 5 times the volume of solution B. The eluent (containing recombinant sheep growth hormone hisogh) was collected.

[0147] SDS-PAGE analysis of the eluent yielded the following results: Figure 8 As shown, 1 is the eluent obtained from the above three (containing recombinant sheep growth hormone Trx-hisogh with the Trx tag), and 2 is the eluent (containing recombinant sheep growth hormone hisogh). It can be seen that a crude extract containing approximately 22KD of recombinant sheep growth hormone hisogh was obtained.

[0148] Recombinant sheep growth hormone hisogh ( Figure 10 The amino acid sequence shown in the image below is positions 125-322 of sequence 2.

[0149] 2. Ultrafiltration

[0150] Using a Millipore ultrafilter with a molecular weight cutoff of 10 kDa, the function is to remove small molecule impurities and salts and concentrate proteins.

[0151] Take the eluent obtained in step 1 above, dilute it with 10 times the volume of solution C, perform ultrafiltration, and repeat 4 times to obtain the purified recombinant sheep growth hormone hisogh solution.

[0152] Bradford's analysis and calculation showed that the purified recombinant sheep growth hormone hisogh soluble protein yield prepared by engineered bacteria BAC ID NO:2 was 21.9 mg / L, where L is the volume of the bacterial culture of BAC ID NO:2 after 20 hours of IPTG induction.

[0153] Bradford's analysis and calculation showed that the purified recombinant sheep growth hormone hisogh soluble protein yield prepared by engineered strain BAC ID NO:1 using the same method was 2.4 mg / L, where L is the volume of the bacterial culture of BAC ID NO:1 after 20 hours of IPTG induction.

[0154] 3. Western Bolt and mass spectrometry detection

[0155] The purified recombinant sheep growth hormone hisogh obtained in step 2 above was detected by Western bolus assay using a His-tagged primary antibody (Sangon Biotech (Shanghai) Co., Ltd., catalog number: D191001), and the molecular weight of the purified recombinant sheep growth hormone hisogh was detected by LC-MS.

[0156] Western Bolt test results are as follows: Figure 9 As shown in Figure a, 1 is the BSA blank control, and 2-4 are the purified recombinant sheep growth hormone hisogh obtained from three repeated experiments; it can be seen that approximately 22KD of recombinant sheep growth hormone (positions 125-322 of sequence 2) was obtained.

[0157] LC-MS detection results are as follows Figure 9 As shown in b, it can be seen that 22749.22 Da of recombinant sheep growth hormone was obtained. SEQUENCE LISTING <110> China Agricultural University <120> A method for efficiently expressing sheep growth hormone <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 637 <212> DNA <213> Artificial sequence <400> 1 tggccatggt ggaggtggcg gtctggtgcc acgcggttct caccatcatc atcatcattt 60 tccggccatg agtctgagcg gtctgtttgc caatgccgtt ctgcgcgcac agcatctgca 120 tcagctggca gcagatacct ttaaagaatt tgaacgtacc tatatcccgg aaggtcagcg 180 ctatagtatt cagaataccc aggttgcatt ttgttttagc gaaaccattc cggccccgac 240 cggtaaaaat gaagcacagc agaaaagtga tctggaactg ctgcgcatta gtctgctgct 300 gattcagagc tggctgggtc cgctgcagtt tctgagccgc gtgtttacca atagcctggt 360 gtttggcacc agcgatcgtg tgtatgaaaa actgaaagat ctggaagaag gtattctggc 420 actgatgcgc gaactggaag atgttacccc gcgcgccggt cagattctga aacagaccta 480 tgataaattt gacaccaata tgcgtagcga tgatgcactg ctgaaaaatt atggcctgct 540 gagttgtttt cgtaaagatc tgcataaaac cgaaacctat ctgcgtgtga tgaaatgtcg 600 ccgctttggc gaagccagtt gcgcctttta aggatcc 637 <210> 2 <211> 322 <212> PRT <213> Artificial sequence <400> 2 Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly 100 105 110 Ser Gly His Gly Gly Gly Gly Gly Leu Val Pro Arg Gly Ser His His 115 120 125 His His His His Phe Pro Ala Met Ser Leu Ser Gly Leu Phe Ala Asn 130 135 140 Ala Val Leu Arg Ala Gln His Leu His Gln Leu Ala Ala Asp Thr Phe 145 150 155 160 Lys Glu Phe Glu Arg Thr Tyr Ile Pro Glu Gly Gln Arg Tyr Ser Ile 165 170 175 Gln Asn Thr Gln Val Ala Phe Cys Phe Ser Glu Thr Ile Pro Ala Pro 180 185 190 Thr Gly Lys Asn Glu Ala Gln Gln Lys Ser Asp Leu Glu Leu Leu Arg 195 200 205 Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Gly Pro Leu Gln Phe Leu 210 215 220 Ser Arg Val Phe Thr Asn Ser Leu Val Phe Gly Thr Ser Asp Arg Val 225 230 235 240 Tyr Glu Lys Leu Lys Asp Leu Glu Glu Gly Ile Leu Ala Leu Met Arg 245 250 255 Glu Leu Glu Asp Val Thr Pro Arg Ala Gly Gln Ile Leu Lys Gln Thr 260 265 270 Tyr Asp Lys Phe Asp Thr Asn Met Arg Ser Asp Asp Ala Leu Leu Lys 275 280 285 Asn Tyr Gly Leu Ser Cys Phe Arg Lys Asp Leu His Thr Glu 290,295,300 Thr Tyr Leu Arg Will Met Cys Arg Arg Phe Gly Glu Ala Ser Cys 305 310 315 320 Ala Phe

Claims

1. A method for improving the expression of soluble growth hormone in sheep prokaryotes, comprising the following steps: 1) The recombinant vector and the vector expressing the molecular chaperone were co-transformed into Escherichia coli to obtain recombinant bacteria; The recombinant vector contains a Trx tag and sheep growth hormone; 2) Inducing the expression of the recombinant bacteria to enhance the soluble expression of sheep growth hormone; The recombinant vector is obtained by inserting a nucleic acid molecule containing the sheep growth hormone encoding gene into an expression vector with a Trx tag; The expression vector with the Trx tag is a vector that... Msc 1 and BamH 1. The linear vector backbone obtained after double digestion of pET-32a(+) vector; The vector for expressing the molecular chaperone is pGro7; The nucleic acid molecule containing the sheep growth hormone encoding gene includes a restriction enzyme recognition site, a thrombin cleavage site, a purification tag, the sheep growth hormone encoding gene, and another restriction enzyme recognition site. The restriction enzyme recognition site and the other restriction enzyme recognition site are MScI and BamHI, respectively; The purification tag is a His tag; The nucleic acid molecule containing the sheep growth hormone encoding gene is sequence 1.

2. A method for preparing sheep growth hormone, comprising the following steps: 1) The recombinant vector and the vector expressing the molecular chaperone were co-transformed into Escherichia coli to obtain recombinant bacteria; The recombinant vector contains a Trx tag and sheep growth hormone; 2) Induce expression of the recombinant bacteria to obtain sheep growth hormone with the Trx tag; 3) Remove the Trx tag from the sheep growth hormone with the Trx tag to obtain sheep growth hormone; The recombinant vector is obtained by inserting a nucleic acid molecule containing the sheep growth hormone encoding gene into an expression vector with a Trx tag; The expression vector with the Trx tag is a vector that... Msc 1 and BamH 1. The linear vector backbone obtained after double digestion of pET-32a(+) vector; The vector for expressing the molecular chaperone is pGro7; The nucleic acid molecule containing the sheep growth hormone encoding gene includes a restriction enzyme recognition site, a thrombin cleavage site, a purification tag, the sheep growth hormone encoding gene, and another restriction enzyme recognition site. The restriction enzyme recognition site and the other restriction enzyme recognition site are MScI and BamHI, respectively; The purification tag is a His tag; The nucleic acid molecule containing the sheep growth hormone encoding gene is sequence 1.

3. The method according to claim 1 or 2, characterized in that: The conditions for inducing expression were: 0.025mM-1mM IPTG, induction culture at 20℃-37℃ for 20 hours.

4. The method according to claim 2, characterized in that: The removal of the Trx tag from the sheep growth hormone is achieved by thrombin enzymatic digestion of the Trx-tagged sheep growth hormone.

5. The method according to claim 4, characterized in that: The enzyme digestion conditions are 0-4℃ for 8 minutes.

6. The method according to claim 1 or 2, characterized in that: The method further includes the following steps: purifying the pre-digestion product or the post-digestion product using the purification tag before or after the thrombin digestion.

7. The use of sheep growth hormone, Trx-tagged expression vector, expression molecular chaperone vector, and thrombin as described in any one of claims 1-6 in the preparation of sheep growth hormone or in increasing sheep growth hormone production.

8. The use of the recombinant vector or the recombinant bacteria of any one of claims 1-6 in the preparation of sheep growth hormone or in increasing the yield of sheep growth hormone.

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