Engineered bacterium for producing KGF-2 protein, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention is an engineered bacterium for producing KGF-2 protein. The engineered bacterium is Escherichia coli transfected with a recombinant vector. The recombinant vector is a pET-30a vector, pET-28a vector or pET-42a vector carrying a KGF-2 protein coding sequence. The Escherichia coli is OverExpress C43(DE3) Escherichia coli, Rosetta(DE3) Escherichia coli or BL21 Star(DE3) Escherichia coli. The engineered bacterium, when used for producing KGF-2 protein, has high expression level of KGF-2, and the plasmid can remain stable during the fermentation production of KGF-2 protein, thereby stably expressing KGF-2 protein and achieving the purpose of long-term fermentation production of KGF-2 protein. In addition, the yield of KGF-2 protein produced by fermentation with the engineered bacterium is up to 40%-50%, and the engineered bacterium has a good application value.

Description

An engineered bacterium for producing KGF-2 protein, and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to an engineering bacterium for producing KGF-2 protein, a preparation method thereof and an application thereof. Background Art

[0002] Keratinocyte Growth Factor-2 (KGF-2), also known as fibroblast growth factor-10 (FGF-10), is the tenth member of the fibroblast growth factor (FGF) family and is an endogenous polypeptide growth factor. KGF-2 is a single-copy gene consisting of three exons and two introns, located on chromosome 15. It is a single-chain polypeptide with five cysteine ​​residues, four of which form two pairs of disulfide bonds, and the other cysteine ​​is located within the folded peptide, forming a cloverleaf pattern. The mature human KGF-2 protein consists of 171 amino acids with a theoretical molecular weight of 19.4 kDa. The human KGF-2 gene sequence published in GenBank is accession number NM_004465.1.

[0003] KGF-2 is highly specific for epithelial tissue and possesses physical, chemical, and biological stability. It not only promotes the healing of corneal epithelial lesions but also does not promote the growth of new blood vessels, making it suitable for the treatment of corneal epithelial lesions. KGF-2 promotes the growth of epidermal keratinocytes, repairs damaged cells, and accelerates cell metabolism. Through KGF-2's specific keratinocyte repair, it increases skin immunity and resistance, restoring skin to a healthy, brighter state. It is also widely used in the preparation of cosmetics and pharmaceuticals that can repair tissue.

[0004] Currently, KGF-2 is typically produced through genetic engineering to produce recombinant human keratinocyte growth factor-2 (rhKGF-2), typically expressed in Escherichia coli. Expression has been demonstrated in a variety of ways, including inclusion body expression, soluble expression, and secretory expression. A prior art study (Study on Fermentation Conditions for Recombinant Human Keratinocyte Growth Factor-2) described a KGF-2 protein produced by fermentation in an engineered E. coli strain [pET26b / KGF-2 / BL(DE)21]. After scaled-up fermentation, the yield of rhKGF-2 protein was only approximately 30%. Furthermore, the addition of ampicillin to the fermentation medium indicated that the E. coli strain producing the rhKGF-2 protein was ampicillin-resistant.

[0005] However, the use of engineered bacteria carrying ampicillin to produce KGF-2 no longer meets the requirements of the Chinese Pharmacopoeia. In addition, the protein expression level of the engineered bacteria used to produce KGF-2 protein in the existing technology is low, with a yield of only about 30%. At the same time, its plasmid stability is poor, and the plasmid loss rate in the late fermentation period reaches more than 50%, which cannot achieve stable production of KGF-2 protein through generations.

[0006] Therefore, there is an urgent need for an engineered bacterium that does not require ampicillin screening and, more importantly, has high KGF-2 protein expression and excellent plasmid stability, so as to achieve efficient production of KGF-2 protein. Summary of the Invention

[0007] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide an engineered bacterium for producing KGF-2 protein and a preparation method and application thereof.

[0008] The first object of the present invention is to provide an engineered bacterium for producing KGF-2 protein.

[0009] The second object of the present invention is to provide the use of the above-mentioned engineered bacteria in producing KGF-2 protein.

[0010] The third object of the present invention is to provide a method for producing KGF-2 protein.

[0011] In order to achieve the above object, the present invention is achieved through the following scheme:

[0012] An engineered bacterium for producing KGF-2 protein, wherein the engineered bacterium is Escherichia coli transfected with a recombinant vector;

[0013] The recombinant vector is a pET-30a vector, a pET-28a vector or a pET-42a vector carrying a KGF-2 protein coding sequence, and the amino acid sequence of the KGF-2 protein is shown in SEQ ID NO: 2;

[0014] The Escherichia coli is OverExpress C43 (DE3) Escherichia coli, Rosetta (DE3) Escherichia coli or BL21Star (DE3) Escherichia coli.

[0015] Preferably, the recombinant vector is a pET-30a vector carrying the KGF-2 protein coding sequence, and the Escherichia coli is Rosetta (DE3) Escherichia coli or OverExpress C43 (DE3) Escherichia coli.

[0016] More preferably, the nucleotide sequence of the KGF-2 protein coding sequence is shown as SEQ ID NO: 1.

[0017] More preferably, the recombinant vector is a pET-30a vector carrying the KGF-2 protein coding sequence, and the Escherichia coli is Rosetta (DE3) Escherichia coli.

[0018] The present invention also seeks to protect the use of any of the above-mentioned engineering bacteria in producing KGF-2 protein.

[0019] The above-mentioned engineered bacteria not only do not require the use of ampicillin for screening, but also have a high expression level of KGF-2 protein. At the same time, the plasmid remains stable during the fermentation process of producing KGF-2 protein, and can stably express KGF-2 protein. The yield of KGF-2 protein produced by the above-mentioned engineered bacteria is as high as 40% to 50%.

[0020] The present invention also seeks to protect a method for producing KGF-2 protein, wherein the method utilizes any of the above-mentioned engineered bacteria to ferment and produce KGF-2 protein.

[0021] Preferably, the following steps are included:

[0022] S1. Inoculate any of the above-described engineered bacteria into LB medium at a volume ratio of 1:20 to 200, and fully culture to obtain a primary seed solution. Inoculate the primary seed solution into LB medium at a volume ratio of 1:20 to 200, and fully culture to obtain a secondary seed culture solution.

[0023] S2. The secondary seed culture medium obtained in step S1 is mixed with the fermentation base material in a volume ratio of 2 to 10:20 to 100 to obtain a culture medium, the pH of the culture medium is adjusted to 6.2 to 8.2, and culture is carried out under the conditions of dissolved oxygen>10%, temperature of 25 to 45°C, rotation speed of 100 to 1600 rpm and ventilation volume of 5 to 100 L / min. The OD of the culture medium is 600 When the pH value is 10 to 50, IPTG with a final concentration of 0.1 to 3.0 mM and a feed solution accounting for 1.0 to 30% of the total volume are added to the culture solution to fully culture the fermented engineered bacterial cells;

[0024] The fermentation base material is obtained by mixing base material 1 culture medium, base material 2 culture medium and 1-20g / L thiamine solution in the ratio of 15-25L:0.5-1.5L:6-10mL;

[0025] The base material 1 medium contains peptone at a final concentration of 15-25 g / L, yeast extract at a final concentration of 15-25 g / L, sodium chloride at a final concentration of 3-7 g / L, potassium dihydrogen phosphate at a final concentration of 1-3 g / L, potassium hydrogen phosphate at a final concentration of 2-4 g / L, and ammonium chloride at a final concentration of 1-3 g / L; the base material 2 medium contains glucose monohydrate at a final concentration of 2-10 g / L, magnesium sulfate anhydrous at a final concentration of 0.2-0.8 g / L, and calcium chloride at a final concentration of 0.01-0.09 g / L.

[0026] The feed solution contains peptone at a final concentration of 15 to 25 g / L, yeast powder at a final concentration of 15 to 25 g / L, sodium chloride at a final concentration of 3 to 7 g / L, potassium dihydrogen phosphate at a final concentration of 1 to 3 g / L, and potassium dihydrogen phosphate trihydrate at a final concentration of 2 to 4 g / L.

[0027] S3. The fermented engineered bacterial cells obtained in step S2 are homogenized and broken, and the supernatant is collected for solid-liquid separation and subjected to CM chromatography to obtain crude KGF-2 protein. The crude KGF-2 protein is subjected to heparin chromatography to obtain KGF-2 protein.

[0028] Preferably, the LB medium in step S1 is an LB medium containing yeast extract powder, peptone and sodium chloride at a final concentration of 2-10 g / L, 9-21 g / L, and a pH value of 6.2-8.2.

[0029] More preferably, the LB medium in step S1 is an LB medium containing yeast extract powder, peptone and sodium chloride at a final concentration of 5.0 g / L, and a pH value of 6.2 to 8.2.

[0030] Preferably, the thiamine solution in step S2 is an aqueous thiamine solution.

[0031] Preferably, in step S2: the OD of the culture medium to be cultured 600 When the pH value is 40, IPTG with a final concentration of 0.1 to 3.0 mM and a feed solution of 1.0 to 30% of the total volume are added to the culture solution.

[0032] Preferably, the homogenization and disruption in step S3 is specifically as follows: the fermented engineered bacterial cells obtained in step S2 and Buffer A are fully mixed at a ratio of 1 g: 1-100 mL, and homogenized and disrupted at 4-40° C.;

[0033] The formula of the Buffer A is: 15-45 mM phosphate buffer (PB), 0.2-1.8 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.2.

[0034] More preferably, the formula of Buffer A is: 30mM phosphate buffer (PB), 0.2M sodium chloride and 20M EDTA-2Na, pH=6.2-8.2.

[0035] Preferably, the CM chromatography in step S3 is specifically:

[0036] Equilibrate the CM column, add the supernatant in step S3 to the equilibrated CM column at 0.01-10.0 g / mL, collect the flow-through peak, re-equilibrate the CM column with Buffer A, wash the column thoroughly with Buffer E, and then elute with Buffer C for 0.5-10 column volumes, collect UV 280 The eluate at >20 mAU yielded crude pure KGF-2 protein;

[0037] The formula of Buffer C is: 15-45 mM phosphate buffer (PB), 0.2-1.8 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.2;

[0038] The formula of the Buffer E is: 15-45 mM phosphate buffer (PB), 0.2-1.8 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.2.

[0039] More preferably, the balancing CM chromatography column is specifically as follows: adding Buffer A to the CM chromatography column for 0.5 to 10 column volumes until the baseline of the CM chromatography column is stable and the conductivity value is not greater than 100ms / cm, the pH value of the effluent of the CM chromatography column is 6.2 to 8.2, and UV 280 The absorption baseline is stable, and the CM chromatography column is balanced.

[0040] More preferably, elution is performed using Buffer C for 4 column volumes.

[0041] More preferably, the formula of Buffer C is: 30mM phosphate buffer (PB), 0.6M sodium chloride and 20mM EDTA-2Na, pH = 6.2-8.2;

[0042] The formula of Buffer E is: 30 mM phosphate buffer (PB), 0.3 M sodium chloride and 20 mM EDTA-2Na, pH = 6.2-8.2.

[0043] Preferably, the heparin chromatography in step S3 is specifically:

[0044] Equilibrate the heparin chromatography column, mix the crude KGF-2 protein described in step S3 with an equal volume of Buffer A, load the equilibrated heparin chromatography column at 0.10-2.00 g / mL, collect the flow-through peak, re-equilibrate the heparin chromatography column with Buffer B, thoroughly wash with Buffer C, and then elute with Buffer D for 0.5-10 column volumes, collect UV 280 When the eluate is >20mAU, the KGF-2 protein is obtained;

[0045] The formula of Buffer B is: 15-45 mM phosphate buffer (PB), 0.1-3 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.2;

[0046] The formula of the Buffer D is: 15-45 mM phosphate buffer (PB), 0.1-3 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.2.

[0047] More preferably, the equilibration of the heparin chromatography column is specifically as follows: adding Buffer B to the heparin chromatography column for 0.5 to 10 column volumes until the baseline of the heparin chromatography column is stable and the conductivity value is not greater than 100ms / cm, the pH value of the effluent of the heparin chromatography column is 6.2 to 8.2, and UV 280 The absorption baseline is stable, and the heparin chromatography column is balanced.

[0048] More preferably, elution is performed using Buffer D for 2 column volumes.

[0049] More preferably, the formula of Buffer B is: 30mM phosphate buffer (PB), 0.4M sodium chloride and 20mM EDTA-2Na, pH=6.2-8.2;

[0050] The formula of Buffer D is: 30 mM phosphate buffer (PB), 0.8 M sodium chloride and 20 mM EDTA-2Na, pH = 6.2-8.2.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention provides an engineered bacterium for producing KGF-2 protein. The engineered bacterium is Escherichia coli transfected with a recombinant vector; the recombinant vector is a pET-30a vector, a pET-28a vector, or a pET-42a vector carrying a KGF-2 protein coding sequence; and the Escherichia coli is OverExpress C43 (DE3) Escherichia coli, Rosetta (DE3) Escherichia coli, or BL21 Star (DE3) Escherichia coli. When the engineered bacterium is used to produce KGF-2 protein, KGF-2 expression is high. During fermentation, the plasmid remains stable, thereby stably expressing the KGF-2 protein and achieving long-term fermentation production of the KGF-2 protein. Furthermore, the yield of KGF-2 protein produced by fermentation using the engineered bacterium is as high as 40% to 50%, which is approximately 66% higher than the current yield, thus having excellent application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG1 is a spectrum of 500109-pET-28a(+) in Example 1;

[0054] FIG2 is a spectrum of 500109-pET-30a(+) in Example 1;

[0055] FIG3 is a spectrum of 500109-pET-42a(+) in Example 1;

[0056] FIG4 is an observation diagram of the 500109-S9 engineered bacteria after cultivation in Example 1;

[0057] FIG5 is an electron microscope observation result after culturing the 500109-S9 engineered bacteria in Example 1;

[0058] FIG6 is a graph showing the electrophoresis detection results of the recombinant engineered bacteria 500109-S2 to 500109-S5 in Example 1 after induction for 4 hours;

[0059] FIG7 is a graph showing the electrophoresis detection results of the recombinant engineered bacteria 500109-S6 to 500109-S9 in Example 1 after induction for 4 hours;

[0060] FIG8 is a graph showing the electrophoresis detection results of the recombinant engineered bacteria 500109-S10 to 500109-S13 in Example 1 after induction for 4 hours;

[0061] FIG9 is a graph showing the electrophoresis detection results of the recombinant engineered bacteria 500109-S2 to 500109-S5 in Example 1 after induction for 16 hours;

[0062] Figure 10 is a graph showing the electrophoresis results of the recombinant engineered bacteria 500109-S6 to 500109-S9 in Example 1 after induction for 16 hours;

[0063] Figure 11 is a graph showing the electrophoresis results of the recombinant engineered bacteria 500109-S10 to 500109-S13 in Example 1 after induction for 16 hours;

[0064] FIG12 is a graph showing the SDS-PAGE electrophoresis results when the induction temperature is changed in Example 1;

[0065] Figure 13 is a graph showing the results of SDS-PAGE electrophoresis when the inducer concentration is changed in Example 1;

[0066] FIG14 is a growth curve of the recombinant engineered bacteria at different inoculation amounts in Example 3;

[0067] FIG15 is a graph showing the SDS-PAGE results of recombinant engineered bacteria with different inoculation amounts in Example 3;

[0068] FIG16 is a growth curve of the recombinant engineered bacteria with different fermentation substrates in Example 3;

[0069] FIG17 is a graph showing the SDS-PAGE results of the recombinant engineered bacteria with different fermentation substrates in Example 3;

[0070] FIG18 is a growth curve of the recombinant engineered bacteria to which the inducer was added at different times in Example 3;

[0071] FIG19 is a graph showing the SDS-PAGE results of the recombinant engineered bacteria to which the inducer was added at different times in Example 3;

[0072] Figure 20 is a growth curve of the recombinant engineered bacteria at different inducer concentrations in Example 3;

[0073] FIG21 is a graph showing the SDS-PAGE results of the recombinant engineered bacteria at different inducer concentrations in Example 3;

[0074] FIG22 is a growth curve of the recombinant engineered bacteria at different temperatures in Example 3;

[0075] FIG23 is a graph showing the SDS-PAGE results of the recombinant engineered bacteria at different temperatures in Example 3;

[0076] FIG24 is a low column high cation exchange chromatography spectrum in Example 3;

[0077] Figure 25 is a high-column high-cation exchange chromatography spectrum in Example 3;

[0078] Figure 26 is a low-column high-affinity chromatography spectrum in Example 3;

[0079] Figure 27 is a high-column high-affinity chromatography spectrum in Example 3;

[0080] Figure 28 is a graph showing the SDS-PAGE results of different column heights in Example 3;

[0081] Figure 29 is a cation exchange chromatography profile at a purification temperature of 10°C in Example 3;

[0082] Figure 30 is an affinity chromatography profile of the purification at a temperature of 10°C in Example 3;

[0083] FIG31 is a diagram showing the SDS-PAGE results of purification at a temperature of 10° C. in Example 3;

[0084] FIG32 is a cation exchange chromatography spectrum after washing in Example 3;

[0085] FIG33 is an affinity chromatography spectrum after washing in Example 3;

[0086] FIG34 is a diagram showing the SDS-PAGE results after washing in Example 3;

[0087] FIG35 is a cation exchange chromatography spectrum after urea washing in Example 3;

[0088] FIG36 is an affinity chromatography spectrum after urea washing in Example 3;

[0089] Figure 37 is a graph showing the SDS-PAGE results of control group 3 in Example 3;

[0090] Figure 38 is a cation exchange chromatography profile at pH 7.2 in Example 3;

[0091] Figure 39 is an affinity chromatography profile at pH 6.8 in Example 3;

[0092] Figure 40 is an affinity chromatography profile at pH 7.0 in Example 3;

[0093] Figure 41 is an affinity chromatography profile at pH 7.4 in Example 3;

[0094] Figure 42 is a graph showing the SDS-PAGE results at pH 6.8 in Example 3;

[0095] Figure 43 is a graph showing the SDS-PAGE results at pH 7.0 in Example 3;

[0096] Figure 44 is a graph showing the SDS-PAGE results at pH 7.4 in Example 3;

[0097] Figure 45 is an affinity chromatography profile of control group 7 in Example 3;

[0098] FIG46 is an SDS-PAGE result of the purified rhKGF-2 protein of Control Group 7 in Example 3;

[0099] Figure 47 is a cation exchange chromatography spectrum of control group 8 in Example 3;

[0100] Figure 48 is an affinity chromatography profile of control group 8 in Example 3;

[0101] Figure 49 is a graph showing the SDS-PAGE results of control group 8 in Example 3;

[0102] Figure 50 is a cation exchange chromatography profile of Experimental Group 5 in Example 3;

[0103] Figure 51 is the affinity chromatography profile of Experimental Group 5 in Example 3;

[0104] Figure 52 is an affinity chromatography profile of control group 9 in Example 3;

[0105] Figure 53 is an affinity chromatography profile of control group 10 in Example 3;

[0106] FIG54 is a graph showing the SDS-PAGE results of different sample loading amounts in Example 3;

[0107] Figure 55 is a cation exchange chromatography spectrum of control group 11 in Example 3;

[0108] Figure 56 is an affinity chromatography profile of control group 11 in Example 3;

[0109] Figure 57 is a graph showing the SDS-PAGE results of different elution buffers in Example 3;

[0110] Figure 58 is a cation exchange chromatography profile of control group 12 in Example 3;

[0111] Figure 59 is an affinity chromatography profile of control group 12 in Example 3;

[0112] Figure 60 is a graph showing the SDS-PAGE results of Buffers of different pH values ​​in Example 3;

[0113] Figure 61 is a cation exchange chromatography spectrum of Experimental Group 7 in Example 3;

[0114] Figure 62 is the affinity chromatography profile of Experimental Group 7 in Example 3;

[0115] FIG63 is an SDS-PAGE result of adding displacement envelope after chromatography in Example 3;

[0116] Figure 64 is a cation exchange chromatography profile of Experimental Group 8 in Example 3;

[0117] Figure 65 is the affinity chromatography profile of Experimental Group 8 in Example 3;

[0118] Figure 66 is a graph showing the SDS-PAGE results of different rhKGF-2 protein concentrations in Example 3;

[0119] Figure 67 is a cation exchange chromatography profile of Experimental Group 9 in Example 3;

[0120] Figure 68 is the affinity chromatography profile of Experimental Group 9 in Example 3;

[0121] FIG69 is the SP chromatogram of Experimental Group 9 in Example 3;

[0122] FIG70 is a graph showing the SDS-PAGE results of the heparin chromatography eluate and the replacement fluid of Experimental Group 9 in Example 3;

[0123] FIG71 is a graph showing the SDS-PAGE results of the SP eluate in Experimental Group 9 in Example 3;

[0124] Figure 72 is a diagram of the SDS-PAGE results in Example 4;

[0125] Figure 73 is the SDS-PAGE result of the purified rhKGF-2 protein in Comparative Example 1. DETAILED DESCRIPTION

[0126] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0127] Example 1 Screening and Construction of Engineering Bacteria for Producing KGF-2 Protein

[0128] The human KGF-2 gene sequence (NM_004465.2) published in NCBI was used as the reference genome, and the nucleotide sequence of gene 500109 (KGF-2 protein coding sequence) was synthesized as shown in SEQ ID NO: 1. The amino acid sequence of the KGF-2 protein encoded by it is shown in SEQ ID NO: 2.

[0129] 1. Construction of expression vector

[0130] Using the 500109 gene with the nucleotide sequence shown in SEQ ID NO: 1 as a template, an Xba1I restriction enzyme site was introduced at its 5' end and an XhoI restriction enzyme site was introduced at its 3' end to obtain an inserted gene 1 with the nucleotide sequence shown in SEQ ID NO: 3; using the 500109 gene with the nucleotide sequence shown in SEQ ID NO: 1 as a template, an NdeI restriction enzyme site was introduced at its 5' end and an XhoI restriction enzyme site was introduced at its 3' end to obtain an inserted gene 2 with the nucleotide sequence shown in SEQ ID NO: 4.

[0131] The insert gene 1 (SEQ ID NO: 3) and the pET-28a(+) vector were digested with Xba1I restriction enzyme and XhoI restriction enzyme, respectively, and then ligated with T4 ligase to obtain a pET-28a vector carrying the gene with the nucleotide sequence shown in SEQ ID NO: 1, i.e., 500109-pET-28a(+), as shown in FIG1 .

[0132] The insert gene 2 (SEQ ID NO: 4) and the pET-30a(+) vector were digested with NdeI restriction enzyme and XhoI restriction enzyme, respectively, and then ligated with T4 ligase to obtain a pET-30a(+) vector carrying the gene with the nucleotide sequence shown in SEQ ID NO: 1, i.e., 500109-pET-30a(+), as shown in FIG2 ;

[0133] The pET-30a(+) vector was replaced with the pET-42a(+) vector and subjected to the same treatment to obtain the pET-42a(+) vector carrying the gene with the nucleotide sequence shown in SEQ ID NO: 1, i.e., 500109-pET-42a(+), as shown in FIG3 .

[0134] 2. Construction of Engineered Bacteria

[0135] 1. Method

[0136] BL21(DE3) plysS competent cells (Weidi Biotech EC1003), BL21 Star(DE3) competent cells (Thermo 44-00049), Rosetta(DE3) competent cells (Solerbo C1420) and Over Express C43(DE3) competent cells (Weidi Biotech EC1040) were used as host bacteria, respectively.

[0137] Taking BL21 (DE3) plysS competent cells as host bacteria as an example: the host bacteria was placed in a centrifuge tube and ice-bathed for 15 minutes, and then the 500109-pET-28a (+) obtained in step 1 was mixed with the host bacteria at a volume ratio of 100:6. After mixing evenly, the mixture was ice-bathed for 20 minutes. After the ice bath, the mixture was shaken and placed in a 42 ° C water bath for 90 seconds, and then returned to the ice bath for 2 minutes. After the ice bath, anti-antibody LB liquid medium (containing 5 g / L yeast extract, 10 g / L peptone and 10 g / L sodium chloride) was added to the centrifuge tube manifold. After mixing evenly, the mixture was placed in a 37 ° C constant temperature shaking incubator for 50 minutes. The bacterial liquid was collected and spread on an LB solid medium plate containing 30 mg / L kanamycin sulfate resistance (containing 5 g / L yeast extract, 10 g / L peptone and 10 g / L sodium chloride), sealed with a sealing film, and inverted in a 37 ° C constant temperature incubator for 16 hours.

[0138] After the culture was completed, two monoclonal strains of uniform size were selected and inoculated into two LB liquid media containing 30 mg / L kanamycin sulfate resistance. The cells were cultured in a constant temperature shaking incubator at 37°C and 220 rpm until the OD 600 The cultured bacterial solution is obtained between 2 and 3, glycerol is added to the cultured bacterial solution to a final glycerol concentration of 20% (v / v), and after uniform mixing, a glycerol strain containing recombinant engineered bacteria with a vector of 500109-pET-28a(+) and a host bacteria of BL21(DE3)plysS competent cells is obtained.

[0139] The host bacteria were replaced with BL21 Star (DE3) competent cells, Rosetta (DE3) competent cells and Over Express C43 (DE3) competent cells in sequence and treated identically to obtain recombinant engineered bacteria using different competent cells as host bacteria.

[0140] 500109-pET-28a(+) was replaced with 500109-pET-30a(+) and 500109-pET-42a(+) obtained in step 1, and the same treatment was performed to obtain the corresponding recombinant engineered bacteria. The specific information of different recombinant engineered bacteria is shown in Table 1. A total of 12 recombinant engineered bacteria were prepared.

[0141] Table 1 Recombinant engineering bacteria information

[0142] The 12 recombinant engineered bacteria shown in Table 1 were sequenced, and the sequencing results showed that all of the recombinant engineered bacteria carried the gene with the nucleotide sequence shown in SEQ ID NO: 1.

[0143] Taking the constructed 500109-S9 engineered bacteria as an example, it was spread on an LB agar culture plate (containing 5 g / L yeast extract, 10 g / L peptone and 10 g / L sodium chloride) containing 30 mg / L kanamycin sulfate and cultured at 37°C for 16 hours. After the culture was completed, photos were taken and the cultured strain was stained with crystal violet and the morphological characteristics were observed under an electron microscope.

[0144] 2. Experimental results

[0145] The observation picture of the 500109-S9 engineered bacteria after cultivation is shown in Figure 4, and the observation result of the electron microscope is shown in Figure 5. The results show that after cultivation, the 500109-S9 engineered bacteria showed typical Escherichia coli colony morphology and no growth of other bacteria was observed; the electron microscope observation results showed no impurity contamination.

[0146] 3. Engineering bacteria screening

[0147] 1. Recombinant engineering bacteria induced 4h protein expression test

[0148] (1) Experimental methods

[0149] The 12 recombinant engineering bacteria shown in Table 1 of step 2 were used to ferment and express KGF-2 protein. One clone was set up in parallel for each recombinant engineering bacteria (two groups in total). Taking 500109-S2 as an example, the details are as follows:

[0150] Glycerol was added to 500109-S2 to a final glycerol concentration of 20% (v / v), and the mixture was evenly mixed to obtain RCB glycerol bacteria. The RCB glycerol bacteria was inoculated into LB liquid culture medium containing 30 mg / L kanamycin sulfate resistance at an inoculum size of 0.5% (v / v), and cultured in a constant temperature shaking incubator at 37°C and 150 rpm for 16 h to obtain a level 1 bacterial solution.

[0151] The first-level bacterial solution was inoculated into LB liquid medium containing 30 mg / L kanamycin sulfate resistance at 1% (v / v) and cultured in a constant temperature shaking incubator at 37°C and 200 rpm until the OD 600 The pH value was 0.8-1.2 (i.e., the strain was in the logarithmic growth phase), IPTG was added to the culture medium to a final concentration of 1.5 mM, and the induction culture was continued at 40°C and 200 rpm for 4 h. After the culture was completed, the bacteria were fully centrifuged and the protein expression level was detected by SDS-PAGE electrophoresis.

[0152] The cloned strain 500109-S2 was treated in the same manner, and 500109-S3 to 500109-S13 shown in Table 1 were treated in the same manner, and the protein expression levels were detected by SDS-PAGE electrophoresis.

[0153] (2) Experimental results

[0154] The results of SDS-PAGE electrophoresis detection of the recombinant engineered bacteria after 4 hours of induction are shown in Figures 6 to 8; Figure 6 is the SDS electrophoresis detection result of 500109-S2 to 500109-S5, lanes 1 and 2 are the electrophoresis results of 500109-S2, lanes 3 and 4 are the electrophoresis results of 500109-S3, lanes 5 and 6 are the electrophoresis results of 500109-S4, lanes 7 and 8 are the electrophoresis results of 500109-S5, and lane 9 is the electrophoresis result of the marker; Figure 7 is the SDS electrophoresis detection result of 500109-S6 to 500109-S9, lanes 10 and 11 are the electrophoresis results of 500109-S6, lanes 12 and Lane 13 is the electrophoresis result of 500109-S7, lanes 14 and 15 are the electrophoresis results of 500109-S8, lane 16 is the electrophoresis result of marker, and lanes 17 and 18 are the electrophoresis results of 500109-S9; Figure 8 is the SDS electrophoresis detection results of 500109-S10 to 500109-S13, lanes 19 and 20 are the electrophoresis results of 500109-S10, lane 21 is the electrophoresis result of marker, lanes 22 and 23 are the electrophoresis results of 500109-S11, lanes 24 and 25 are the electrophoresis results of 500109-S12, and lanes 26 and 27 are the electrophoresis results of 500109-S13.

[0155] The size of KGF-2 protein is about 20KD. The results showed that all 12 recombinant engineered bacteria were able to express KGF-2 protein after 4 hours of induction. However, the KGF-2 protein expression levels of three recombinant engineered bacteria (500109-S2, 500109-S6 and 500109-S10) using BL21 (DE3) plysS competent cells as host bacteria were lower than those of other recombinant engineered bacteria, and they were not suitable as KGF-2 protein expression production strains; while the KGF-2 protein expression levels of 500109-S3, 500109-S9, 500109-S11, 500109-S12 and 500109-S13 were relatively high, and the KGF-2 protein expression levels of 500109-S9 and 500109-S13 engineered bacteria were even higher (the protein bands were more obvious in the electrophoresis detection results).

[0156] 2. Protein expression test induced by recombinant engineering bacteria for 16 hours

[0157] (1) Experimental methods

[0158] The 12 recombinant engineered bacteria shown in Table 1 of step 2 were used to ferment and express KGF-2 protein. The difference was that the experimental method described in 1.(1) of step 3 was as follows: IPTG was added to a final concentration of 1.5 mM, and the induction culture was continued at 40°C and 200 rpm for 16 hours. After the culture was completed, the bacteria were fully centrifuged and the protein expression level was detected by SDS-PAGE electrophoresis; the rest of the experimental steps were the same.

[0159] (2) Experimental results

[0160] The results of SDS-PAGE electrophoresis detection of the recombinant engineered bacteria after 16 hours of induction are shown in Figures 9 to 11; Figure 9 is the SDS electrophoresis detection result of 500109-S2 to 500109-S5, lanes 1 and 2 are the electrophoresis results of 500109-S2, lanes 3 and 4 are the electrophoresis results of 500109-S3, lanes 5 and 6 are the electrophoresis results of 500109-S4, lanes 7 and 8 are the electrophoresis results of 500109-S5, and lane 9 is the electrophoresis result of the marker; Figure 10 is the SDS electrophoresis detection result of 500109-S6 to 500109-S9, lanes 10 and 11 are the electrophoresis results of 500109-S6, lanes 12 and 13 are the electrophoresis results of 500109-S7, and lanes 14 and 15 are the electrophoresis results of 500109-S8. Figure 11 shows the SDS electrophoresis detection results of 500109-S10 to 500109-S13, lanes 19 and 20 show the electrophoresis results of 500109-S10, lane 21 shows the electrophoresis result of marker, lanes 22 and 23 show the electrophoresis results of 500109-S11, lanes 24 and 25 show the electrophoresis results of 500109-S12, and lanes 26 and 27 show the electrophoresis results of 500109-S13; the highlighted bands in the electrophoresis result diagram shown in Figure 10 are caused by gel rupture during the decolorization process and do not affect the interpretation of the electrophoresis results. The electrophoresis detection result diagrams shown in Figures 9 to 11 are only for visual observation of the target protein expression and are not used for quantitative analysis.

[0161] The results showed that all 12 recombinant engineered bacteria were able to express KGF-2 protein after 16 hours of induction; however, the expression levels of KGF-2 protein in 500109-S11 and 500109-S12 were not high; while the expression levels of KGF-2 protein in 500109-S3, 500109-S9 and 500109-S13 were significantly higher than those in other recombinant engineered bacteria.

[0162] 3. Detection of plasmid loss rate of recombinant engineered bacteria

[0163] (1) Experimental methods

[0164] 500109-S3, 500109-S9, 500109-S13 and 500109-S6 were used as test strains, and the bacterial cultures of the four recombinant engineered bacteria were collected after induction for 8 h (according to the method shown in step 3, 1, the induction culture time was changed to 8 h) and 16 h (according to the method shown in step 3, 2) (a total of 8 bacterial cultures), and each bacterial culture was diluted to 10 6After 100 single colonies were collected from each medium, they were inoculated into LB agar medium containing 30 mg / L kanamycin sulfate resistance and LB agar medium without antibiotics. Each medium was inoculated with 50 single colonies and cultured at 37°C for 16 h. The number of colonies in each medium was calculated to obtain the number of colonies growing on the resistant medium and the number of colonies growing on the non-resistant medium.

[0165] The plasmid loss rate was calculated based on the number of colonies grown on the resistant medium and the number of colonies grown on the non-resistant medium, combined with the plasmid loss rate inspection method (based on Appendix IX G of Part III of the 2015 edition of the Chinese Pharmacopoeia).

[0166] (2) Experimental results

[0167] The results of plasmid loss rate detection are shown in Table 2.

[0168] Table 2 Plasmid loss rate detection results

[0169] The results showed that the plasmids of the four recombinant engineered bacteria were all stable, and the plasmid loss rates of the strains induced for 16 hours were all less than 50%; and the plasmid loss rates of 500109-S9 and 500109-S13 were both 0% after 8 hours of induction, indicating that the plasmids of 500109-S9 and 500109-S13 were relatively stable.

[0170] 4. Optimization of conditions for expression and production of KGF-2 protein by recombinant engineering bacteria

[0171] (1) Experimental methods

[0172] 1) Induction temperature optimization, taking 500109-S6 as an example, the details are as follows:

[0173] Glycerol was added to 500109-S6 to a final glycerol concentration of 20% (v / v), and the mixture was evenly mixed to obtain RCB glycerol bacteria 1. RCB glycerol bacteria 1 was inoculated into LB liquid culture medium containing 30 mg / L kanamycin sulfate resistance at an inoculum size of 0.5% (v / v), and cultured in a constant temperature shaking incubator at 30°C and 150 rpm for 16 h to obtain level 1 bacterial solution 1.

[0174] The first-level bacterial solution 1 was inoculated into LB liquid medium containing 30 mg / L kanamycin sulfate resistance at 1% (v / v), and cultured in a constant temperature shaking incubator at 37°C and 200 rpm until the OD 600The pH value was 0.5 to 2.0 (i.e., the strain was in the logarithmic growth phase), IPTG was added to the culture medium to a final concentration of 1.5 mM, and the culture was continued at 40°C and 200 rpm for 12 h. After the culture was completed, the bacteria were fully centrifuged and the protein expression level was detected by SDS-PAGE electrophoresis.

[0175] The culture temperature after adding IPTG was changed to 45°C, and the other steps remained unchanged. The protein expression level was detected by SDS-PAGE electrophoresis. 500109-S6 was replaced by 500109-S9 and 500109-S13, respectively. The other processing steps remained unchanged, and the protein expression level was detected by SDS-PAGE electrophoresis.

[0176] 2) Optimize the inducer concentration as follows:

[0177] Glycerol was added to 500109-S9 to a final glycerol concentration of 20% (v / v), and the mixture was evenly mixed to obtain RCB glycerol bacteria 2. RCB glycerol bacteria 2 was inoculated into LB liquid culture medium containing 30 mg / L kanamycin sulfate resistance at an inoculum size of 0.5% (v / v), and cultured in a constant temperature shaking incubator at 30°C and 150 rpm for 16 h to obtain level 1 bacterial liquid 2.

[0178] Inoculate 1% (v / v) of the first-level bacterial solution 2 into LB liquid medium containing 30 mg / L kanamycin sulfate resistance, and culture in a constant temperature shaking incubator at 37°C and 200 rpm until the OD 600 The pH value was 0.5 to 2.0 (i.e., the strain was in the logarithmic growth phase), IPTG was added to the culture medium to a final concentration of 1.5 mM, and the culture was continued at 40°C and 200 rpm for 10 h. After the culture was completed, the bacteria were fully centrifuged and the protein expression level was detected by SDS-PAGE electrophoresis.

[0179] The final concentration of IPTG was changed to 2.0 mM, and the other steps remained unchanged. The protein expression level was detected by SDS-PAGE electrophoresis.

[0180] (2) Experimental results

[0181] The SDS-PAGE electrophoresis detection results when the induction temperature was changed are shown in Figure 12; wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of 500109-S6 induced at 40°C, lane 3 is the electrophoresis result of 500109-S6 induced at 45°C, lane 4 is the electrophoresis result of 500109-S9 induced at 40°C, lane 5 is the electrophoresis result of 500109-S9 induced at 45°C, lane 6 is the electrophoresis result of 500109-S13 induced at 40°C, and lane 7 is the electrophoresis result of 500109-S13 induced at 45°C.

[0182] The results showed that when the induction temperature was 40℃, the expression level of KGF-2 protein in 500109-S9 and 500109-S13 was higher than that when the induction temperature was 45℃; and the expression level of KGF-2 protein in 500109-S9 at the induction temperature of 40℃ was significantly higher than that in other recombinant engineered bacteria.

[0183] The SDS-PAGE electrophoresis detection results when the inducer concentration was changed are shown in Figure 13; wherein lane 1 is the electrophoresis result of the marker, lanes 2 and 3 are the electrophoresis results of 500109-S9 induced when the final IPTG concentration was 1.5 mM, and lanes 4 and 5 are the electrophoresis results of 500109-S9 induced when the final IPTG concentration was 2.0 mM.

[0184] The results showed that whether the final concentration of IPTG (inducer) was 1.5 mM or 2.0 mM, there was no significant difference in the expression level of KGF-2 protein, and an increase in the concentration of IPTG (inducer) did not increase the expression level of KGF-2 protein.

[0185] Based on the above results, the 500109-S9 recombinant engineered bacteria, that is, the recombinant engineered bacteria using Rosetta (DE3) as the host bacteria and transformed with the 500109-pET-30a(+) vector, not only contain the kanamycin resistance gene, but also have a higher expression level of KGF-2 protein compared with other recombinant engineered bacteria, and also have excellent plasmid stability, and can be used as an engineered bacteria for the production and expression of KGF-2 protein; and the protein expression level of 500109-S9 is the highest when the induction temperature is 40°C and the final concentration of the inducer (IPTG) is 1.5mM.

[0186] Example 2 A method for producing and purifying rhKGF-2 protein

[0187] 1. KGF-2 protein production method

[0188] S1. Preparation of primary seed liquid: 500109-S9 constructed in Example 1 was mixed with glycerol to a final glycerol concentration of 20% (v / v) to obtain glycerol bacteria; the pH of LB medium (containing 5 g / L yeast extract, 10 g / L peptone and 10 g / L sodium chloride) was adjusted to 7.2 and transferred to a shake flask, the glycerol bacteria and LB medium were mixed in a volume ratio of 1:50, and cultured in a shaker at 40°C and 150 rpm for 18 h to obtain a primary seed liquid.

[0189] S2. Preparation of secondary seed solution: The primary seed solution obtained in step S1 was inoculated into LB culture medium at a volume ratio of 1:200, and cultured in a shaker at 40°C and 150 rpm for 8 h to obtain a secondary seed solution.

[0190] S3. Fermentation: After the substrate 1 culture medium was sterilized by heat sterilization in the main fermentation tank, a sterile thiamine aqueous solution (20 g / L) was added, and then the substrate 2 culture medium was added to the main fermentation tank and mixed to obtain a fermentation substrate; wherein the volume ratio of the substrate 1 culture medium, the substrate 2 culture medium and thiamine was 20 to 40 L: 0.5 to 1.5 L: 6 to 10 mL;

[0191] The secondary seed solution (containing the 500109-S9 strain) obtained in step S2 was inoculated into the main fermentation tank at an inoculum volume of 8% (v / v), and the initial fermentation volume was recorded. The pH in the main fermentation tank was controlled to 7.2, and fermentation was carried out under the conditions of a temperature of 40°C, a dissolved oxygen content of 10%, a ventilation volume of 50 L / min, and a rotation speed of 1000 rpm. When the OD 600 =40, IPTG with a final concentration of 1.5 mM (based on the initial fermentation broth) and a feed solution accounting for 8% of the total volume (containing 20 g / L peptone, 23 g / L yeast powder, 4 g / L sodium chloride, 1 g / L potassium dihydrogen phosphate and 3 g / L potassium hydrogen phosphate trihydrate) were added, and the culture was continued for 6 h. After the culture was completed, the fermented engineered bacteria were obtained.

[0192] The base material 1 medium contains peptone with a final concentration of 20 g / L, yeast powder with a final concentration of 23 g / L, sodium chloride with a final concentration of 4 g / L, potassium dihydrogen phosphate with a final concentration of 1 g / L, potassium hydrogen phosphate with a final concentration of 3 g / L, and ammonium chloride with a final concentration of 2 g / L; the base material 2 medium contains glucose monohydrate with a final concentration of 5 g / L, magnesium sulfate with a final concentration of 0.6 g / L, and calcium chloride with a final concentration of 0.013 g / L.

[0193] S4. Purification:

[0194] Prepare Buffer A to Buffer E for column chromatography in sequence and control the temperature of Buffer A to Buffer E to be 15°C;

[0195] The formula of Buffer A is: 30mM PB, 0.2M NaCl and 20mM EDTA-2Na, pH 6.2;

[0196] The formula of Buffer B is: 30mM PB, 0.4M NaCl and 20mM EDTA-2Na, pH 6.2;

[0197] The formula of Buffer C is: 30mM PB, 0.6M NaCl and 20mM EDTA-2Na, pH 6.2;

[0198] The formula of Buffer D is: 30mM PB, 0.8M NaCl and 20mM EDTA-2Na, pH 6.2;

[0199] The formula of Buffer E is: 30mM PB, 0.3M NaCl and 20mM EDTA-2Na, pH 6.2;

[0200] The fermented engineered bacteria obtained in step S3 were mixed with Buffer A at a ratio of 1 g: 20 mL, and stirred thoroughly to obtain a mixed solution. The mixture was homogenized at 200 Bar and then at 700 Bar at 15°C for one time. After the mixture was completely broken (i.e., there were no intact bacteria in the mixture, OD 600 <30), collect the homogenized liquid and centrifuge it at 7000 rpm at 10℃ for 20 min, collect the supernatant and filter it, and collect the filtrate.

[0201] CM chromatography: Connect a CM chromatography column (Cytiva CM Sepharose Fast Flow, column height: 20.2 cm, CV: 107 mL) and equilibrate with Buffer A for 3 column volumes at a linear velocity of 100 cm / h until the CM chromatography column baseline is stable and the conductivity value is ≤100 ms / cm. The pH of the effluent of the CM chromatography column is 7.2. UV 280 When the absorption baseline is stable, the filtrate (CM loading solution) is loaded at 0.5 g / mL, the flow-through is collected and the CM column is re-equilibrated with Buffer A until UV 280 When the absorption baseline is stable, wash with Buffer E for 2 column volumes. Start collecting when UV>20mAU and stop collecting when UV<20mAU to obtain the washing solution. Observe the peak shape, UV and conductivity, and record the impurity peak volume. After washing, elute with Buffer C for 2 column volumes. When UV 280 When the elution volume is >100mAU, the eluate is collected and UV 280 When the concentration is less than 100 mAU, the collection is stopped and the CM chromatography eluate is collected. The CM chromatography eluate is the crude pure rhKGF-2 protein, and the CM chromatography column is regenerated, cleaned and stored.

[0202] Heparin chromatography: Connect a Heparin chromatography column (Cytiva Heparin Sepharose Fast Flow, column height: 15 cm, CV: 80 mL) and equilibrate with Buffer B for 3 column volumes at a linear velocity of 60 cm / h until the baseline of the Heparin chromatography column is stable and the conductivity value is ≤100 ms / cm. The pH of the effluent of the heparin chromatography column is 7.2. UV 280 When the absorption baseline is stable, mix the CM chromatography eluate with equal volumes of Buffer A, load the sample at 0.5 g / mL, collect the flow-through peak, and then re-equilibrate the Heparin chromatography column with Buffer B until UV 280 The absorption baseline is stable, then Buffer C is used to wash the impurities and wait for UV280 After the absorption baseline is flushed and stabilized, the heparin washing solution is collected and eluted with Buffer D for 2 column volumes. 280 When the elution volume is >100mAU, the eluate is collected and UV 280 When the concentration is less than 100 mAU, the collection is stopped to obtain the Heparin chromatography eluate (heparin eluate), which is the purified rhKGF-2. The quality of the purified rhKGF-2 is determined. The Heparin chromatography column is then regenerated, cleaned and stored.

[0203] 2. Detection of rhKGF-2 protein expression

[0204] The protein content of the purified rhKGF-2 obtained in S4 of step 1 was detected by Lowry method, and the purified rhKGF-2 obtained in S4 of step 1 was subjected to high performance liquid chromatography (HPLC) and SDS-PAGE gel electrophoresis to obtain the purity of the purified rhKGF-2 obtained in S4 of step 1.

[0205] The mass of the rhKGF-2 protein is calculated based on the purity of the purified ruKGF-2 and the mass of the purified rhKGF-2 protein obtained in step 1.

[0206] 3. Experimental results

[0207] The results of the expression level detection of the purified rhKGF-2 protein obtained in S4 in step 1 are shown in Table 3.

[0208] Table 3 Detection results of rhKGF-2 protein expression after purification

[0209] The results showed that the 500109-S8 constructed in Example 1 was combined with the method shown in step 1 to produce and purify KGF-2 protein, and a high-purity KGF-2 protein was obtained; and the mass of the purified rhKGF-2 protein was 7 g, and the yield reached 50% (theoretical yield was 14 g).

[0210] Example 3 Optimization of a method for producing and purifying rhKGF-2 protein

[0211] 1. Experimental Methods

[0212] 1. Effect of the inoculum size of recombinant engineered bacteria on the expression of rhKGF-2 protein

[0213] According to the production method of KGF-2 protein shown in step 1 of Example 2, the inoculation amount of the secondary seed liquid in S3 was changed to 15% (v / v), and rhKGF-2 protein was fermented and purified (denoted as F2023040218).

[0214] During the fermentation process, the OD value of the material in the fermentation tank was recorded every 2 hours. 600 , with fermentation time as the horizontal axis, OD 600 The growth curve of the strain when the inoculation amount was 15% was plotted as the vertical axis; the strains were collected after fermentation for 2h, 4h, 6h and 8h, and the protein expression level was detected by SDS-PAGE electrophoresis; the plasmid loss rate was detected by the plasmid loss rate test method.

[0215] The method shown in step 1 of Example 2 was used as a control group to detect the growth curve, protein expression level and plasmid loss rate of the strain (denoted as F2023042321) when the inoculation amount was 8%.

[0216] 2. Effect of fermentation substrate on rhKGF-2 protein expression

[0217] According to the production method of KGF-2 protein described in step 1 of Example 2, the base medium 1 in step S3 was replaced with M9Y medium, and the base medium 2 was replaced with 4 g / L carbon source medium, and fermentation was carried out to produce rhKGF-2 protein and purified (denoted as F2023042317); wherein the components of M9Y medium are: 10 g / L peptone, 10 g / L yeast extract, 12 g / L potassium dihydrogen phosphate, 3 g / L dipotassium hydrogen phosphate, 2 g / L ammonium sulfate, 1 g / L citric acid monohydrate, 0.02 g / L ferrous sulfate heptahydrate, and 3 g / L anhydrous magnesium sulfate.

[0218] During the fermentation process, the OD value of the material in the fermentation tank was recorded every 2 hours. 600 , with fermentation time as the horizontal axis, OD 600 The growth curve of the strain was drawn with the vertical axis; the strains were collected after fermentation for 2h, 4h, 6h and 8h, and the protein expression level was detected by SDS-PAGE electrophoresis; the plasmid loss rate was detected by the plasmid loss rate test method.

[0219] The method shown in step 1 of Example 2 was used as a control group (denoted as F2023042321), and the strain growth curve, protein expression level and plasmid loss rate when fermentation culture was carried out using the fermentation base material shown in step 1 of Example 2 were detected.

[0220] 3. Effect of inducer (IPTG) addition time on rhKGF-2 protein expression

[0221] According to the production method of KGF-2 protein shown in step 1 of Example 2, the addition time of IPTG in step S3 was changed to: OD 600 =50, fermentation was performed to produce rhKGF-2 protein and then purified (denoted as F2023042318).

[0222] During the fermentation process, the OD value of the material in the fermentation tank was recorded every 2 hours. 600 , with fermentation time as the horizontal axis, OD 600 The growth curve of the strain was drawn with the vertical axis; the strains were collected after fermentation for 2h, 4h, 6h and 8h, and the protein expression level was detected by SDS-PAGE electrophoresis; the plasmid loss rate was detected by the plasmid loss rate test method.

[0223] The method shown in step 1 of Example 2 was used as the control group to detect the 600 =40 when IPTG was added for fermentation culture, the strain growth curve, protein expression level and plasmid loss rate (denoted as F2023042321).

[0224] 4. Effect of inducer (IPTG) concentration on rhKGF-2 protein expression

[0225] According to the production method of KGF-2 protein described in step 1 of Example 2, the final concentration of IPTG in S3 was changed to 2.0 mM, and rhKGF-2 protein was fermented and purified (denoted as F2023043019).

[0226] During the fermentation process, the OD value of the material in the fermentation tank was recorded every 2 hours. 600 , with fermentation time as the horizontal axis, OD 600 The growth curve of the strain was drawn with the vertical axis; the strains were collected after fermentation for 2h, 4h, 6h and 8h, and the protein expression level was detected by SDS-PAGE electrophoresis; the plasmid loss rate was detected by the plasmid loss rate test method.

[0227] The method shown in step 1 of Example 2 was used as a control group (denoted as F2023042321), and the strain growth curve, protein expression level and plasmid loss rate were detected when the final IPTG concentration was 1.5 mM.

[0228] 5. Effect of induction temperature on rhKGF-2 protein expression

[0229] According to the production method of KGF-2 protein shown in step 1 of Example 2, the temperature of the fermentation tank in step S3 was adjusted to 45°C (i.e., the induction temperature was maintained at 45°C), and rhKGF-2 protein was fermented and purified (denoted as F2023042622).

[0230] During the fermentation process, the OD value of the material in the fermentation tank was recorded every 2 hours. 600 , with fermentation time as the horizontal axis, OD 600 The growth curve of the strain was drawn as the vertical axis; the strains were collected after fermentation for 2h, 4h, 6h and 8h, and the protein expression level was detected by SDS-PAGE electrophoresis, and the plasmid loss rate was detected by the plasmid loss rate test method.

[0231] The method shown in step 1 of Example 2 was used as a control group (denoted as F2023042321), and the growth curve, protein expression level and plasmid loss rate of the fermentation strain at an induction temperature of 40°C were detected.

[0232] 6. Effect of column height on rhKGF-2 protein purification during purification

[0233] Experimental Group 1: According to the production method of KGF-2 protein described in step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 13.3 cm, and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 7.5 cm; the linear velocity in the CM chromatography and heparin chromatography was adjusted to 150 cm / h, and no washing solution (Buffer E) was used for washing impurities during the CM chromatography process. The remaining treatments were the same to obtain low-column, highly purified rhKGF-2 protein, and the chromatograms during the CM chromatography process (low-column, high cation exchange chromatography spectrum) and the chromatograms during the heparin chromatography process (low-column, high affinity chromatography spectrum) were recorded.

[0234] According to the method shown in step 2 of Example 2, the protein content and purity of the rhKGF-2 protein after low-column high-purification were detected (HPLC and SDS-PAGE).

[0235] Control group 1 was as follows: the CM chromatography column with a column height of 13.3 cm was replaced with a CM chromatography column with a column height of 20.2 cm, and the Heparin chromatography column with a column height of 7.5 cm was replaced with a Heparin chromatography column with a column height of 15 cm, and the linear speed of the CM chromatography was controlled at 120 cm / h, and the linear speed of the heparin chromatography was controlled at 120 cm / h to obtain high-column and highly purified rhKGF-2 protein, and the chromatographic spectrum during the CM chromatography process (high-column high cation exchange chromatography spectrum) and the chromatographic spectrum during the heparin chromatography process (high-column high affinity chromatography spectrum) were recorded.

[0236] According to the method shown in step 2 of Example 2, the protein content and purity of the rhKGF-2 protein after high column purification were detected (HPLC and SDS-PAGE).

[0237] 7. Effect of purification process temperature on the purification effect of rhKGF-2 protein

[0238] Control group 2 was performed according to the method for producing KGF-2 protein described in step 1 of Example 2, except that the temperature of Buffer A to Buffer D used for column chromatography in S4 was controlled at 10°C, and no washing solution (Buffer E) was used for washing during CM chromatography. The remaining treatments were the same, resulting in crudely purified rhKGF-2 protein at a purification temperature of 10°C and finely purified rhKGF-2 protein at a purification temperature of 10°C. The heparin chromatography eluate obtained during heparin chromatography was collected in sections to obtain heparin elution 1 (80-791-500 mAU), heparin elution 2 (500-200 mAU), and heparin elution 3 (200-800 mAU).

[0239] The chromatograms during the CM chromatography process (cation exchange chromatography at a purification temperature of 10°C) and the chromatograms during the heparin chromatography process (affinity chromatography at a purification temperature of 10°C) were recorded. According to the method shown in step 2 of Example 2, the protein content and purity (HPLC and SDS-PAGE) of the crudely purified rhKGF-2 protein at a purification temperature of 10°C and the refined rhKGF-2 protein at a purification temperature of 10°C were detected. Heparin elution 1, heparin elution 2, heparin elution 3, and KGF-2 stock solution (a sample containing only KGF-2 protein) were subjected to SDS-PAGE gel electrophoresis.

[0240] 8. Effect of the washing step during CM chromatography on the purification of rhKGF-2 protein

[0241] Experimental Group 2: According to the production method of KGF-2 protein described in step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 21 cm (Cytiva CM Sepharose Fast Flow, column height: 21 cm; CV: 422 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 31 cm (Cytiva Heparin Sepharose Fast Flow, column height: 31 cm; CV: 165 mL). The remaining treatments were the same to obtain crudely purified rhKGF-2 protein after washing and finely purified rhKGF-2 protein after washing.

[0242] The chromatograms during the CM chromatography process (cation exchange chromatography after washing) and the chromatograms during the heparin chromatography process (affinity chromatography after washing) were recorded. The protein content and purity (HPLC and SDS-PAGE) of the crudely purified rhKGF-2 protein after washing and the refined purified rhKGF-2 protein after washing were detected according to the method shown in step 2 of Example 2.

[0243] The difference between control group 3 and experimental group 2 is that Buffer E also contains 2M urea. The remaining steps are exactly the same. RhKGF-2 protein crudely purified by urea washing and rhKGF-2 protein finely purified by urea washing are obtained, and the chromatograms during CM chromatography (cation exchange chromatography after urea washing) and the chromatograms during heparin chromatography (affinity chromatography after urea washing) are recorded; the protein content and purity of the rhKGF-2 protein finely purified by urea washing are detected by HPLC and SDS-PAGE according to the method shown in step 2 of Example 2; SDS-PAGE purity detection is performed on the CM load solution, flow-through solution, washing solution, rhKGF-2 protein crudely purified by urea washing, and rhKGF-2 protein finely purified by urea washing during CM chromatography, and SDS-PAGE electrophoresis detection is performed on the blank sample containing no protein.

[0244] 9. Effect of pH value on rhKGF-2 protein purification during heparin chromatography

[0245] Experimental group 3 was set up as follows: according to the production method of KGF-2 protein in step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 19 cm (Cytiva CM Sepharose Fast Flow, column height: 19 cm; CV: 100 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 25 cm (Cytiva Heparin Sepharose Fast Flow, column height: 25 cm; CV: 100 mL). Flow, column height: 25 cm; CV: 50 mL), and the remaining treatment was the same to obtain crudely purified rhKGF-2 protein at a pH of 7.2 during heparin chromatography and purified rhKGF-2 protein at a pH of 7.2 during heparin chromatography; the chromatograms during CM chromatography (cation exchange chromatography at pH 7.2) and the chromatograms during heparin chromatography (affinity chromatography at pH 7.2) were recorded, and the protein content and purity of the purified rhKGF-2 protein at pH 7.2 were detected (HPLC and SDS-PAGE) according to the method shown in step 2 of Example 2.

[0246] The difference between control group 4 and experimental group 3 is that the pH of Buffer A to Buffer D is changed to 7.0, and the other treatments remain unchanged. The crudely purified rhKGF-2 protein at pH 7.0 during the heparin chromatography process and the rhKGF-2 protein after being purified at pH 7.0 during the heparin chromatography process are obtained; the chromatogram during the CM chromatography process (cation exchange chromatography at pH 7.0) and the chromatogram during the heparin chromatography process (affinity chromatography at pH 7.0) are recorded, and the protein content and purity of the rhKGF-2 protein after being purified at pH 7.0 during the heparin chromatography process are detected by HPLC and SDS-PAGE according to the method shown in step 2 of Example 2; and the heparin wash solution during the heparin chromatography process and the rhKGF-2 protein after being purified at pH 7.0 during the heparin chromatography process are simultaneously subjected to SDS-PAGE purity detection.

[0247] The difference between control group 5 and experimental group 3 is that the pH of Buffer A to Buffer D is changed to 6.8, and the other treatments remain unchanged. The crudely purified rhKGF-2 protein at a pH of 6.8 during the heparin chromatography process and the rhKGF-2 protein purified at a pH of 6.8 during the heparin chromatography process are obtained; the chromatogram during the heparin chromatography process (affinity chromatography spectrum at a pH of 6.8) is recorded, and the protein content and purity of the rhKGF-2 protein purified at a pH of 6.8 during the heparin chromatography process are detected by HPLC and SDS-PAGE according to the method shown in step 2 of Example 2; at the same time, the CM load solution, flow-through solution, wash solution during the CM chromatography process, the crudely purified rhKGF-2 protein at a pH of 6.8 during the heparin chromatography process, the heparin wash solution during the heparin chromatography process, and the rhKGF-2 protein purified at a pH of 6.8 during the heparin chromatography process are subjected to SDS-PAGE purity detection.

[0248] The difference between control group 6 and experimental group 3 is that the pH of Buffer A to Buffer D is changed to 7.4, and the other treatments remain unchanged. The crude purified rhKGF-2 protein at a pH of 7.4 during the heparin chromatography process and the purified rhKGF-2 protein at a pH of 7.4 during the heparin chromatography process are obtained; the chromatogram during the heparin chromatography process (affinity chromatography at a pH of 7.4) is recorded, and the protein content and purity of the purified rhKGF-2 protein at a pH of 7.4 during the heparin chromatography process are detected (HPLC and SDS-PAGE) according to the method shown in step 2 of Example 2; and the heparin wash solution during the heparin chromatography process is subjected to SDS-PAGE purity detection.

[0249] 10. Effect of Buffer on the Purification of rhKGF-2 Protein during Heparin Chromatography

[0250] Experimental Group 5 was set up as follows: according to the production method of KGF-2 protein described in step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 19 cm (Cytiva CM Sepharose Fast Flow, column height: 19 cm; CV: 100 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 25 cm (Cytiva Heparin Sepharose Fast Flow, column height: 25 cm; CV: 50 mL). The other treatments remained unchanged to obtain the purified rhKGF-2 protein of Experimental Group 5.

[0251] The difference between control group 7 and experimental group 5 is that in the heparin chromatography, Buffer C was used to balance 3 column volumes at a linear speed of 100 cm / h until the baseline of the Heparin chromatography column was stable, and then the CM chromatography eluate was added at a rate of 0.01 to 10.0 g. (菌泥) / mL (CM填料) The sample was loaded to obtain the purified rhKGF-2 protein of control group 7; the chromatogram during the heparin chromatography process (affinity chromatography spectrum of control group 7) was recorded, and the protein content and purity (HPLC and SDS-PAGE) of the purified rhKGF-2 protein of control group 7 were detected according to the method shown in step 2 of Example 2.

[0252] The difference between control group 8 and control group 7 is that the CM chromatography eluate is adjusted to 0.01-10.0 g (菌泥) / mL (CM填料) After loading, the heparin flow-through was collected and then re-equilibrated with Buffer C for 3 column volumes. The re-equilibrated heparin flow-through was collected and then eluted with Buffer F (Buffer C and Buffer D were mixed in a volume ratio of 1:3, and the NaCl concentration was 0.8 M) for 2 column volumes. 100mAU<UV 280 The eluate when the concentration was less than 400 mAU was used to obtain the purified rhKGF-2 protein of control group 8; the chromatogram during CM chromatography (cation exchange chromatography of control group 8) and the chromatogram during heparin chromatography (affinity chromatography of control group 8) were recorded, and the protein content and purity (HPLC and SDS-PAGE) of the purified rhKGF-2 protein of control group 8 were detected according to the method shown in step 2 of Example 2.

[0253] 11. Effect of sample loading on rhKGF-2 protein purification during heparin chromatography

[0254] The heparin chromatography loading capacity of experimental group 5 was 1 g / mL (肝素填料)Specifically, according to the production method of KGF-2 protein shown in step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 19 cm (Cytiva CM Sepharose Fast Flow, column height: 19 cm; CV: 100 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 25 cm (Cytiva Heparin Sepharose Fast Flow, column height: 25 cm; CV: 50 mL). The other treatments remained unchanged to obtain the crudely purified rhKGF-2 protein of experimental group 5 and the purified rhKGF-2 protein of experimental group 5; and the chromatograms during the CM chromatography process (cation exchange chromatography spectrum of experimental group 5) and the chromatograms during the heparin chromatography process (affinity chromatography spectrum of experimental group 5) were recorded.

[0255] The difference between control group 9 and experimental group 5 is that during the heparin chromatography, the CM eluate was mixed with equal volumes of Buffer A and the mixture was heated at 0.6 g / mL. (肝素填料) The sample was loaded and the rest of the treatment was the same to obtain the purified rhKGF-2 protein of control group 9, and the chromatogram during the heparin chromatography process was recorded (affinity chromatography spectrum of control group 9).

[0256] The difference between control group 10 and experimental group 5 is that during the heparin chromatography, the CM eluate was mixed with equal volumes of Buffer A and the mixture was heated at 0.9 g / mL. (肝素填料) The sample was loaded and the rest of the treatment was the same to obtain the purified rhKGF-2 protein of the control group 10, and the chromatogram during the heparin chromatography process (affinity chromatography spectrum of the control group 10) was recorded.

[0257] According to the method shown in step 2 of Example 2, the protein content and purity (HPLC and SDS-PAGE) of the crudely purified rhKGF-2 protein of experimental group 5, the purified rhKGF-2 protein of experimental group 5, the purified rhKGF-2 protein of control group 9, and the purified rhKGF-2 protein of control group 10 were respectively detected.

[0258] 12. Effect of elution buffer on rhKGF-2 protein purification during heparin chromatography

[0259] The difference between the control group 11 and the method shown in Example 2 is that: in the heparin chromatography, Buffer G was used to elute 2 column volumes, and when UV 280 When the elution volume is >100mAU, the eluate is collected and UV 280When the concentration was less than 100 mAU, collection was stopped to obtain the purified rhKGF-2 protein of control group 11, wherein Buffer G differed from Buffer D in that Buffer G did not contain 20 mM EDTA-2Na. The chromatograms during the CM chromatography process (cation exchange chromatography of control group 11) and the chromatograms during the heparin chromatography process (affinity chromatography of control group 11) were recorded. The protein content and purity of the purified rhKGF-2 protein of control group 11 were detected by HPLC and SDS-PAGE according to the method described in step 2 of Example 2. At the same time, the CM loading solution, flow-through solution, wash solution, crude purified rhKGF-2 protein of control group 11, heparin wash solution, and purified rhKGF-2 protein of control group 11 during the CM chromatography process were detected by SDS-PAGE electrophoresis.

[0260] 13. Effects of Buffer pH and Tween 80 Addition on the Purification of rhKGF-2 Protein during Heparin Chromatography

[0261] Experimental Group 6 was set up as follows: according to the production method of KGF-2 protein described in step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 19 cm (Cytiva CM Sepharose Fast Flow, column height: 19 cm; CV: 100 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 25 cm (Cytiva Heparin Sepharose Fast Flow, column height: 25 cm; CV: 50 mL). The other treatments remained unchanged to obtain the purified rhKGF-2 protein of Experimental Group 6.

[0262] The difference between control group 12 and experimental group 6 is that the pH value of Buffer A to Buffer E was adjusted to 7.2, and Tween 80 was added to Buffer A to Buffer E to a final concentration of 0.05% (w / w). The other treatments remained unchanged. The purified rhKGF-2 protein of control group 12 was obtained. The chromatogram during CM chromatography (cation exchange chromatography of control group 12) and the chromatogram during heparin chromatography (affinity chromatography of control group 12) were recorded. The protein content and purity (HPLC) of the purified rhKGF-2 protein of control group 12 were detected according to the method shown in step 2 of Example 2. At the same time, SDS-PAGE electrophoresis was performed on the CM sample solution, flow-through solution, wash solution, crude purified rhKGF-2 protein of control group 12, heparin wash solution, and purified rhKGF-2 protein of control group 12 during CM chromatography.

[0263] 14. Effect of post-chromatographic membrane replacement on rhKGF-2 protein purification

[0264] Experimental Group 7 was set up as follows: according to the production method of KGF-2 protein shown in Step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 19 cm (Cytiva CM Sepharose Fast Flow, column height: 19 cm; CV: 373 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 25 cm (Cytiva Heparin Sepharose Fast Flow, column height: 25 cm; CV: 165 mL). The other treatments remained unchanged to obtain the crudely purified rhKGF-2 protein of Experimental Group 7 and the purified rhKGF-2 protein of Experimental Group 7; and the chromatograms during the CM chromatography process (cation exchange chromatography spectrum of Experimental Group 7) and the chromatograms during the heparin chromatography process (affinity chromatography spectrum of Experimental Group 7) were recorded.

[0265] The control group 13 was set up as follows: according to the production method of KGF-2 protein shown in step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 19 cm (Cytiva CM Sepharose Fast Flow, column height: 19 cm; CV: 373 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 25 cm (Cytiva Heparin Sepharose Fast Flow, column height: 25 cm; CV: 165 mL). CM chromatography and heparin chromatography were performed, and the Heparin chromatography eluate was collected. Buffer H (pH = 7.2, 30 mM PB) was used as the replacement membrane liquid. After mixing with an equal volume of the Heparin chromatography eluate, the mixture was replaced three times using a 2KD dialysis membrane to obtain the purified rhKGF-2 protein of the control group 13 and the permeate of the control group 13 was collected.

[0266] The difference between the control group 14 and the control group 13 is that the 2KD dialysis membrane is replaced with a 5KD dialysis membrane, and the purified rhKGF-2 protein and the permeate of the control group 14 are obtained.

[0267] According to the method shown in step 2 of Example 2, the protein content and purity (HPLC and SDS-PAGE) of the crudely purified rhKGF-2 protein of experimental group 7, the purified rhKGF-2 protein of experimental group 7, the purified rhKGF-2 protein of control group 13, the permeate of control group 13, the purified rhKGF-2 protein of control group 14, and the permeate of control group 14 were respectively detected.

[0268] 15. Effect of rhKGF-2 protein concentration on stability

[0269] Experimental Group 8 was set up as follows: according to the production method of KGF-2 protein shown in Step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 19 cm (Cytiva CM Sepharose Fast Flow, column height: 19 cm; CV: 373 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 25 cm (Cytiva Heparin Sepharose Fast Flow, column height: 25 cm; CV: 165 mL). CM chromatography and heparin chromatography were performed, the Heparin chromatography eluate was collected, and the chromatographic profile during the CM chromatography process (cation exchange chromatography profile of Experimental Group 8) and the chromatographic profile during the heparin chromatography process (affinity chromatography profile of Experimental Group 8) were recorded.

[0270] Buffer H (pH = 7.2, 30mM PB) was used as the replacement membrane fluid. The Heparin chromatography eluate and the replacement membrane fluid were mixed in equal volumes, and then replaced five times using a 2KD dialysis membrane. The replacement fluid was collected and diluted with Buffer H to rhKGF-2 protein concentrations of 3.3 mg / mL, 3.0 mg / mL, 2.0 mg / mL, 1.0 mg / mL, and 0.5 mg / mL. The dilutions of different concentrations were allowed to stand at 4°C for 1 day, and then subjected to high performance liquid chromatography (HPLC) and SDS-PAGE detection.

[0271] 16. Effect of adding SP chromatography after heparin chromatography on the purification of rhKGF-2 protein

[0272] Experimental Group 9 was set up as follows: according to the production method of KGF-2 protein shown in Step 1 of Example 2, the CM chromatography column in S4 was replaced with a CM chromatography column with a column height of 21.5 cm (Cytiva CM Sepharose Fast Flow, column height: 21.5 cm; CV: 422 mL), and the Heparin chromatography column in S4 was replaced with a Heparin chromatography column with a column height of 31 cm (Cytiva Heparin Sepharose Fast Flow, column height: 31 cm; CV: 165 mL). CM chromatography and heparin chromatography were performed to obtain the purified rhKGF-2 protein of Experimental Group 9, and the chromatographic profiles during the CM chromatography process (cation exchange chromatography profile of Experimental Group 9) and the chromatographic profiles during the heparin chromatography process (affinity chromatography profile of Experimental Group 9) were recorded.

[0273] After heparin chromatography, the heparin chromatography eluate was collected and mixed with an equal volume of PB (pH=7.2, 20 mM). After replacement 5 times using a 3KD dialysis membrane, the replacement fluid was collected; the heparin chromatography eluate and the replacement fluid were respectively detected by SDS-PAGE gel electrophoresis.

[0274] Connect an SP chromatography column (Borgron Diamond SP Mustang, column height: 25 cm; CV: 50 mL) and equilibrate with 30 mM PB (pH = 7.2) for 2 column volumes at a linear velocity of 150 cm / h until the baseline of the SP chromatography column is stable and the conductivity value is ≤100 ms / cm. The pH of the effluent of the CM chromatography column is 7.2. UV 280 When the absorption baseline was stable, the replacement solution was loaded at 3 mg / mL, the flow-through solution was collected, and the SP column was re-equilibrated with 30 mM PB (pH = 7.2) until UV 280 The absorption baseline was stable.

[0275] Then, 30 mM PB (pH = 7.2) and Buffer I (20 mM PB + 1 M NaCl, pH 7.2) were mixed for 40 column volumes of gradient elution, and eluate 1 was collected in sections; then, Buffer I was used for another 3 column volumes of elution, and eluate 2 was collected in sections (10 mAu < UV 280 <10mAu), and SP chromatography sample 1 was obtained.

[0276] After mixing equal volumes of SP chromatography sample 1 and 30 mM PB (pH = 7.2), the mixture was diluted with 30 mM PB (pH = 7.2) to a NaCl concentration of 0.6 M to obtain a loading solution;

[0277] The SP column was equilibrated with 30 mM PB (pH = 7.2) at a linear velocity of 150 cm / h for 3 column volumes until the baseline of the SP column was stable and the conductivity value was ≤ 100 ms / cm. The pH of the effluent of the CM column was 7.2. UV 280 When the absorption baseline was stable, the sample solution was loaded at 3 mg / mL, and the SP chromatography column was re-equilibrated with 30 mM PB (pH = 7.2) until UV 280 The absorption baseline was stable, and 3 column volumes of Buffer I was used for washing, followed by 15 column volumes of gradient elution using 30 mM PB (pH = 7.2) mixed with Buffer I, and the SP eluate was collected in sections to obtain the rhKGF-2 protein after SP chromatography; wherein the SP eluate was collected in sections to collect UV 280The SP eluate at 5-10 mAu, the SP eluate at 18-38 mAu, the SP eluate at 230-560 mAu, the SP eluate at 560-peak-560 mAu, the SP eluate at 560-350 mAu, the SP eluate at 130-45 mAu and the SP eluate at 20-10 mAu were collected, and the SP eluates collected in segments were subjected to SDS-PAGE gel electrophoresis.

[0278] Protein content (Lowry), high performance liquid chromatography (HPLC) and SDS-PAGE purity were tested for heparin chromatography fluid, replacement fluid, SP eluate at 230-560 mAu, SP eluate at 560-peak-560 mAu and SP eluate at 560-350 mAu.

[0279] 2. Experimental Results

[0280] 1. Effect of the inoculum size of recombinant engineered bacteria on the expression of rhKKG-2 protein

[0281] When the recombinant engineered bacteria with different inoculum amounts produced rhKGF-2 protein according to the method shown in step 1 of Example 2, the growth curve of the strain is shown in Figure 14; the SDS-PAGE detection results are shown in Figure 15, wherein lanes 1 and lane 6 are the electrophoresis results of the marker, lanes 2 to 5 are the electrophoresis results of the recombinant engineered bacteria with an inoculum amount of 8% at 2h, 4h, 6h and 8h of fermentation, respectively, and lanes 7 to 10 are the electrophoresis results of the recombinant engineered bacteria with an inoculum amount of 15% at 2h, 4h, 6h and 8h of fermentation; the results of the plasmid loss rate detection are shown in Table 4.

[0282] Table 4 Plasmid loss rate detection results when recombinant engineering bacteria with different inoculation amounts produced rhKGF-2 protein

[0283] The results showed that during the production of rhKGF-2 protein, there was no significant difference in the growth curve of the strain regardless of whether the inoculation size of the recombinant engineered bacteria (500109-S9) was 8% or 15%; and there was no significant difference in the expression level of rhKGF-2 protein and the plasmid loss rate; this indicated that increasing the inoculation size of the recombinant engineered bacteria would not increase the expression level of rhKGF-2 protein, and the fermentation conditions (temperature, pH and fermentation substrate) had reached the optimal state for cell growth and rhKGF-2 protein expression, so an inoculation size of 8% was selected as the optimal inoculation size for the production of rhKGF-2 protein.

[0284] 2. Effect of fermentation substrate on rhKKG-2 protein expression

[0285] When rhKGF-2 protein was produced using different fermentation substrates according to the method shown in step 1 of Example 2, the growth curve of the strain was shown in Figure 16; the SDS-PAGE detection results were shown in Figure 17, wherein lanes 1 and 6 were the electrophoresis results of the marker, lanes 2 to 5 were the electrophoresis results of the recombinant engineered bacteria using M9Y medium at 2h, 4h, 6h and 8h of fermentation, and lanes 7 to 10 were the electrophoresis results of the recombinant engineered bacteria using the fermentation substrate shown in step 1 of Example 2 at 2h, 4h, 6h and 8h of fermentation; the results of the plasmid loss rate detection were shown in Table 5.

[0286] Table 5 Plasmid loss rate test results of recombinant engineering bacteria producing rhKGF-2 protein using different fermentation substrates

[0287] The results showed that during the production of rhKGF-2 protein, after the fermentation substrate shown in step 1 of Example 2 was replaced with M9Y medium, the early growth trend of the recombinant engineered bacteria significantly deteriorated; when two different fermentation substrates were used in combination with the recombinant engineered bacteria to produce rhKGF-2 protein, there was no significant difference in protein expression; although the plasmid loss rate was lower when the recombinant engineered bacteria were cultured using M9Y medium as the fermentation substrate to produce rhKGF-2 protein, the growth trend of the strain when the engineered bacteria were cultured using M9Y medium was significantly worse than that when the recombinant engineered bacteria were cultured using the fermentation substrate shown in step 1 of Example 2; therefore, the fermentation substrate shown in step 1 of Example 2 was selected for the fermentation of the recombinant engineered bacteria to produce rhKGF-2 protein.

[0288] 3. Effect of inducer addition time on rhKGF-2 protein expression

[0289] According to the method shown in step 1 of Example 2, the inducer (IPTG) was added at different times to produce rhKGF-2 protein. The growth curve of the strain is shown in FIG18 ; the SDS-PAGE detection results are shown in FIG19 , wherein lanes 1 to 4 are the growth curves of the strain at OD 600 =50, the electrophoresis results of the recombinant engineering bacteria after adding IPTG at 2h, 4h, 6h and 8h of fermentation, lane 5 is the electrophoresis result of the marker, lanes 6 to 9 are the electrophoresis results of the marker at OD 600 =40, and the electrophoresis results of the recombinant engineering bacteria after adding IPTG at 2h, 4h, 6h and 8h of fermentation; the results of the plasmid loss rate detection are shown in Table 6.

[0290] Table 6 Plasmid loss rate test results of recombinant engineering bacteria producing rhKGF-2 protein by adding IPTG at different times

[0291] The results showed that: whether in OD 600 =40 or OD600 When the inducer (IPTG) was added at pH = 50, no significant difference was found in the growth curves of the strains; and SDS-PAGE detection results showed no significant difference in protein expression levels.

[0292] The higher OD 600 The bacterial cells will be in a state of stress, facing more metabolic load, and the metabolic balance will be broken, resulting in a decrease in growth rate and the production of undesirable metabolites and waste; at the same time, the lower OD 600 It can control the expression level of rhKGF-2 protein, keep it at a lower synthesis rate, and reduce the occurrence of protein aggregation or degradation. 600 When pH = 40, inducer (IPTG) was added for induction.

[0293] 4. Effects of different inducer concentrations on rhKGF-2 protein expression

[0294] According to the method shown in step 1 of Example 2, different final concentrations of inducer (IPTG) were added to produce rhKGF-2 protein, and the growth curve of the strain is shown in Figure 20; the SDS-PAGE detection results are shown in Figure 21, wherein lanes 1 and lane 6 are the electrophoresis results of the marker, lanes 2 to 5 are the electrophoresis results of the recombinant engineered bacteria at a final IPTG concentration of 1.5 mM at 2 h, 4 h, 6 h and 8 h of fermentation, respectively, and lanes 7 to 10 are the electrophoresis results of the recombinant engineered bacteria at a final IPTG concentration of 2.0 mM at 2 h, 4 h, 6 h and 8 h of fermentation; the results of the plasmid loss rate detection are shown in Table 7.

[0295] Table 7 Plasmid loss rate test results of recombinant engineering bacteria producing rhKGF-2 protein by adding different final concentrations of inducers

[0296] The results showed that after the final concentration of IPTG in step 1 of Example 2 was changed from 1.5 mM to 2.0 mM, the growth trend of the recombinant engineered bacteria increased; the SDS-PAGE detection results showed that after the final concentration of IPTG in step 1 of Example 2 was changed from 1.5 mM to 2.0 mM, the expression level of rhKGF-2 protein decreased, but there was no significant difference; after the final concentration of IPTG in step 1 of Example 2 was changed from 1.5 mM to 2.0 mM, the plasmid loss rate during the fermentation production of rhKGF-2 protein also showed an increasing trend, but the difference was not significant; and excessively high concentrations of IPTG would have a toxic effect on cells, resulting in cell growth inhibition or metabolic disorders, so TPTG with a final concentration of 1.5 mM was selected as an inducer to induce the recombinant engineered bacteria to produce rhKGF-2 protein.

[0297] 5. Effects of different induction temperatures on rhKGF-2 protein expression

[0298] According to the method shown in step 1 of Example 2, the growth curve of the recombinant engineered bacteria when producing rhKGF-2 protein at different temperatures is shown in Figure 22; the SDS-PAGE detection results are shown in Figure 23, wherein lane 1 is the electrophoresis result of the marker, lanes 2 to 5 are the electrophoresis results of the recombinant engineered bacteria when the induction temperature is 40°C for 2h, 4h, 6h and 8h, respectively, and lanes 1 to 9 are the electrophoresis results of the recombinant engineered bacteria when the induction temperature is 45°C for 2h, 4h, 6h and 8h; the plasmid loss rate detection results are shown in Table 8.

[0299] Table 8 Plasmid loss rate test results of recombinant engineering bacteria producing rhKGF-2 protein at different induction temperatures

[0300] The results showed that after the temperature of the fermentation tank in step 1 of Example 2 was changed to 45°C (i.e., the induction temperature was 45°C), the growth trend of the strain during the production of rhKGF-2 protein by the recombinant engineered bacteria decreased, and the expression level of the rhKGF-2 protein decreased significantly.

[0301] Therefore, the induction temperature of 40°C was selected to induce the recombinant engineered bacteria to ferment and produce rhKGF-2 protein.

[0302] 6. Effect of column height on rhKGF-2 protein purification

[0303] The low-column high cation exchange chromatography spectrum is shown in FIG24 , the high-column high cation exchange chromatography spectrum is shown in FIG25 , the low-column high-affinity chromatography spectrum is shown in FIG26 , and the high-column high-affinity chromatography spectrum is shown in FIG27 .

[0304] The SDS-PAGE detection results of different column heights are shown in Figure 28, where lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the rhKGF-2 protein after high column and high purification, and lane 3 is the electrophoresis result of the rhKGF-2 protein after low column and high purification; the detection results of the rhKGF-2 protein after high purification on different columns are shown in Table 9.

[0305] Table 9 rhKGF-2 protein detection results after high purification by different columns

[0306] The results showed that increasing the column height of the CM chromatography column and the Heparin chromatography column during the purification process did not significantly improve the purity of the purified rhKGF-2 protein, indicating that increasing the column height of the chromatography column had little effect on the purification effect of the rhKGF-2 protein.

[0307] 7. Effect of purification temperature on rhKGF-2 protein purification results

[0308] The cation exchange chromatography spectrum at a purification temperature of 10°C is shown in Figure 29, and the affinity chromatography spectrum at a purification temperature of 10°C is shown in Figure 30; the SDS-PAGE detection results at a purification temperature of 10°C are shown in Figure 31, wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the rhKGF-2 protein (i.e., CM elution) after crude purification at a purification temperature of 10°C, lane 3 is the electrophoresis result of the heparin elution 1 (80-791-500mAU) sample, lane 4 is the electrophoresis result of the heparin elution 2 (500-200mAU) sample, lane 5 is the electrophoresis result of the heparin elution 3 (200-800mAU) sample, lane 6 is the electrophoresis result of the rhKGF-2 protein after fine purification at a purification temperature of 10°C, and lane 7 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein).

[0309] The protein detection results of the crudely purified rhKGF-2 protein at a purification temperature of 10°C and the finely purified rhKGF-2 protein at a purification temperature of 10°C are shown in Table 10.

[0310] Table 10 Protein detection results of rhKGF-2 protein after purification at 10°C

[0311] The results showed that the SDS-PAGE purity of the rhKGF-2 protein after purification at a purification temperature of 10°C was 93.8%, and the HPLC purity was 96.57%. Compared with the results of the method shown in Example 2, the purity of the heparin elution peak (purified rhKGF-2 protein) was basically unchanged, indicating that controlling the buffer temperature (4-12°C) during the purification chromatography process and further controlling the purification temperature did not significantly affect the purification effect of the KGF-2 protein.

[0312] 8. The effect of the washing step during CM chromatography on the purification of rhKGF-2 protein

[0313] The cation exchange chromatography spectrum after washing is shown in Figure 32, and the affinity chromatography spectrum after washing is shown in Figure 33; the SDS-PAGE detection results after washing are shown in Figure 34, wherein lane 1 is the electrophoresis result of the rhKGF-2 protein after washing and purification, lane 2 is the electrophoresis result of the marker, lane 3 is the electrophoresis result of the rhKGF-2 protein after washing and purification, and lane 4 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein).

[0314] The protein detection results of the crudely purified rhKGF-2 protein after washing and the finely purified rhKGF-2 protein after washing are shown in Table 11.

[0315] Table 11 Protein detection results of the crudely purified rhKGF-2 protein after washing and the refined purified rhKGF-2 protein after washing

[0316] The results showed that adding a washing step (using Buffer E for washing) during CM chromatography significantly weakened the impurity bands of the crudely purified rhKGF-2 protein, thereby improving the purity of the crudely purified rhKGF-2 protein (CM chromatography eluate).

[0317] The cation exchange chromatography spectrum after urea washing is shown in Figure 35, and the affinity chromatography spectrum after urea washing is shown in Figure 36; the SDS-PAGE detection results of control group 3 are shown in Figure 37, wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the CM load solution, lane 3 is the electrophoresis result of the flow-through solution, lane 4 is the electrophoresis result of the washing solution, lane 5 is the electrophoresis result of the rhKGF-2 protein after crude purification by urea washing, lane 6 is the electrophoresis result of the Blank sample, lane 7 is the electrophoresis result of the rhKGF-2 protein after fine purification by urea washing, and lane 8 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein).

[0318] The protein detection results of the rhKGF-2 protein after urea washing and purification are shown in Table 12.

[0319] Table 12 Protein detection results of rhKGF-2 protein after urea washing and purification

[0320] The results showed that compared with experimental group 2, the impurity bands and aggregate bands of the crude rhKGF-2 protein obtained by washing with Buffer E containing 2 M urea during CM chromatography were weakened ( Figure 37 , lane 5). In addition, the HPLC purity of the purified rhKGF-2 protein reached 97.17%, and the SDS-PAGE purity was 94.2%.

[0321] Urea, as a denaturant, can change the hydrophobicity of proteins at low concentrations, thereby affecting protein interactions. However, high concentrations of urea can disrupt the tertiary and quaternary structures of proteins by breaking hydrophobic bonds and hydrogen bonds, thereby improving protein solubility. Therefore, adding urea to Buffer E may destroy the structure of rhKGF-2 protein and does not significantly improve protein purity. Therefore, urea is not added to the wash buffer (Buffer E).

[0322] 9. Effect of pH during heparin chromatography on the purification of rhKGF-2 protein

[0323] The cation exchange chromatography spectrum at pH 7.2 is shown in Figure 38 , the affinity chromatography spectrum at pH 6.8 is shown in Figure 39 , the affinity chromatography spectrum at pH 7.0 is shown in Figure 40 , and the affinity chromatography spectrum at pH 7.4 is shown in Figure 41 .

[0324] The SDS-PAGE detection results at pH 6.8 are shown in Figure 42, wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein), lane 3 is the electrophoresis result of the rhKGF-2 protein after purification at pH 6.8 during heparin chromatography, lane 4 is the electrophoresis result of the Blank control, lane 5 is the electrophoresis result of the wash solution, lane 6 is the electrophoresis result of the CM loading solution, lane 7 is the electrophoresis result of the flow-through solution, lane 8 is the electrophoresis result of the rhKGF-2 protein after crude purification at pH 6.8 during heparin chromatography, and lane 9 is the electrophoresis result of the heparin wash solution;

[0325] The SDS-PAGE detection results at pH 7.0 are shown in Figure 43, wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the heparin wash solution, lane 3 is the electrophoresis result of the rhKGF-2 protein after purification at pH 7.0 during the heparin chromatography process, and lane 4 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein);

[0326] The SDS-PAGE detection results at pH 7.4 are shown in Figure 44, where lane 1 is the electrophoresis result of the purified rhKGF-2 protein at pH 7.4 during the heparin chromatography process, lane 2 is the electrophoresis result of the heparin elution peak, lane 3 is the electrophoresis result of the marker, and lane 4 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein).

[0327] The protein detection results of the purified rhKGF-2 protein obtained by heparin chromatography at different pH values ​​are shown in Table 12.

[0328] Table 12 Protein detection results of purified rhKGF-2 protein obtained by heparin chromatography at different pH values

[0329] The results showed that there was no significant difference in the HPLC purity of the purified rhKGF-2 protein when the pH value during the heparin chromatography process was changed; however, when the pH value during the heparin chromatography process was 7.2, the SDD-PAGE purity of the purified rhKGF-2 protein was the highest and significantly higher than that of other samples; therefore, the optimal pH value during the heparin chromatography process was 7.2.

[0330] 10. Effect of Buffer on rhKGF-2 Protein Purification During Heparin Chromatography

[0331] The affinity chromatography spectrum of the control group 7 is shown in Figure 45; the SDS-PAGE detection results of the rhKGF-2 protein after purification of the control group 7 are shown in Figure 46, wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the heparin elution peak, lane 3 is the electrophoresis result of the rhKGF-2 protein after purification of the control group 7, and lane 4 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein); the protein detection results of the rhKGF-2 protein after purification of the control group 7 are shown in Table 13.

[0332] Table 13 Protein detection results of rhKGF-2 protein after purification in control group 7

[0333] The results showed that compared with experimental group 5, during the heparin chromatography process, the CM chromatography eluate was directly loaded after equilibration of the Heparin chromatography column with 0.9 M NaCl (Buffer C), and no significant difference was observed in the SDS-PAGE purity; however, loading the sample directly after equilibration of the column with 0.9 M NaCl (Buffer C) would cause the target protein (rhKGF-2 protein) and impurity proteins with similar electronegativity to the target protein to be attached to the chromatography column at the same time, and the high concentration of NaCl would weaken the binding ability between the target protein and the chromatography column, resulting in a low recovery rate.

[0334] The cation exchange chromatography spectrum of control group 8 is shown in Figure 47, and the affinity chromatography spectrum of control group 8 is shown in Figure 48; the SDS-PAGE detection results of control group 8 are shown in Figure 49, wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the rhKGF-2 protein after purification of control group 8, lane 3 is the electrophoresis result of the customer's original solution, lane 4 is the electrophoresis result of the CM loading solution, lane 5 is the electrophoresis result of the flow-through liquid, lane 6 is the electrophoresis result of the wash solution, lane 7 is the electrophoresis result of the rhKGF-2 protein after crude purification of control group 8, lane 8 is the electrophoresis result of the heparin flow-through liquid, and lane 9 is the electrophoresis result of the re-equilibrium heparin flow-through liquid; the protein detection results of the rhKGF-2 protein after purification of control group 8 are shown in Table 14.

[0335] Table 14 Protein detection results of the purified rhKGF-2 protein in control group 8

[0336] The results showed that the purity of rhKGF-2 protein did not improve after the Heparin chromatography column was equilibrated with 0.9 M NaCl (Buffer C), the CM chromatography eluate was directly loaded, and then eluted at a concentration of 0.8 M NaCl. In addition, the SDS-PAGE test results showed that there were still impurity bands below the purified rhKGF-2 protein in control group 8, indicating that elution with Buffer F containing 0.8 M NaCl would not improve the purity of rhKGF-2.

[0337] 11. Effect of sample loading on rhKGF-2 protein purification during heparin chromatography

[0338] The cation exchange chromatography spectrum of experimental group 5 is shown in Figure 50, the affinity chromatography spectrum of experimental group 5 is shown in Figure 51, the affinity chromatography spectrum of control group 9 is shown in Figure 52, and the affinity chromatography spectrum of control group 10 is shown in Figure 53; the SDS-PAGE detection results of different loading amounts are shown in Figure 54, wherein lane 1 is the electrophoresis result of the crude purification of rhKGF-2 protein in experimental group 5, lane 2 is the electrophoresis result of the rhKGF-2 protein in control group 9 after fine purification, lane 3 is the electrophoresis result of the rhKGF-2 protein in experimental group 5 after fine purification, lane 4 is the electrophoresis result of the rhKGF-2 protein in control group 10 after fine purification, lane 5 is the electrophoresis result of the customer 02 stock solution, and lane 6 is the electrophoresis result of the marker.

[0339] The protein detection results of the purified rhKGF-2 protein in experimental group 5, the purified rhKGF-2 protein in control group 9, and the purified rhKGF-2 protein in control group 10 are shown in Table 15.

[0340] Table 15 Protein detection results

[0341] The results showed that the sample loading during heparin chromatography had a certain influence on the separation degree of the purified rhKGF-2 protein. When the sample loading during heparin chromatography was 1 g / ml, the purity of the purified rhKGF-2 protein was the highest.

[0342] 12. Effect of elution buffer on rhKGF-2 protein purification during heparin chromatography

[0343] The cation exchange chromatography spectrum of the control group 11 is shown in Figure 55, and the affinity chromatography spectrum of the control group 11 is shown in Figure 56; the SDS-PAGE detection results of different elution buffers are shown in Figure 57, wherein lane 1 is the electrophoresis result of the marker, lane 2 and lane 4 are the electrophoresis results of the rhKGF-2 protein after purification of the control group 11, lane 3 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein), lane 5 is the electrophoresis result of the CM loading solution, lane 6 is the electrophoresis result of the flow-through solution, lane 7 is the electrophoresis result of the rhKGF-2 protein after crude purification of the control group 11, lane 8 is the electrophoresis result of the wash solution, and lane 9 is the electrophoresis result of the heparin wash solution.

[0344] The protein detection results of the purified rhKGF-2 protein in the control group 11 are shown in Table 16.

[0345] Table 16 Protein detection results of rhKGF-2 protein after purification of control group 11

[0346] The results showed that when the rhKGF-2 protein was purified according to the method shown in control group 11 and Buffer G (i.e., without adding EDTA-2Na) was used for heparin chromatography elution, the HPLC purity of the obtained rhKGF-2 decreased to 94.12% and the SDS-PAGE purity was 95.6% compared with the experimental results of Example 2; this indicates that adding EDTA-2Na to the buffer during heparin chromatography elution can make the purity of the obtained rhKGF-2 higher.

[0347] 13. Effects of Buffer pH and Tween 80 Addition on the Purification of rhKGF-2 Protein During Heparin Chromatography

[0348] The cation exchange chromatography spectrum of the control group 12 is shown in Figure 58, and the affinity chromatography spectrum of the control group 12 is shown in Figure 59; the SDS-PAGE detection results of buffers with different pH values ​​are shown in Figure 60, wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein), lane 3 is the electrophoresis result of the rhKGF-2 protein after purification of the control group 12, lane 4 is the electrophoresis result of the CM loading solution, lane 5 is the electrophoresis result of the flow-through solution, lane 6 is the electrophoresis result of the wash solution, lane 7 is the electrophoresis result of the rhKGF-2 protein after crude purification of the control group 12, and lane 8 is the electrophoresis result of the heparin wash solution.

[0349] The protein detection results of the purified rhKGF-2 protein in the control group 12 are shown in Table 17.

[0350] Table 17 Protein detection results of rhKGF-2 protein after purification of control group 12

[0351] The results showed that when the pH value of Buffer A to Buffer E during the purification process was adjusted to 7.2 and Tween 80 was added to Buffer A to Buffer E to a final concentration of 0.05% (w / w), electrophoresis results showed that the target protein band was degraded and the impurity protein band was significantly darkened. In addition, the purity of the obtained rhKGF-2 protein decreased significantly, and the HPLC test results were abnormal. This shows that Tween 80 is not suitable for all protein purifications. Adding Tween 80 to the purification buffer and adjusting the pH will significantly reduce the final purification effect.

[0352] 14. Effect of post-chromatographic membrane replacement on rhKGF-2 protein purification

[0353] The cation exchange chromatography spectrum of experimental group 7 is shown in Figure 61, and the affinity chromatography spectrum of experimental group 7 is shown in Figure 62; the SDS-PAGE detection results of adding displacement envelope after chromatography are shown in Figure 63, wherein lane 1 is the electrophoresis result of the rhKGF-2 protein after crude purification of experimental group 7, lane 2 is the electrophoresis result of the marker, lane 3 is the electrophoresis result of the rhKGF-2 protein after purification of experimental group 7, lane 4 is the electrophoresis result of the KGF-2 stock solution (a sample containing only KGF-2 protein), lane 5 is the electrophoresis result of the rhKGF-2 protein after purification of control group 14, lane 6 is the electrophoresis result of the permeate of control group 14, lane 7 is the electrophoresis result of the rhKGF-2 protein after purification of control group 13, and lane 8 is the electrophoresis result of the permeate of control group 13.

[0354] The protein detection results are shown in Table 18.

[0355] Table 18 Protein detection results

[0356] The results showed that compared with experimental group 7, the concentration, HPLC purity and SDS-PAGE purity of the purified rhKGF-2 protein in the control group 13 obtained by using Buffer H without NaCl and EDTA as the replacement membrane liquid in combination with a 2KD dialysis membrane for replacement had no significant difference; however, after replacing the 2KD dialysis membrane with a 5KD dialysis membrane, the rhKGF-2 protein would pass through the 5KD dialysis membrane, and the concentration of the purified rhKGF-2 protein in the control group 14 decreased significantly; therefore, there is no need to use a dialysis membrane for replacement after heparin chromatography.

[0357] 15. Effect of rhKGF-2 protein concentration on stability

[0358] The cation exchange chromatography spectrum of experimental group 8 is shown in Figure 64, and the affinity chromatography spectrum of experimental group 8 is shown in Figure 65; the SDS-PAGE detection results of different rhKGF-2 protein concentrations are shown in Figure 66, wherein lane 1 is the electrophoresis result of the marker, lane 2 is the electrophoresis result of the blank, lane 3 is the electrophoresis result of the customer's original solution, lane 4 is the electrophoresis result of the 3.3 mg / mL dilution solution, lane 5 is the electrophoresis result of the 3.0 mg / mL dilution solution, lane 6 is the electrophoresis result of the 2.0 mg / mL dilution solution, and lane 7 is the electrophoresis result of the 1.0 mg / mL dilution solution.

[0359] The protein detection results of the dilutions with different concentrations are shown in Table 19.

[0360] Table 19 Protein detection results of dilutions of different concentrations

[0361] The results showed that the lower the rhKGF-2 protein concentration in the dilution, the lighter the KGF-2 protein and impurity protein bands in the SDS-PAGE electrophoresis results, and the lower the HPLC purity; and too high rhKGF-2 protein concentration will cause protein aggregation.

[0362] Therefore, when the concentration of rhKGF-2 protein was controlled at 2.0 mg / mL, the purity of rhKGF-2 protein by SDS-PAGE and HPLC was higher.

[0363] 16. Effect of adding SP chromatography after heparin chromatography on the purification of rhKGF-2 protein

[0364] The cation exchange chromatography spectrum of experimental group 9 is shown in Figure 67, the affinity chromatography spectrum of experimental group 9 is shown in Figure 68, and the SP chromatography spectrum of experimental group 9 is shown in Figure 69; the SDS-PAGE electrophoresis of the heparin chromatography eluate and the replacement fluid of experimental group 9 is shown in Figure 70, where lane 1 is the electrophoresis result of the heparin chromatography eluate and lane 2 is the electrophoresis result of the replacement fluid; the SDS-PAGE electrophoresis result of the SP eluate in experimental group 9 is shown in Figure 71, where lane 1 is marker Lane 2 is the customer 02 stock solution, lane 3 is the SP eluate at 5-10 mAu, lane 4 is the SP eluate at 18-38 mAu, lane 5 is the SP eluate at 230-560 mAu, lane 6 is the SP eluate at 560-peak-560 mAu, lane 7 is the SP eluate at 560-350 mAu, lane 8 is the SP eluate at 130-45 mAu, and lane 9 is the SP eluate at 20-10 mAu.

[0365] The protein detection results of experimental group 9 are shown in Table 20.

[0366] Table 20 Protein detection results of experimental group 9

[0367] The results showed that when SP chromatography was added after CM chromatography and heparin chromatography, the target protein (KGF-2 protein) and the impurity bands could not be further separated, and the purity of KGF-2 protein could not be further effectively improved.

[0368] Example 4: Scale-up of a method for producing and purifying rhKGF-2 protein

[0369] 1. Experimental methods

[0370] (1) 1L process scale-up and purification of rhKGF-2 protein

[0371] Fermentation was performed according to the method shown in S1 to S3 of step 1 of Example 2 to obtain fermented engineered bacteria.

[0372] As shown in S4 of Step 1 of Example 2, Buffer A to Buffer E were prepared for column chromatography, and the pH value of Buffer A to Buffer E was adjusted to 7.2.

[0373] The fermented engineered bacteria were mixed with Buffer A at a ratio of 1 g: 20 mL, and the mixture was stirred thoroughly to obtain a mixture. The mixture was homogenized at 200 Bar and then at 700 Bar at 15 ° C for one time and then twice. After the mixture was completely broken (i.e., there were no intact bacteria in the mixture, OD 600 <30), collect the homogenized liquid and centrifuge it at 7000 rpm at 10℃ for 20 min, collect the supernatant and filter it, and collect the filtrate.

[0374] Connect a CM chromatography column (Borgron CM Bestarose Fast Flow, CV: 1.4 L) and equilibrate with Buffer A for 3 column volumes at a linear velocity of 100 cm / h until the baseline of the CM chromatography column is stable and the conductivity value is ≤100 ms / cm. The pH of the effluent of the CM chromatography column is 7.2. UV 280 When the absorption baseline is stable, the filtrate (CM loading solution) is loaded at 0.8 g / mL, the flow-through liquid is collected, and the CM chromatography column is re-equilibrated with Buffer A until UV 280 When the absorption baseline is stable, wash with Buffer E for 2 column volumes, start collecting when UV>20mAU, stop collecting when UV<20mAU, and obtain the washing solution. Then elute with Buffer C for 2 column volumes. When UV 280 When the elution volume is >100mAU, the eluate is collected and UV 280When the concentration is less than 100 mAU, the collection is stopped and the CM chromatography eluate is collected. The CM chromatography eluate is the crude pure rhKGF-2 protein. After the elution is completed, the CM chromatography column is regenerated, cleaned and stored.

[0375] Connect a Heparin chromatography column (Borgron Heparin Bestarose Fast Flow, CV: 450 mL) and equilibrate with Buffer B for 3 column volumes at a linear velocity of 60 cm / h until the baseline of the Heparin chromatography column is stable and the conductivity value is ≤100 ms / cm. The pH of the effluent of the heparin chromatography column is 7.2. UV 280 When the absorption baseline is stable, mix the CM chromatography eluate with equal volumes of Buffer A, load the sample at 1 g / mL, collect the flow-through peak, and then re-equilibrate the Heparin chromatography column with Buffer B until UV 280 The absorption baseline is stable, then Buffer C is used to wash the impurities and wait for UV 280 After the absorption baseline is flushed and stabilized, the heparin washing solution is collected and eluted with Buffer D for 2 column volumes. 280 When the elution volume is >100mAU, the eluate is collected and UV 280 When the concentration is less than 100 mAU, the collection is stopped to obtain the Heparin chromatography eluate, which is the purified rhKGF-2; after the elution is completed, the Heparin chromatography column is regenerated, cleaned and stored.

[0376] Buffer H (pH=7.2, 30 mM PB) was used as the membrane replacement solution. After being mixed with an equal volume of purified rhKGF-2, the solution was replaced three times using a 2KD dialysis membrane, and the rhKGF-2 replacement solution was collected.

[0377] (2) Protein expression detection

[0378] The purified rhKGF-2, rhKGF-2 replacement solution and KGF-2 stock solution (sample containing only KGF-2 protein) were subjected to SDS-PAGE gel electrophoresis detection, and the rhKGF-2 replacement solution was subjected to HPLC detection and protein content detection.

[0379] 2. Experimental results

[0380] The results of SDS-PAGE gel electrophoresis are shown in Figure 72, where lane 1 is a marker, lane 2 is a blank lane, lane 3 is rhKGF-2 replacement fluid, lane 4 is purified rhKGF-2, and lane 5 is KGF-2 stock solution (a sample containing only KGF-2 protein); the rhKGF-2 replacement fluid detection results are shown in Table 21.

[0381] Table 21 rhKGF-2 replacement fluid test results

[0382] The results showed that when the 1L process was scaled up to produce rhKGF-2 protein, the HPLC purity and SDS-PAGE purity of the obtained rhKGF-2 protein were not significantly different from those of the rhKGF-2 produced by the method shown in Example 2, indicating that the method for producing rhKGF-2 protein shown in Example 2 can be scaled up.

[0383] Comparative Example 1 Expression and purification of recombinant human KGF-2 protein

[0384] 1. Experimental methods

[0385] The fermented engineered bacteria were obtained according to the method shown in steps S1 to S3 of Example 2, and Buffer A to Buffer E for column chromatography were prepared according to the method described in step S4.

[0386] The fermented engineered bacteria were mixed with Buffer A at a ratio of 1 g: 20 mL, and the mixture was stirred thoroughly to obtain a mixture. At 15°C, the mixture was homogenized once at 200 Bar and then twice at 700 Bar. After the mixture was completely broken (i.e., there were no intact bacteria in the mixture, OD 600 <30), collect the homogenized liquid and centrifuge it at 7000 rpm at 10℃ for 20 min, collect the supernatant and filter it, and collect the filtrate.

[0387] CM chromatography: Connect a CM chromatography column (Cytiva CM Sepharose Fast Flow, CV: 70 mL) and equilibrate with Buffer A for 3 column volumes at a linear velocity of 60 cm / h until the baseline of the CM chromatography column is stable and the conductivity value is ≤100 ms / cm. The pH of the effluent of the CM chromatography column is 7.2. UV 280 When the absorption baseline is stable, the filtrate (CM loading solution) is loaded at 0.5 g / mL, the flow-through is collected and the CM column is re-equilibrated with Buffer A until UV 280 The absorption baseline is stable, and then Buffer C is eluted for 2 column volumes. 280 When the eluate is >50mAU, UV 280 When the concentration is less than 50 mAU, the collection is stopped and the CM chromatography eluate is collected. The CM chromatography eluate is the crude pure rhKGF-2 protein, and the CM chromatography column is regenerated, cleaned and stored.

[0388] Heparin chromatography: Connect a Heparin chromatography column (Cytiva Heparin Sepharose Fast Flow, CV: 40 mL) and equilibrate with Buffer B for 3 column volumes at a linear velocity of 60 cm / h until the baseline of the Heparin chromatography column is stable and the conductivity value is ≤100 ms / cm. The pH of the effluent of the heparin chromatography column is 7.2. UV 280 When the absorption baseline is stable, mix the CM chromatography eluate with equal volumes of Buffer A, load the sample at 0.5 g / mL, collect the flow-through peak, and then re-equilibrate the Heparin chromatography column with Buffer B until UV 280 The absorption baseline is stable, then Buffer C is used to wash the impurities and wait for UV 280 After the absorption baseline is flushed and stabilized, the heparin washing solution is collected and eluted with Buffer D for 2 column volumes. 280 When the eluate is >80mAU, UV 280 When the concentration is less than 80 mAU, the collection is stopped to obtain the Heparin chromatography eluate (heparin eluate), which is the purified rhKGF-2 in Comparative Example 1. The Heparin chromatography column is then regenerated, cleaned and stored.

[0389] According to the method shown in step 2 of Example 2, the protein content and purity of the purified rhKGF-2 of Comparative Example 1 were detected (HPLC and SDS-PAGE).

[0390] 2. Experimental results

[0391] The results of the detection of rhKGF-2 protein expression after purification in Comparative Example 1 are shown in Table 22.

[0392] Table 22 Detection results of rhKGF-2 protein expression after purification in Comparative Example 1

[0393] The SDS-PAGE result diagram of the rhKGF-2 protein after purification in Comparative Example 1 is shown in Figure 73. The results show that the protein image (SDS-PAGE result diagram) of the rhKGF-2 after purification in Comparative Example 1 shows that there are several relatively thin miscellaneous protein bands below the target protein band, and the integral shows that the purity of rhKGF-2 is less than 95%.

[0394] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. An engineered bacterium for producing KGF-2 protein, characterized in that: The engineered bacteria is Escherichia coli transfected with a recombinant vector; The recombinant vector is a pET-30a vector, a pET-28a vector or a pET-42a vector carrying a KGF-2 protein coding sequence, and the amino acid sequence of the KGF-2 protein is shown in SEQ ID NO: 2; the Escherichia coli is OverExpress C43 (DE3) Escherichia coli, Rosetta (DE3) Escherichia coli or BL21 Star (DE3) Escherichia coli.

2. The engineered bacteria according to claim 1, characterized in that The recombinant vector is a pET-30a vector carrying a KGF-2 protein coding sequence, and the Escherichia coli is Rosetta (DE3) Escherichia coli or OverExpress C43 (DE3) Escherichia coli.

3. Use of the engineered bacteria according to any one of claims 1 to 2 in producing KGF-2 protein.

4. A method for producing KGF-2 protein, characterized in that: The KGF-2 protein is produced by fermentation using the engineered bacteria described in any one of claims 1 to 2.

5. The method according to claim 4, characterized in that The following steps are involved: S1. The engineered bacteria according to any one of claims 1 to 2 are inoculated into LB medium at a volume ratio of 1:20 to 200, and after sufficient cultivation, a primary seed solution is obtained. The primary seed solution is inoculated into LB medium at a volume ratio of 1:20 to 200, and after sufficient cultivation, a secondary seed culture solution is obtained; S2. The secondary seed culture medium obtained in step S1 is mixed with the fermentation base material in a volume ratio of 2 to 10:20 to 100 to obtain a culture medium, the pH of the culture medium is adjusted to 6.2 to 8.2, and culture is carried out under the conditions of dissolved oxygen>10%, temperature of 25 to 45°C, rotation speed of 100 to 1600 rpm and ventilation volume of 5 to 100 L / min. The OD of the culture medium is 600 When the pH value is 10 to 50, IPTG with a final concentration of 0.1 to 3.0 mM and a feed solution accounting for 1.0 to 30% of the total volume are added to the culture solution to fully culture the fermented engineered bacterial cells; The fermentation base material is obtained by mixing base material 1 culture medium, base material 2 culture medium and 1-20g / L thiamine solution in the ratio of 15-25L:0.5-1.5L:6-10mL; The base material 1 medium contains peptone at a final concentration of 15-25 g / L, yeast extract at a final concentration of 15-25 g / L, sodium chloride at a final concentration of 3-7 g / L, potassium dihydrogen phosphate at a final concentration of 1-3 g / L, potassium hydrogen phosphate at a final concentration of 2-4 g / L, and ammonium chloride at a final concentration of 1-3 g / L; the base material 2 medium contains glucose monohydrate at a final concentration of 2-10 g / L, magnesium sulfate anhydrous at a final concentration of 0.2-0.8 g / L, and calcium chloride at a final concentration of 0.01-0.09 g / L. The feed solution contains peptone at a final concentration of 15 to 25 g / L, yeast powder at a final concentration of 15 to 25 g / L, sodium chloride at a final concentration of 3 to 7 g / L, potassium dihydrogen phosphate at a final concentration of 1 to 3 g / L, and potassium dihydrogen phosphate trihydrate at a final concentration of 2 to 4 g / L. S3. The fermented engineered bacterial cells obtained in step S2 are homogenized and broken, and the supernatant is collected for solid-liquid separation and subjected to CM chromatography to obtain crude KGF-2 protein. The crude KGF-2 protein is subjected to heparin chromatography to obtain KGF-2 protein.

6. The method according to claim 5, characterized in that The LB culture medium in step S1 is an LB culture medium containing yeast extract powder at a final concentration of 2-10 g / L, peptone at a final concentration of 9-21 g / L, and sodium chloride at a final concentration of 9-21 g / L, and a pH value of 6.2-8.

2.

7. The method according to claim 5, characterized in that Step S2: OD of the culture medium 600 When the pH value is 40, IPTG with a final concentration of 0.1 to 3.0 mM and a feed solution accounting for 1.0 to 30% of the total volume are added to the culture solution.

8. The method according to claim 5, characterized in that The homogenization and disruption in step S3 is specifically as follows: the fermented engineered bacterial cells obtained in step S2 and Buffer A are fully mixed at a ratio of 1 g: 1-100 mL, and homogenized and disrupted at 4-40° C.; The formula of the Buffer A is: 15-45 mM phosphate buffer, 0.2-1.8 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.

2.

9. The method according to claim 5, characterized in that The CM chromatography in step S3 is specifically as follows: Equilibrate the CM column, add the supernatant in step S3 to the equilibrated CM column at 0.01-10.0 g / mL, collect the flow-through peak, re-equilibrate the CM column with Buffer A, wash the column thoroughly with Buffer E, and then elute with Buffer C for 0.5-10 column volumes, collect UV 280 The eluate at >20 mAU yielded crude pure KGF-2 protein; The formula of Buffer C is: 15-45 mM phosphate buffer, 0.2-1.8 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.2; The formula of the Buffer E is: 15-45 mM phosphate buffer, 0.2-1.8 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.

2.

10. The method according to claim 5, characterized in that The heparin chromatography in step S3 comprises the following steps: Equilibrate the heparin chromatography column, mix the crude KGF-2 protein described in step S3 with an equal volume of Buffer A, load the equilibrated heparin chromatography column at 0.10-2.00 g / mL, collect the flow-through peak, re-equilibrate the heparin chromatography column with Buffer B, thoroughly wash with Buffer C, and then elute with Buffer D for 0.5-10 column volumes, collect UV 280 When the eluate is >20mAU, the KGF-2 protein is obtained; The formula of Buffer B is: 15-45 mM phosphate buffer, 0.1-3 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.2; The formula of the Buffer D is: 15-45 mM phosphate buffer, 0.1-3 M sodium chloride and 5-35 mM EDTA-2Na, pH = 6.2-8.2.

Images