WO2017205996A1 - 一种重组人隐花色素蛋白I(hCRY1)的生产工艺及其组合物 - Google Patents

一种重组人隐花色素蛋白I(hCRY1)的生产工艺及其组合物 Download PDF

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WO2017205996A1
WO2017205996A1 PCT/CN2016/000641 CN2016000641W WO2017205996A1 WO 2017205996 A1 WO2017205996 A1 WO 2017205996A1 CN 2016000641 W CN2016000641 W CN 2016000641W WO 2017205996 A1 WO2017205996 A1 WO 2017205996A1
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黄文林
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the invention relates to the field of biopharmaceutical technology, in particular to a production process of recombinant human cryptochromin protein I (hCRY1) and a composition thereof.
  • Escherichia coli is the most commonly used engineering bacteria in prokaryotic expression system. It has a clear genetic background, gene safety, high protein expression, and the expression level of exogenous genes is much higher than other gene expression systems. It is rapid in reproduction, easy to operate, and can be rapidly scaled. The production of target proteins; low cost of cultivation; etc.; has been approved by the US FDA as a safe genetic engineering receptor organism. In the process of constructing recombinant engineered bacteria, operons are usually used to control the controllable expression of foreign genes.
  • Isopropylthiogalactoside is the most commonly used high-efficiency inducer in prokaryotic expression systems, and it can stably initiate the lactose operon, so that the target gene can be expressed continuously and efficiently.
  • other auxiliary sequences are inserted into the expression vector to form a fusion expression with the target gene, such as insertion of the purification tag 6-His, GST, CAT, etc.
  • the protein is subjected to one step affinity chromatography to obtain a product having higher homogeneity.
  • the defects of prokaryotic expression system also have certain limitations. For example, the expression of the target gene often forms an insoluble and inactive inclusion body.
  • E. coli As a prokaryotic expression system, when expressing eukaryotic foreign genes, it is often lacking. Post-translational modification, protein activity is greatly affected. Although genetic engineering systems have expanded from E. coli to yeast, insect, plant and mammalian cells, and many new eukaryotic expression systems have emerged, E. coli remains an important tool for gene expression.
  • CRY1 and CRY2 which together with the photolytic enzyme family regulate the adaptive response of organisms to low-intensity irradiation of ultraviolet light and blue light, participate in DNA repair and other functions, and interact with animals.
  • the growth, development, magnetic perception and circadian rhythm are closely related.
  • CRY1 is involved in a variety of light-dependent transcriptional repression in animals, and can directly interact with BmaL/CLOCK protein dimers, Per1, Per2 and other rhythm-related proteins; hCRY1 is involved in transcriptional regulation, DNA damage repair, etc. in humans.
  • cry53-cry2 knockout P53 KO cells showed that the sensitivity of cells to radiation and radiation-like drugs increased, and the proportion of apoptosis increased significantly; the up-regulation and down-regulation of CRYs also correlated with cell carcinogenesis and tumorigenesis. Development has a close relationship.
  • the hCRY2-Flag fusion protein was expressed in Hela cells, and the culture volume was 10L. After purification by FLAG M2 affinity chromatography, only 5-15 ⁇ g of fusion protein was harvested. Wang Huaweing et al. constructed HsCRY1 insect expression system and transfected it. Insect sf9 cells, expressing HsCRY1, obtained 4.8 mg of protein HsCRY1 in a 50 mL culture scale, and the yield after amplification culture was estimated to be about 96 mg/L.
  • the present invention provides a process for producing recombinant human cryptochromin protein I (hCRY1) and a composition thereof in view of the problem of low yield of hCRY1 in the prior art.
  • the present invention is implemented as follows:
  • a process for producing recombinant human cryptochromin protein I comprising the following steps
  • (1) Construction of recombinant expression plasmid The hCRY1 gene fragment was recombined into the prokaryotic expression vector pET28a(+) to obtain the recombinant expression plasmid pET28a(+)-hCRY1. Specifically, the hCRY1 gene was obtained by PCR amplification, and the vector pET28a(+) was digested with restriction endonuclease Xho I and Baml I, respectively, and ligated with T4 DNA ligase to obtain recombinant expression plasmid pET28a(+)-hCRY1. . The amino acid sequence of the hCRY1 protein obtained by translation of the hCRY1 gene is shown in SEQ ID NO: 1.
  • Protein affinity chromatography purification the protein denaturing solution is centrifuged at 12000-14000 rpm for 40-60 min, the supernatant is collected, filtered through a 0.22-0.45 ⁇ m filter, and the imidazole is added to the filtrate to a final concentration of 5-30 mM.
  • the above sample volume is 1-1.5 CV, the loading flow rate is 5-10 ml/min, and the elution flow rate is 10-20 ml/min for HIS label affinity chromatography.
  • the chromatograph Purifier 100; chromatography material: any of ProteinIso Ni-NTA Resin, Ni-NTA His Bind Resin; equilibration: equilibrated with buffer 0-500 mM NaCl, 6-8 M Urea, 0-60 mM imidazole, pH 7.5-8.5 2-4 CV, elution of impurities to baseline; elution: 2-2.5 CV was eluted with buffer 0-500 mM NaCl, 6-8 M Urea, 200-300 mM imidazole, pH 7.5-8.5, and eluted protein peaks were collected.
  • Protein gradient dialysis renaturation The collected eluted protein peak sample was placed in a 30-50 kDa dialysis bag, containing 8M Urea, 0-100 mM Tris, 0-100 mM NaCl, pH 7.5- at 8-10 volumes.
  • Dialysis in 8.5 dialysis buffer at 4 °C for 8-12 h transfer the sample to dialysis buffer containing 4M Urea, 0-100 mM Tris, 0-100 mM NaCl, pH 7.5-8.5, dialysis for 8-12 h at 4 °C; transfer the sample To dialysis containing 2M Urea, 0-100 mM Tris, 0-400 mM Argine, 0-10 mM EDTA, 0-10 mM GSH, 0-5 mM GSSG, pH 7.5-8.5 dialysis buffer, 4 °C for 8-12 h; transfer the sample to Contains 1M Urea, 0-100 mM Tris, 0-100 mM NaCl, 0-2% glycerol, pH 7.5-8.5 dialysis buffer, dialysis at 4 °C for 8-12 h; transfer of sample to 0-100 mM Tris, 0-500 mM NaCl, 0-10% glycerol, pH 7.5
  • Protein ultrafiltration concentration and solution displacement ultrafiltration concentration of the dialysis sample was carried out using a 30-50 kDa ultrafiltration membrane package using an ultrafiltration apparatus Millipore: after concentrating the sample to an original volume of about 1/4 to 1/5, The dilution buffer containing 0-100 mM Tris, 0-200 mM NaCl, 0-20% glycerol, pH 7.5-8.5 was gradually added for dilution, and finally the sample volume was concentrated to 1/5 of the original volume to obtain soluble hCRY1 protein. The solution was filtered through a 0.22 ⁇ m filter to obtain a soluble hCRY1 protein solution.
  • composition comprises the recombinant hCRY1 protein prepared by the above production process and a pharmaceutically acceptable carrier or adjuvant.
  • the present invention optimizes the production process: inducing expression in the fermenter culture system; and successively using two different denatured liquids to dissolve the inclusion bodies.
  • the denatured hCRY1 protein lysate was obtained, it was purified by denaturing affinity chromatography to obtain a higher purity hCRY1 elution peak, and then subjected to dialysis renaturation, and finally concentrated by ultrafiltration using a 30-50 kD ultrafiltration membrane to obtain hCRY1.
  • the yield reached 200-300mg/L.
  • hCRY1 of the present invention The yield of hCRY1 of the present invention is much higher than that of other research methods and our earlier published production methods, and the protein product produced by the present invention has been verified to have the desired radiation absorption function and radiation damage protection activity, and has been developed to be radiotherapy protection.
  • An active protein of a drug-like potential An active protein of a drug-like potential.
  • the hCRY1 yield of the production process is 200-300 mg/L, which is much higher than other hCRY1 production methods in the prior art, which is 10-15 times that of our earlier disclosed process.
  • the chromatographic purification stage of the process is purified by denaturing, and the recovery rate of hCRY1 protein is close to 100%, and the purity is more than 95%; over the previous renaturation and purification process, the protein in the renaturation stage is precipitated with the impurity protein.
  • the column effect is poor and the protein degradation is insufficient.
  • the process uses fermentor culture to induce expression of hCRY1 protein.
  • the wet weight of the bacteria is increased from about 1 g of the original shake flask culture process to about 120 g.
  • the ultrafiltration method is firstly used to super-concentrate the sample to reduce the sample volume, thereby reducing the volume of the dialysis buffer required for dialysis refolding and saving about 5 times of material input.
  • Figure 1 Flow chart of construction of prokaryotic expression plasmid pET28a(+)-hCRY1;
  • Figure 2 The left panel shows the results of agarose gel electrophoresis of the PCR product, and the right panel shows the results of the digestion assay of the prokaryotic expression plasmid, wherein M is DNA Marker;
  • Figure 3 Screening results of high-efficiency expression of hCRY1 strain.
  • the above figure shows the results of different single colony-induced expression.
  • the following figure shows the gray value of the corresponding bands and the ratio of target protein to heteroprotein, where M is the protein Marker;
  • Figure 4 Bacterial growth curve during fermenter induction of expression of hCRY1;
  • Figure 5 Induced expression of BL21-hCRY1-b, the upper panel shows the results of SDS-PAGE, and the lower panel shows the results of Western blot;
  • Figure 7 The upper panel shows the results of the target protein band by mass spectrometry.
  • the underlined part is the part that matches the hCRY1 amino acid sequence.
  • the lower panel shows the alignment of the above sequence in the NCBI database, with the highest hCRY1 score.
  • Figure 8 The above figure shows the results of SDS-PAGE electrophoresis on the sample, the flowthrough, and the elution peak during the affinity chromatography purification process.
  • Figure 9 Use Purification of hCRY1 chromatogram by affinity chromatography on a purifier 100 chromatograph;
  • FIG. 11 The left panel shows the comparison of the intracellular FOCI fluorescence intensity of the control group (cBSA) and the experimental group (hCRY1) after X-ray irradiation.
  • the right panel shows the fluorescence intensity of the above two groups (***P ⁇ 0.001);
  • Figure 12 The upper left panel shows the intracellular FOCI fluorescence intensity of HaCaT cells with different concentration gradients of hCRY1 without X-ray irradiation.
  • the lower left panel shows the intracellular FOCI of HaCaT cells with different concentration gradients of hCRY1 after X-ray irradiation. Fluorescence intensity results, the right panel shows the quantitative results of fluorescence intensity of each group of cells (**P ⁇ 0.05, ***P ⁇ 0.001);
  • Figure 13 Comparison of the effect of hCRY1 and BSA after treatment on apoptosis.
  • a process for producing recombinant human cryptochromin protein I comprising the following steps
  • the desired fragment of interest was amplified by PCR (total system 25 uL, pre-denaturation at 94 ° C for 3 min, 32 cycles: 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 2 min, and finally 72 ° C for 10 min).
  • the restriction endonuclease XhoI and BamlI (TAKARA) were used to digest the target fragment and the prokaryotic expression vector pET28a(+), and then the vector with the sticky end was ligated with the T4 DNA ligase to obtain the prokaryotic expression plasmid pET28a(+). -hCRY1, as shown in Figure 1.
  • the amplified product was detected by agarose gel electrophoresis to obtain a band of about 1900 bp, as shown in the left figure of Fig. 2, and it was confirmed that the target fragment was successfully amplified. Detection of the digested product by electrophoresis, A band of the target fragment of about 1900 bp and a vector band of about 5300 bp in size were obtained, as shown in the right panel of Fig. 2, and the prokaryotic expression plasmid was successfully constructed.
  • the amino acid sequence of the hCRY1 protein obtained by translation of the hCRY1 gene is shown in SEQ ID NO: 1.
  • the collected cells were washed with PBS and resuspended in 30 ml of PBS, and subjected to sonication after adding 1 mM protease inhibitor PMSF. 20 ⁇ l samples were taken from each broken sample for SDS-PAGE analysis. The electrophoresis results are shown in the figure above in Fig. 3. The gray values of the target protein and the heteroprotein bands were measured and the ratio was calculated, and the b clone with the highest proportion of the target protein was selected. That is, the clone with the target protein band is brighter and the heteroprotein band is weaker (Fig. 3), and stored at -80 °C and named BL21-hCRY1-b. All subsequent experiments were performed using this strain.
  • the culture temperature was set at 37 ° C, dissolved oxygen 25%, pH 7.0, stirring speed 200-350 rpm, cultured 5.5 h, OD 600 value reached 7.0, IPTG was added to the final concentration in the tank of about 1.0 mM, and fermentation was completed after 3 hours of induction.
  • the OD 600 was measured for each time period of fermentation, and the growth curve was drawn, as shown in Fig. 4; and the cell samples were taken at various time points after induction, and the expression of the target protein was detected by SDS-PAGE, as shown in Fig. 4. After the end of the fermentation, the cells were collected by centrifugation at 3500 rpm for 10 min, and the wet weight of the cells was about 130 g.
  • the growth curve from Fig. 4 shows that Escherichia coli was in the logarithmic growth phase 4 to 7 h after inoculation into the fermentor, and the introduction of 1.0 mM was initiated at the middle and late logarithmic growth (6 h), and bacterial proliferation began to slow down. From the SDS-PAGE electrophoresis of Fig. 4, hCRY1 expression was already detected after 1 h of induction.
  • hCRY1 In the two stages of inclusion body washing, a large amount of heteroprotein is dissolved in the supernatant and hCRY1 is present as an insoluble inclusion body, which is beneficial to the enrichment and primary purification of hCRY1.
  • the target band was excised and identified by mass spectrometry, and the amino acid sequence of the protein is shown in SEQ ID NO: 1.
  • the results of comparison with the NCBI database are shown in Figure 7, and the inclusion body protein was officially confirmed as hCRY1.
  • Inclusion body denaturing and dissolution 40 ml of newly prepared components containing 6M guanidine hydrochloride, pH 8.0 phosphate buffer, resuspended and washed inclusion body precipitate, with a power of 225W, working time of 5s, intermittent time of 8s Solubilized for 90 min; then diluted 8 times with 8 M Urea, pH 8.0 phosphate buffer, sonicated for 90 min with a power of 225 W, working time 5 s, intermittent time 8 s, sampling for SDS-PAGE detection of inclusion body protein dissolution, see Figure 6. It can be observed from lanes 6, 7, and 8 of Figure 6, that under ultrasound conditions, the hCRY1 inclusion bodies begin to dissolve into the supernatant.
  • Protein affinity chromatography purification the protein denaturing solution was centrifuged at 14000 rpm for 45 min at high speed, the supernatant was collected, filtered through a 0.45 ⁇ m filter, and the imidazole was added to the filtrate to a final concentration of 20 mM in the sample, the above volume was 1 CV, and the above volume was 1 CV.
  • HIS label affinity chromatography chromatograph: sample flow rate 8ml / min, elution flow rate 15ml / min Purifier 100; chromatography material: ProteinIso Ni-NTA Resin 100 ml; equilibration: 2CV equilibrated with buffer 500 mM NaCl, 8 M Urea, 20 mM imidazole, pH 8.0; elution: buffer 500 mM NaCl, 8 M Urea, 200 mM imidazole, pH 8.0 The eluted protein peak was collected by eluting 2 CV. For the sample loading, the breakthrough peak and the elution peak were sampled and subjected to SDS-PAGE electrophoresis. The results are shown in Fig. 8, and the chromatogram is shown in Fig. 9.
  • Protein gradient dialysis renaturation The collected eluted protein peak sample was placed in a 30-50 kDa dialysis bag and placed in 8 volumes of 6 M Urea, 50 mM Tris, 50 mM NaCl, pH 8.0 dialysis buffer.
  • Dialysis for 8-12 h at 4 ° C transfer the sample to 4 M Urea, 50 mM Tris, 50 mM NaCl, pH 8.0 dialysis buffer, dialysis at 4 ° C for 8-12 h; transfer the sample to 2M Urea, 100 mM Tris, 400 mM Argine, 5 mM EDTA, 5 mM GSH, 0.5 mM GSSG, pH 8.0 dialysis buffer, dialysis at 4 ° C for 8-12 h; transfer the sample to dialysis buffer containing 1 M Urea, 50 mM Tris, 50 mM NaCl, 10% glycerol, pH 8.0 Dialysis was carried out at 4 ° C for 8-12 h; the sample was transferred to a dialysis buffer containing 50 mM Tris, 500 mM NaCl, 10% glycerol, pH 8.0, and dialyzed at 4 ° C for 8-12 h; after the dialysis was completed, the sample in the dialysis
  • Ultrafiltration concentration of the dialysis sample was carried out using a 30 kDa ultrafiltration membrane package using an ultrafilter Millipore: After concentrating the sample to about 1/4 of the original volume, gradually adding 50 mM Tris, Dilute with 100 mM NaCl, 10% glycerol, pH 8.0 replacement buffer, and finally concentrate the sample volume to 1/5 of the original volume to obtain hCRY1 protein solution, and then filter through 0.22 ⁇ m filter to obtain sterile soluble hCRY1. Protein solution. The samples before and after concentration were analyzed by SDS-PAGE electrophoresis, and the results are shown in FIG. The protein was quantified using the Bradford Protein Quantitative Assay Kit at a protein concentration of approximately 0.52 mg/ml and an estimated protein yield of approximately 300 mg/L.
  • the purified protein solution was diluted to 500 ⁇ g/ml for use as a backup, and 1 mg/ml BSA protein was dispensed with 300 mM imidazole, and the nickel column was loaded and the ultrafiltration replacement was identical to that of the hCRY1 sample, and then diluted to 500 ⁇ g/ml.
  • Control cBSA
  • the Hela cells were plated and cultured to a cell density of about 80-90%, and after three hours, the prepared hCRY1 and the control protein were separately added to the cell culture solution in a ratio of 1:1 to the culture medium to make each well.
  • the total internal volume of the liquid was 2 ml, and the final concentration of the added protein was 250 ⁇ g/ml.
  • the two groups of cells were irradiated in an X-ray irradiator at a dose of 1.132/Gy per minute, an irradiation time of 530 s, and a total dose of 10 Gy.
  • the cell culture medium was changed to the pre-experimental medium (DMEM), and the cells were cultured for 45 minutes, and then the cells were incubated.
  • the cells were washed 3 times with PBS, fixed cells at 100% methanol -20 ° C for 5 min, washed 3 times with PBS, and blocked with 5% BSA overnight.
  • the cells were incubated with the antibody Phospho-Histone H2A.X at 37 ° C for 1 h, then washed with PBS.
  • the cells were incubated with antibody goat anti-rabbit IgG/FITC at 37 ° C for 1 h, washed with PBS for 3 times, and the fluorescence of the phosphors in the two groups were observed by laser confocal microscopy, and each piece was taken under the same shooting conditions.
  • the average fluorescence intensity was counted for 3 different fields of view.
  • the difference in FOCI fluorescence intensity between the two groups of cells was observed by the naked eye (Fig. 11 left). Three fields of view were randomly selected and the fluorescence intensity was quantified. The difference between the two groups was extremely significant (P ⁇ 0.001) ( Figure 11 right). It can be seen that the hCRY1 prepared by us reduces the fluorescence intensity of FOCI in the cells, which has a certain protective effect on the cells and reduces the damage of X-ray.

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Abstract

一种重组人隐花色素蛋白I(hCRY1)的生产工艺及其组合物，所述生产工艺步骤包括：将hCRY1基因克隆至pET-28a得到重组载体pET-28a(+)-hCRY1；将重组载体导入大肠杆菌E.coli BL21(DE3)并筛选得到基因工程菌BL21-hCRY1-b；将BL21-hCRY1-b在发酵罐中培养及重组蛋白诱导表达；收集和洗涤包涵体；包涵体变性溶解；蛋白亲和层析纯化；蛋白梯度透析复性；蛋白超滤浓缩及溶液置换，得到重组的hCRY1蛋白。该生产工艺的hCRY1产率在200-300mg/L，远高于目前已知的其他hCRY1生产方法。

Description

一种重组人隐花色素蛋白I(hCRY1)的生产工艺及其组合物 技术领域
本发明涉及生物制药工艺领域，具体涉及一种重组人隐花色素蛋白I(hCRY1)的生产工艺及其组合物。
背景技术
大肠杆菌是目前原核表达系统中最常用的工程菌，其具有遗传背景清晰，基因安全；蛋白表达量高，外源基因表达水平远高于其他基因表达系统；繁殖迅速，操作简便，可以快速规模化生产靶蛋白；培养成本低廉等等优势；已被美国FDA批准为安全的基因工程受体生物。在构建重组工程菌的过程中，通常会采用操纵子来控制外源基因的可控表达。异丙基硫代半乳糖苷(IPTG)是原核表达系统中最常用的高效诱导剂，能够稳定地启动乳糖操纵子，使目的基因得到持续高效的表达。除了表达载体的调控性，为了提高蛋白产物的活性以及简化下游纯化操作等往往在表达载体上插入其他辅助序列与目的基因构成融合表达，如在插入纯化标签6-His、GST、CAT等，融合蛋白进过一步亲和层析即可得到均一性较高的产物。原核表达系统缺陷也存在着一定的局限性，如目的基因表达，往往会形成不溶性、无活性的包涵体形式；作为一种原核表达系统，在表达真核生物外源基因时，也往往在于缺少翻译后修饰，蛋白活性受到很大影响。虽然基因工程系统已经从大肠杆菌扩大到酵母、昆虫、植物及哺乳动物细胞，并且也出现了很多新型的真核表达系统，但大肠杆菌仍然是基因表达的重要工具。
迄今为止，动物体内已经发现的隐花色素主要有CRY1和CRY2两种，它们与光裂解酶家族共同调节生物体对紫外线和蓝光小剂量照射的适应性反应，参与DNA修复等功能，并与动物的生长、发育、磁感知及生理节律等有着密切联系。有研究发现，CRY1在动物体内参与多种光依赖型转录抑制，能够与BmaL/CLOCK蛋白二聚体、Per1、Per2等节律相关蛋白直接作用；hCRY1在人体内参与转录调控、DNA损伤修复等多种生理过程；cry1/cry2基因敲除的P53KO细胞体外试验表明，细胞对辐射、类辐射药物的敏感性提高，凋亡比例显著上升；CRYs的上调、下调等还与细胞癌变、肿瘤的发生发展有着密切的关系。
Figure PCTCN2016000641-appb-000001
和Song等在2007年分别构建了来源于D.Plexippus，A.Gambiae，和A.Pernyi的maltose-binding protein-CRY融合蛋白表达载体，在E.coli BL21中诱导表达，培养体积12L约收获2mg融合蛋白。
Figure PCTCN2016000641-appb-000002
等在2009年构建了昆虫细胞表达系统，表达拟南芥、果蝇等植物和非哺乳动物CRYs，经FLAG亲和层析纯化，获得得了2mg/L的产量。对于哺乳动物CRYs，特别是hCRYs的诱导表达方面研究，还没有取得较大进展。由于哺乳动物中天然状态的隐花色素含量低，分离获取大量天然产物十分困难。而多肽合成成本过高，缺乏作为药 物生产的应用前景。
Figure PCTCN2016000641-appb-000003
等在2003年构建hCRY2-Flag融合蛋白在Hela细胞中表达，培养体积10L，经FLAG M2亲和层析纯化后，仅收获5-15μg融合蛋白；王晓明等构建了HsCRY1昆虫表达系统，通过转染昆虫sf9细胞，表达HsCRY1，在50mL培养规模中获得4.8mg蛋白HsCRY1，推算出放大培养后产量约为96mg/L。
此前，我们报道了利用大肠杆菌表达hCRY1的相关研究。其中，我们采用摇瓶培养重组大肠杆菌，超声破菌后，获得以包涵体形式表达的目的蛋白，再采用高浓度尿素重悬包涵体，并超声溶解。在获得了包涵体溶解液后，首先将蛋白复性，后再进行亲和层析纯化，再用10kDa超滤管超滤浓缩和溶液置换获得了10-15mg/L的hCRY1产量。
发明内容
在之前研究的基础上，本发明针对现有技术中hCRY1产量较低的问题，提供一种重组人隐花色素蛋白I(hCRY1)的生产工艺及其组合物。
本发明是这样实现的：
一种重组人隐花色素蛋白I(hCRY1)的生产工艺，包括以下步骤
(1)构建重组表达质粒：将hCRY1基因片段重组至原核表达载体pET28a(+)，获得重组表达质粒pET28a(+)-hCRY1。具体为：通过PCR扩增获得hCRY1基因，再与载体pET28a(+)分别用限制性内切酶Xho I与Baml I酶切后用T4DNA连接酶连接，最终获得重组表达质粒pET28a(+)-hCRY1。所述的hCRY1基因翻译得到的hCRY1蛋白氨基酸序列如SEQ ID NO：1所示。
(2)构建工程菌：将重组表达质粒pET28a(+)-hCRY1转入感受态宿主细胞E.coli BL21(DE3)，构建含有重组表达载体pET-28a(+)-hCRY1基因工程菌，然后通过SDS-PAGE筛选hCRY1表达量高的克隆菌株BL21-hCRY1-b。
(3)工程菌培养及重组蛋白诱导表达：菌种BL21-hCRY1-b过夜培养，次日挑取单菌落接入含卡那霉素的LB液体培养基中，摇菌6h后以1∶100转接到新鲜的含卡那霉素的LB培养基中进行二次放大培养，摇菌过夜后，以1∶100接种量至NBS系列发酵罐中进行发酵，培养温度36.5-37.5℃，溶氧20-40％，pH6.8-7.4，搅拌速度200-350rpm，培养5-8h后，OD600＝7-8时，加入终浓度0.5-3.0mM IPTG诱导培养3-4h；
(4)包涵体收集和洗涤：3000-5000rpm离心收集菌体，以功率200-250W、工作时间3-8s、间歇时间5-10s的条件超声破碎菌体60-90min，3000-5000rpm离心50-80min，收集包涵 体沉淀，用组分含10-100mM Tris、0.1-1.0mM EDTA、0-100mM NaCl、1-10％Triton X-100、1-2M Urea，pH7.5-8.5的洗涤缓冲液重悬包涵体，以功率100-150W、工作时间3-8s、间歇时间5-10s的条件超声洗涤60-90min，12000-14000rpm离心60-90min收集沉淀；
(5)包涵体变性溶解：用组分含4-8M盐酸胍、pH7.5-8.5磷酸缓冲液重悬洗涤后的包涵体沉淀，以功率200-250W、工作时间3-8s、间歇时间5-10s的条件超声促溶60-90min；再用含6-8M Urea、pH7.5-8.5磷酸缓冲液稀释6-10倍，以功率200-250W、工作时间3-8s、间歇时间5-10s的条件超声促溶60-90min，得到蛋白变性溶解溶液；
(6)蛋白亲和层析纯化：将蛋白变性溶解溶液12000-14000rpm高速离心40-60min，收集上清，经0.22-0.45μm滤膜过滤，添加咪唑至滤液中咪唑终浓度为5-30mM，以上样体积1-1.5CV、上样流速5-10ml/min、洗脱流速10-20ml/min进行HIS标签亲和层析，层析仪：
Figure PCTCN2016000641-appb-000004
Purifier 100；层析填料：ProteinIso Ni-NTA Resin、Ni-NTA His Bind Resin中任一种；平衡：用缓冲液0-500mM NaCl、6-8M Urea、0-60mM咪唑、pH7.5-8.5平衡2-4CV，洗去杂质至基线平；洗脱：用缓冲液0-500mM NaCl、6-8M Urea、200-300mM咪唑、pH7.5-8.5洗脱2-2.5CV，收集洗脱蛋白峰。
(7)蛋白梯度透析复性：将收集的洗脱蛋白峰样品装入30-50kDa透析袋中，于8-10倍体积包含6M Urea，0-100mM Tris，0-100mM NaCl，pH7.5-8.5透析缓冲液中，4℃透析8-12h；将样品转移至包含4M Urea，0-100mM Tris，0-100mM NaCl，pH 7.5-8.5透析缓冲液中，4℃透析8-12h；将样品转移至包含2M Urea，0-100mM Tris，0-400mM Argine，0-10mM EDTA，0-10mM GSH，0-5mM GSSG，pH 7.5-8.5透析缓冲液中，4℃透析8-12h；将样品转移至包含1M Urea，0-100mM Tris，0-100mM NaCl，0-2％甘油，pH7.5-8.5透析缓冲液中，4℃透析8-12h；将样品转移至包含0-100mM Tris，0-500mM NaCl，0-10％甘油，pH7.5-8.5透析缓冲液中，4℃透析8-12h；透析完成后，收集透析袋内样品。
(8)蛋白超滤浓缩及溶液置换：使用超滤仪Millipore，用30-50kDa超滤膜包对透析所得样品进行超滤浓缩：将样品浓缩至原体积约1/4-1/5后，逐步添加包含0-100mM Tris，0-200mM NaCl，0-20％甘油，pH7.5-8.5的置换缓冲液进行稀释，最后将样品体积浓缩至原体积的1/5，得到可溶的hCRY1蛋白溶液，再经0.22μm滤膜过滤后，即得到可溶性hCRY1蛋白溶液成品。
组合物，含有上述生产工艺制备的重组hCRY1蛋白以及药学上可接受的载体或辅料。
与之前的研究相比，本发明对生产工艺进行了优化：在发酵罐培养体系中诱导表达；先后联合使用两种不同变性液溶解包涵体。获得了变性的hCRY1蛋白溶解液后，先进行变性亲和层析纯化，得到较高纯度的hCRY1洗脱峰后，再进行透析复性，最后用30～50kD超滤膜包超滤浓缩获得hCRY1，产量达到了200-300mg/L。本发明所述hCRY1得率远高于其他研究所述方法以及我们早期公开的生产方法，由本发明生产得到的蛋白产物经验证具有预期的射线吸收功能和放射损伤防护活性，是具有开发成为放疗保护类药物潜质的活性蛋白。
本发明的有益效果是：
(1)该生产工艺的hCRY1产率在200-300mg/L，远高于现有技术中其他hCRY1生产方法，是我们早期公开工艺的10-15倍。
(2)本工艺层析纯化阶段，采用变性后纯化，hCRY1蛋白纯化回收率接近100％，纯度大于95％；克服了此前先复性后纯化工艺中，复性阶段蛋白随杂蛋白析出，挂柱效果差，蛋白降解的不足。
(3)本工艺采用发酵罐培养诱导表达hCRY1蛋白，发酵结束，菌体湿重从原摇瓶培养工艺的约1g提高至约120g。
(4)本工艺包涵体溶解部分，采用了4-8M盐酸胍初溶，再用6-8M尿素二次进行溶解，提高了包涵体蛋白的溶解效率。
(5)本工艺蛋白复性阶段，首先采用超滤技术对样品进行超滤浓缩，减少样品体积，从而减少了透析复性所需对透析缓冲液体积，节约约5倍的物料投入。
附图说明
图1：原核表达质粒pET28a(+)-hCRY1的构建流程图；
图2：左图为PCR产物的琼脂糖凝胶电泳检测结果，右图为原核表达质粒的酶切检测结果，其中M为DNA Marker；
图3：高效表达hCRY1菌株的筛选结果，上图为不同单菌落诱导表达效果检测结果，下图为对应条带灰度值以及目标蛋白与杂蛋白比例结果，其中M为蛋白质Marker；
图4：发酵罐诱导表达hCRY1过程中，细菌生长曲线；
图5：BL21-hCRY1-b的诱导表达结果，上图为SDS-PAGE结果，下图为Western blot结果；
图6：包涵体变性溶解过程SDS-PAGE检测；
图7：上图为质谱鉴定目标蛋白条带结果，下划线部分为与hCRY1氨基酸序列相匹配的部分，下图为以上序列在NCBI数据库中比对结果，其中hCRY1得分最高；
图8：上图为亲和层析纯化过程中，对上样样品(Sample)，上样穿透(Flowthrough)，洗脱回收峰(Elution)进行SDS-PAGE电泳分析结果；
图9：使用
Figure PCTCN2016000641-appb-000005
purifier 100层析仪进行亲和层析纯化hCRY1层析图谱；
图10：超滤浓缩前后取样进行SDS-PAGE电泳分析结果；
图11：左图为经X-ray照射后的对照组(cBSA)与实验组(hCRY1)细胞内FOCI荧光强度比对结果，右图为以上两组细胞荧光强度量化结果(***P＜0.001)；
图12：左上图为加入不同浓度梯度hCRY1的HaCaT细胞未经X-ray照射时细胞内的FOCI荧光强度结果，左下图为加入不同浓度梯度hCRY1的HaCaT细胞经X-ray照射后细胞内的FOCI荧光强度结果，右图为每组细胞荧光强度量化结果(**P＜0.05，***P＜0.001)；
图13：hCRY1和处理之后的BSA对细胞凋亡的影响比对结果。
具体实施方式
以下结合附图和实施例描述本发明，以下实施例以发明最优效果进行解释说明。
实施例1：
一种重组人隐花色素蛋白I(hCRY1)的生产工艺，包括以下步骤
(1)构建重组表达质粒：以实验室保存的hCRY1真核表达质粒pcDNA3.1-hCRY1为模版，根据hCRY1的编码区序列设计上下游特异性引物：hCRY1FW(5′-ATACTCGAGGCTAAGCCTTCC-3′)，hCRY1RV(5′-CGCGGATCCTACGTTTATACT-3′)(上下游引物均由北京奥科鼎盛生物科技有限公司合成)。PCR扩增出所需目的片段(总体系25uL，94℃预变性3min，32个循环：94℃ 30s，55℃30s，72℃ 2min，最后72℃延伸10分钟)。用限制性内切酶XhoI与BamlI(TAKARA)酶切目的片段与原核表达载体pET28a(+)，然后用T4DNA连接酶连接带有粘性末端的载体与目的片段，最终获得原核表达质粒pET28a(+)-hCRY1，如图1所示。用琼脂糖凝胶电泳对扩增产物进行检测，得到大小约为1900bp的条带，如图2左图所示，证实目的片段已成功扩增。用电泳检测酶切产物，可 得到大小约为1900bp的目的片段条带和大小约为5300bp的载体条带，如图2右图所示，证实原核表达质粒构建成功。所述的hCRY1基因翻译得到的hCRY1蛋白氨基酸序列如SEQ ID NO：1所示。
(2)构建工程菌：将上述重组质粒pET28a(+)-hCRY1转化至感受态细胞E.coli BL21(DE3)中，涂板培养，次日挑取5个不同的单菌落克隆，分别接入含50mg/L卡那霉素的LB培养基中，摇菌过夜后以1∶100转接到新鲜的含50mg/L卡那霉素的LB培养基中进行放大培养。于三角烧瓶中摇菌至菌液OD600值0.6后，加入1mM诱导剂IPTG，24℃、200rpm诱导6h后离心收菌。将所收菌体用PBS洗涤并重悬于30ml PBS中，加入1mM蛋白酶抑制剂PMSF后进行超声破碎。从每份破碎样品中取20μl样品进行SDS-PAGE分析，电泳结果如图3上图所示，分别测量目标蛋白与杂蛋白条带灰度值并计算比例，挑选目标蛋白比例最高的b克隆(即目标蛋白条带较亮、杂蛋白条带较弱的克隆)(图3下图)，-80℃保菌储存并命名为BL21-hCRY1-b。之后的所有实验均使用该菌株完成。
(3)工程菌培养及重组蛋白诱导表达：将BL21-hCRY1-b接种至5ml含卡那霉素的LB培养基中，摇菌6h后以1∶100接种100ml含卡那霉素的LB培养基进行扩种，摇菌12h后，以1∶100接种至含9L培养基的NBS系列发酵罐中进行发酵。培养基配方酵母提取物24g/L，胰蛋白胨12g/L，甘油4mL/L，KH2PO4 2.3g/L，K2HPO4·3H2O 12.5g/L，消泡剂0.2mL/L。设定培养温度37℃，溶氧25％，pH 7.0，搅拌速度200-350rpm，培养5.5h OD600值达7.0以后，加入IPTG至罐内终浓度约1.0mM，诱导3h后完成发酵。对发酵各时间段取样进行OD600测定，绘制生长曲线，见附图4；并取诱导前，诱导后各时间段菌体样品，进行SDS-PAGE检测目的蛋白表达情况，见附图4。发酵结束后，3500rpm离心10min收集菌体，菌体湿重约130g。
从附图4生长曲线显示，在接种至发酵罐4至7h，大肠杆菌处于对数生长期，在对数生长中后期(6h)时开始加入1.0mM诱导，细菌增殖开始趋缓。从图4SDS-PAGE电泳显示，在开始诱导1h后，已经能够检测到hCRY1表达。
(4)包涵体收集和洗涤：取其中10g菌体，按4ml/g湿菌体加入PBS重悬，以功率225W、工作时间5s、间歇时间8s的条件超声破碎菌体80min，取不同破碎阶段的悬液、上清和沉淀进行SDS-PAGE电泳检测，结果见附图5。从附图5可以观察到，在超声破菌之后，hCRY1均以沉淀形式存在，表明hCRY1以不溶形式的包涵体表达，破菌过程中hCRY1不溶 于PBS，有助于目标蛋白的富集。4000rpm离心60min收集沉淀，用40ml新配制的组分含50mM Tris、0.5mM EDTA、50mM NaCl、1％Triton X-100、2M Urea，pH 8.0的洗涤缓冲液重悬包涵体，以功率125W、工作时间5s、间歇时间8s的条件超声洗涤60min，14000rpm离心80min收集沉淀，重复洗涤一次；超声洗涤过程中，取样进行SDS-PAGE，检测洗涤效果，见附图6。从附图6前5个泳道可以观察得到，在包涵体洗涤的两个阶段，大量杂蛋白溶于上清中被除去，hCRY1以不溶包涵体形式存在，有利于hCRY1的富集和初级提纯。将目标条带切下，进行质谱鉴定，蛋白的氨基酸序列如SEQ ID NO：1所示。与NCBI数据库对比结果如附图7所示，该包涵体蛋白正式确认为hCRY1。
(5)包涵体变性溶解：加入40ml新配制的组分含6M盐酸胍、pH8.0磷酸缓冲液重悬洗涤后的包涵体沉淀，以功率225W、工作时间5s、间歇时间8s的条件超声促溶90min；再用含8M Urea、pH8.0磷酸缓冲液稀释8倍，以功率225W、工作时间5s、间歇时间8s的程序超声促溶90min，取样进行SDS-PAGE检测包涵体蛋白溶解情况，见附图6。从附图6第6、7、8泳道可以观察到，在超声条件下，hCRY1包涵体开始溶解至上清中。
(6)蛋白亲和层析纯化：将蛋白变性溶解溶液14000rpm高速离心45min，收集上清，经0.45μm滤膜过滤，添加咪唑至滤液中样品中咪唑终浓度为20mM，以上样体积1CV、上样流速8ml/min、洗脱流速15ml/min进行HIS标签亲和层析，层析仪：
Figure PCTCN2016000641-appb-000006
Purifier 100；层析填料：ProteinIso Ni-NTA Resin 100ml；平衡：用缓冲液500mM NaCl、8M Urea、20mM咪唑、pH 8.0平衡2CV；洗脱：用缓冲液500mM NaCl、8M Urea、200mM咪唑、pH 8.0洗脱2CV，收集洗脱蛋白峰。对上样样品，穿透峰以及洗脱峰取样，进行SDS-PAGE电泳检测，结果如附图8所示，层析图谱如附图9所示。
(7)蛋白梯度透析复性：将收集的洗脱蛋白峰样品装入30-50kDa透析袋中，置于8倍体积的含6M Urea，50mM Tris，50mM NaCl，pH8.0透析缓冲液中，4℃透析8-12h；再将样品转移至含4M Urea，50mM Tris，50mM NaCl，pH 8.0透析缓冲液中，4℃透析8-12h；将样品转移至包含2M Urea，100mM Tris，400mM Argine，5mM EDTA，5mM GSH，0.5mM GSSG，pH8.0透析缓冲液中，4℃透析8-12h；将样品转移至包含1M Urea，50mM Tris，50mM NaCl，10％甘油，pH8.0透析缓冲液中，4℃透析8-12h；将样品转移至包含50mM Tris，500mM NaCl，10％甘油，pH8.0透析缓冲液中，4℃透析8-12h；透析完成后，收集透析袋内样品。
(8)蛋白超滤浓缩及溶液置换：使用超滤仪Millipore，用30kDa超滤膜包对透析所得样品进行超滤浓缩：将样品浓缩至原体积约1/4后，逐步添加包含50mM Tris，100mM NaCl，10％甘油，pH8.0的置换缓冲液进行稀释，最后将样品体积浓缩至原体积的1/5，获得hCRY1蛋白溶液，再经0.22μm滤膜过滤后，即得到无菌可溶性hCRY1蛋白溶液。取浓缩前后样品进行SDS-PAGE电泳分析，结果如图10所示。用Bradford蛋白定量检测试剂盒对样品进行蛋白定量，蛋白浓度约为0.52mg/ml，推算蛋白产率约为300mg/L。
重组蛋白活性及功能检测：
取纯化后的蛋白溶液稀释至500μg/ml以作备用，同时用300mM咪唑配置1mg/ml BSA蛋白，经镍柱上样流出以及与hCRY1样品完全相同的超滤置换后，稀释至500μg/ml作为对照(cBSA)。将Hela细胞爬片铺板培养至约80-90％细胞密度时换液，三小时后，分别将备好的hCRY1与对照蛋白按照与培养基1∶1的比例加入细胞培养液，使每个孔内液体总体积为2ml，加入蛋白终浓度为250μg/ml。之后立刻将两组细胞置于X-ray照射机内进行照射，每分钟剂量为1.132/Gy，照射时间530s，总剂量为10Gy。照射之后立刻将细胞培养液均换为实验前的培养基(DMEM)继续培养45分钟后收细胞爬片。用PBS洗片3次，100％甲醇-20℃固定细胞5min，再用PBS洗3次，5％BSA封闭过夜后，用抗体Phospho-Histone H2A.X于37℃孵育细胞1h，再用PBS洗3次，再用抗体山羊抗兔IgG/FITC于37℃孵育细胞1h，PBS3次洗后封片，激光共聚焦显微镜观察两组细胞内磷光体亮度，在完全相同的拍摄条件下每片分别取3个不同视野统计平均荧光强度。在完全相同的观察条件下，肉眼可见两组细胞中形成的FOCI荧光强度差异十分显著(图11左图)，随机选取3个视野并将荧光强度量化，两组之间差异极其显著(P＜0.001)(图11右图)。由此可知，我们制备的hCRY1降低了细胞中FOCI的荧光强度，对细胞起到了一定的保护作用，减少了X-ray的侵害。
为进一步证实细胞内FOCI的减少是由于hCRY1的射线防护作用，我们在HaCaT细胞中进一步进行hCRY1浓度梯度实验并设置未经X-ray处理的对照组细胞，用上述经处理后的BSA溶液稀释hCRY1溶液来设立浓度梯度，用同样方法处理细胞，制片并观察。结果表明，当细胞培养基中的hCRY1浓度达到50μg/ml时，FOCI的荧光强度明显降低(图12)。
我们还分别检测了hCRY1和处理之后的BSA对细胞凋亡的影响。在两组细胞中分别加入250μg/ml hCRY1和250μg/ml处理后的BSA，1h后与另一组未加任何蛋白的细胞一起检测细胞凋亡情况，用细胞凋亡检测试剂盒(由南京凯基生物物质发展有限公司购买)对细胞进行双染后用流式细胞仪进行分析。结果表明1h之内，不论是hCRY1还是BSA都不会造成细胞凋亡，该结果进一步证明FOCI荧光强度的降低是因为细胞内DNA所受损伤的减少而非 细胞的凋亡(图13)。
最后应说明的是：显然，上述实施例仅仅是为了清楚的说明本发明所作的举例，而并非对实施的限定。对于所述领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式子以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围之中。

Claims (7)

  1. 一种重组人隐花色素蛋白I(hCRY1)的生产工艺，其特征在于，包括以下步骤：
    (1)构建重组表达质粒：将hCRY1基因片段重组至原核表达载体pET28a(+)，获得重组表达质粒pET28a(+)-hCRY1；
    (2)构建工程菌：将重组表达质粒pET28a(+)-hCRY1转入感受态宿主细胞E.coli BL21(DE3)，然后通过SDS-PAGE筛选hCRY1表达量高的克隆菌株BL21-hCRY1-b；
    (3)工程菌培养及重组蛋白诱导表达：使用发酵罐培养BL21-hCRY1-b，培养温度36.5-37.5℃，溶氧20-40％，pH6.8-7.4，搅拌速度200-350rpm，培养5-8h后，OD600＝7-8时，加入IPTG诱导培养3-4h；
    (4)包涵体收集和洗涤：3000-5000rpm离心收集菌体，以功率200-250W、工作时间3-8s、间歇时间5-10s的条件超声破碎菌体60-90min，3000-5000rpm离心50-80min，收集包涵体沉淀，用组分含10-100mM Tris、0.1-1.0mM EDTA、0-100mM NaCl、1-10％Triton X-100、1-2M Urea，pH7.5-8.5的洗涤缓冲液重悬包涵体，以功率100-150W、工作时间3-8s、间歇时间5-10s的条件超声洗涤60-90min，12000-14000rpm离心60-90min收集沉淀；
    (5)包涵体变性溶解：用组分含4-8M盐酸胍、pH7.5-8.5磷酸缓冲液重悬洗涤后的包涵体沉淀，以功率200-250W、工作时间3-8s、间歇时间5-10s的条件超声促溶60-90min；再用含6-8M Urea、pH7.5-8.5磷酸缓冲液稀释6-10倍，以功率200-250W、工作时间3-8s、间歇时间5-10s的条件超声促溶60-90min，获得蛋白变性溶解溶液；
    (6)蛋白亲和层析纯化：将蛋白变性溶解溶液12000-14000rpm高速离心40-60min，收集上清，经0.22-0.45μm滤膜过滤，添加咪唑至滤液中咪唑终浓度为5-30mM，以上样体积1-1.5CV、上样流速5-10ml/min、洗脱流速10-20ml/min进行HIS标签亲和层析，用缓冲液0-500mM NaCl、6-8M Urea、0-60mM咪唑、pH7.5-8.5平衡2-4CV，用缓冲液0-500mM NaCl、6-8M Urea、200-300mM咪唑、pH7.5-8.5洗脱2-2.5CV，收集洗脱蛋白峰。
    (7)蛋白梯度透析复性：将收集的蛋白洗脱峰样品进行梯度透析复性，缓冲液包含0-6M Urea、0-20％甘油、0-1M NaCl、0-100mM Tris、0-400mM Arginine、0-10mM EDTA、0-10mM GSH，0-5mM GSSG，pH7.5-8.5。
    (8)蛋白超滤浓缩及溶液置换：使用30-50KDa超滤膜包对透析所得样品进行超滤浓 缩：将样品浓缩至原体积1/4-1/5后，逐步添加包含0-100mM Tris，0-200mM NaCl，0-20％甘油，pH7.5-8.5的置换缓冲液进行稀释，最后将样品体积浓缩至原体积的1/5，得到可溶的hCRY1蛋白溶液，再经0.22μm滤膜过滤后，即得到可溶性hCRY1蛋白溶液；
    所述hCRY1的氨基酸序列如SEQID NO：1所示。
  2. 根据权利要求1所述的重组人隐花色素蛋白I(hCRY1)的生产工艺，其特征在于，所述发酵罐为NBS系列发酵罐。
  3. 根据权利要求2所述的重组人隐花色素蛋白I(hCRY1)的生产工艺，其特征在于，所述IPTG的终浓度为0.5-3.0mM。
  4. 根据权利要求1所述的重组人隐花色素蛋白I(hCRY1)的生产工艺，其特征在于：所述亲和层析的填料为ProteinIso Ni-NTA Resin、Ni-NTA His Bind Resin中任一种，层析所用仪器为
    Figure PCTCN2016000641-appb-100001
    Purifier100。
  5. 根据权利要求1所述的重组人隐花色素蛋白I(hCRY1)的生产工艺，其特征在于：所述梯度透析复性的程序为：将收集的洗脱蛋白峰样品装入30-50kDa透析袋中，于8-10倍体积包含6M Urea，0-100mM Tris，0-100mM NaCl，pH7.5-8.5透析缓冲液中，4℃透析8-12h；将样品转移至包含4M Urea，0-100mM Tris，0-100mM NaCl，pH7.5-8.5透析缓冲液中，4℃透析8-12h；将样品转移至包含2M Urea，0-100mM Tris，0-400mM Argine，0-10mM EDTA，0-10mM GSH，0-5mM GSSG，pH7.5-8.5透析缓冲液中，4℃透析8-12h；将样品转移至包含1M Urea，0-100mM Tris，0-100mM NaCl，5-15％甘油，pH7.5-8.5透析缓冲液中，4℃透析8-12h；将样品转移至包含0-100mM Tris，0-500mM NaCl，0-20％甘油，pH7.5-8.5透析缓冲液中，4℃透析8-12h；透析完成后，收集透析袋内样品。
  6. 根据权利要求1所述的重组人隐花色素蛋白I(hCRY1)的生产工艺，其特征在于：所述超滤使用超滤仪Millipore。
  7. 组合物，含有权利要求1-6任一项所述生产工艺制备的重组hCRY1蛋白以及药学上可接受的载体或辅料。
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