分子伴侣协助的基因工程药物重组人干扰素-γ体外重折叠研究
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摘要
干扰素(Interferon，IFN)是一种高活性、多功能诱生蛋白，具有抗病毒、抗肿瘤及免疫调节等生理功能，因而在医药、临床医学等领域有着重要的应用。与IFN-α／β相比，IFN-γ具有更强的免疫调节和细胞抑制活性，因而更加受到人们的重视。目前，人们不仅成功地构建了能够大量表达IFN-γ的基因工程菌，而且利用该基因工程菌生产的IFN-γ已被批准为临床用药。但是，在利用E.coli进行大量表达IFN-γ时，往往形成无活性的包涵体，这就要求对其进行复性，使其重新具有生物活性。
小分子伴侣sht GroEL 191-345是GroEL顶端区域氨基端残基191～345片段克隆表达产物。它是一种新型的协助蛋白质折叠体系。本文研究了在小分子伴侣的协助条件下，对由E.coli基因工程菌产生的重组人IFN-γ(rhIFN-γ)包涵体进行复性技术研究，探索较佳的复性条件，以期得到具有更高蛋白收率及活性的rhIFN-γ。
首先建立了rhIFN-γ的生物活性检测方法，测定细胞为人羊膜细胞Wish株，攻击病毒为水泡性口炎病毒(VSV)株。对以细胞病变(CPE)效应为基础的结晶紫染色法进行了条件优化，考察了结晶紫染液的用量及作用时间、溶媒提取时间、孔板效应及边缘效应等条件的影响。结果表明结晶紫染液的加入量在30～50μL之间较适宜，同时应当与染液作用时间相关联，用量大时作用时间相对减少；有机溶液脱色30min后测定较好，此时细胞核中的结晶紫已被提取完全，而结晶紫在脱色液中尚能稳定存在；为了最大程度利用微量板，同时考虑到边缘效应和孔板效应的存在，第1和第12列加细胞对照，第6和第7列加病毒对照。
其次在摇瓶的基础上进行了小分子伴侣sht GroEL 191-345的发酵罐培养。操作条件为：初始葡萄糖浓度为5g／L，接种量2.5％，转速为350r／min，通气量200L／h，装液量2.1L／3.7L，发酵温度33℃。经过超声波破碎、高速离心分离、亲和层析纯化及脱盐后得到sht GroEL 191-345 216.2mg／L发酵液。
然后考察了游离和固定化小分子伴侣sht GroEL 191-345对rhIFN-γ体外重折叠复性的作用。sht GroEL191-345的加入有效地促进了rhIFN-γ的复性，用含脲1.0mol／L，pH7.7的复性缓冲液将rhIFN-γ包涵体稀释至初始蛋白浓度100μg／mL，15℃复性4h后，加入sht GroEL 191-345的一组(sht GroEL 191-345同rhIFN-γ的摩尔比为2:1)蛋白收率提高了2.2倍，总活性提高了近3倍；将小分子伴侣固定化在NHS-activated Sepharose Fast Flow凝胶后，不但能重复利用，而且进一步提高了rhIFN-γ复性效率，在初始蛋白浓度为400μg／mL时，仍使蛋白质收率达到46.29％和比活达到9.05×10~6IU／mg。
浙江大学硕士学位论文
摘要
最后初步研究了人工分子伴侣(cTAB+p一CD)系统对thIFN一丫体外重折叠
复性的作用。实验结果表明，人工分子伴侣系统也可以提高thIFN一丫的复性效果，
较优复性操作条件:复性体系中cTAB浓度1 00林mol几，15℃下复性30min后，
加入p一CD(p一CD与cTAB的摩尔比为18:l)，继续复性至4h终止，复性缓冲液
pH7.7，脉浓度1.5 mol/L。在此操作条件下，当初始蛋白浓度为100林g/mL时，
蛋白收率 29.76%，比活6.34、IO6IU/ing。
The interferons are Cytokines with antiviral, antiproliferative and imrnnnomodulatory activities. Among three major distinguished types, interferon-γ (IFN- γ) is more hailed for their specific properties in inhibition of cell growth and modulation of immune functions. Now the recombinant DNA techniques make it possible to mass express recombinant IFN-γ in E. coli; however, this may result in the formation of inclusion bodies, and the recovery of biologically active products becomes significant.
Molecular chaperone GroEL-GroES is a ubiquitous class of proteins in E. coli that play an essential role in protein folding by helping other polypeptides reach a proper conformation or cellular location without becoming part of the final structure. As the minimal mechanism of GroEL-mediated protein folding, minichaperones, fragments encompassing the apical domain of GroEL, can facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL.
Here, we reported the minichaperone sht GroEL191-345 mediated refolding of recombinant human IFN- γ (rhIFN-γy) produced in E. coli. SDS-PAGE was performed with Laemmli's Tris-glycine buffer system for protein analysis. Protein concentrations and rhIFN-γ total activity were determined by a modification of Bradford's .method and cytopatic effect (CPE) method, respectly.
Firstly, the colorimetric CPE method has been optimized to measure the biological activity during the refolding of rhIFN-γ inclusion bodies.The dosage and the absorbance of crystalline violet, the extraction time of the solvent, plate effect and marginal effect were investigated.
Secondly, we successfully transferred a batch culture from a 500ml shaldng flask to a 3.7L bioreactor and explored the soluble expression and efficient purification of recombinant sht GroEL191-345, and 216.2 mg/L homogeneous protein was obtained. Control parameters: operating volume 2.1L, inoculation percent 2.5%, temperature 33℃, the stirred speed 350 r/mih and the airflow rate 200 L/h.
Thirdly, we optimized the refolding condition of rhIFN-γ assisted by sht GroEL191-345. Compared with the conventional method, such as dilution and dialysis, the presence of sht GroEL191-345 in the refolding buffer not only enhanced the specific activity, but also raised the refolding efficiency. With the initial protein concentration of 100μg/mL, the protein yield and specific activity, of rhIFN- γ
assisted by sht GroEL191-345 was 2.2 and 3 folds of that under spontaneous condition respectively. Optimal operating parameters in refolding of rhlFN-γy assisted by sht GroEL191-345 were as follows: refolding temperature 15 ℃, refolding time 4 h, pH 7.7, initial concentration of IFN- y 100-200μg/mL and the mol ratio of sht GroEL191-345 versus IFN- y 1:1-2:1. Furthermore, the immobilization of sht GroEL191-345 on NHS-activated sepharose fast flow made it possible to be recycled. The protein yield and the specific activity of rhlFN- y were 46.29% and 9.05 x 106 IU/mg even the initial protein concentration was up to 400μg/mL.
Finally, preliminary results were obtained in the refolding of rhlFN- y assisted by artificial chaperone system (CTAB and P-CD). Optimal operating parameters in refolding of rhlFN- y assisted by CTAB and P-CD were as follows: refolding temperature 15℃, refolding time 4 h, pH=7.7, urea concentration 1.5mol/L, CTAB concentration 100μmol/L, the mol ratio of P-CD versus CTAB 18:1 and the point of adding beta-CD 30 min after the refolding process began. With the initial protein concentration of 100 μg/mL, the protein yield and specific activity of rhlFN- y assisted by CTAB and p-CD was 29.76% and 6.34 x106 IU/mg correspondingly.
小分子伴侣sht GroEL 191-345是GroEL顶端区域氨基端残基191～345片段克隆表达产物。它是一种新型的协助蛋白质折叠体系。本文研究了在小分子伴侣的协助条件下，对由E.coli基因工程菌产生的重组人IFN-γ(rhIFN-γ)包涵体进行复性技术研究，探索较佳的复性条件，以期得到具有更高蛋白收率及活性的rhIFN-γ。
首先建立了rhIFN-γ的生物活性检测方法，测定细胞为人羊膜细胞Wish株，攻击病毒为水泡性口炎病毒(VSV)株。对以细胞病变(CPE)效应为基础的结晶紫染色法进行了条件优化，考察了结晶紫染液的用量及作用时间、溶媒提取时间、孔板效应及边缘效应等条件的影响。结果表明结晶紫染液的加入量在30～50μL之间较适宜，同时应当与染液作用时间相关联，用量大时作用时间相对减少；有机溶液脱色30min后测定较好，此时细胞核中的结晶紫已被提取完全，而结晶紫在脱色液中尚能稳定存在；为了最大程度利用微量板，同时考虑到边缘效应和孔板效应的存在，第1和第12列加细胞对照，第6和第7列加病毒对照。
其次在摇瓶的基础上进行了小分子伴侣sht GroEL 191-345的发酵罐培养。操作条件为：初始葡萄糖浓度为5g／L，接种量2.5％，转速为350r／min，通气量200L／h，装液量2.1L／3.7L，发酵温度33℃。经过超声波破碎、高速离心分离、亲和层析纯化及脱盐后得到sht GroEL 191-345 216.2mg／L发酵液。
然后考察了游离和固定化小分子伴侣sht GroEL 191-345对rhIFN-γ体外重折叠复性的作用。sht GroEL191-345的加入有效地促进了rhIFN-γ的复性，用含脲1.0mol／L，pH7.7的复性缓冲液将rhIFN-γ包涵体稀释至初始蛋白浓度100μg／mL，15℃复性4h后，加入sht GroEL 191-345的一组(sht GroEL 191-345同rhIFN-γ的摩尔比为2:1)蛋白收率提高了2.2倍，总活性提高了近3倍；将小分子伴侣固定化在NHS-activated Sepharose Fast Flow凝胶后，不但能重复利用，而且进一步提高了rhIFN-γ复性效率，在初始蛋白浓度为400μg／mL时，仍使蛋白质收率达到46.29％和比活达到9.05×10~6IU／mg。
浙江大学硕士学位论文
摘要
最后初步研究了人工分子伴侣(cTAB+p一CD)系统对thIFN一丫体外重折叠
复性的作用。实验结果表明，人工分子伴侣系统也可以提高thIFN一丫的复性效果，
较优复性操作条件:复性体系中cTAB浓度1 00林mol几，15℃下复性30min后，
加入p一CD(p一CD与cTAB的摩尔比为18:l)，继续复性至4h终止，复性缓冲液
pH7.7，脉浓度1.5 mol/L。在此操作条件下，当初始蛋白浓度为100林g/mL时，
蛋白收率 29.76%，比活6.34、IO6IU/ing。
The interferons are Cytokines with antiviral, antiproliferative and imrnnnomodulatory activities. Among three major distinguished types, interferon-γ (IFN- γ) is more hailed for their specific properties in inhibition of cell growth and modulation of immune functions. Now the recombinant DNA techniques make it possible to mass express recombinant IFN-γ in E. coli; however, this may result in the formation of inclusion bodies, and the recovery of biologically active products becomes significant.
Molecular chaperone GroEL-GroES is a ubiquitous class of proteins in E. coli that play an essential role in protein folding by helping other polypeptides reach a proper conformation or cellular location without becoming part of the final structure. As the minimal mechanism of GroEL-mediated protein folding, minichaperones, fragments encompassing the apical domain of GroEL, can facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL.
Here, we reported the minichaperone sht GroEL191-345 mediated refolding of recombinant human IFN- γ (rhIFN-γy) produced in E. coli. SDS-PAGE was performed with Laemmli's Tris-glycine buffer system for protein analysis. Protein concentrations and rhIFN-γ total activity were determined by a modification of Bradford's .method and cytopatic effect (CPE) method, respectly.
Firstly, the colorimetric CPE method has been optimized to measure the biological activity during the refolding of rhIFN-γ inclusion bodies.The dosage and the absorbance of crystalline violet, the extraction time of the solvent, plate effect and marginal effect were investigated.
Secondly, we successfully transferred a batch culture from a 500ml shaldng flask to a 3.7L bioreactor and explored the soluble expression and efficient purification of recombinant sht GroEL191-345, and 216.2 mg/L homogeneous protein was obtained. Control parameters: operating volume 2.1L, inoculation percent 2.5%, temperature 33℃, the stirred speed 350 r/mih and the airflow rate 200 L/h.
Thirdly, we optimized the refolding condition of rhIFN-γ assisted by sht GroEL191-345. Compared with the conventional method, such as dilution and dialysis, the presence of sht GroEL191-345 in the refolding buffer not only enhanced the specific activity, but also raised the refolding efficiency. With the initial protein concentration of 100μg/mL, the protein yield and specific activity, of rhIFN- γ
assisted by sht GroEL191-345 was 2.2 and 3 folds of that under spontaneous condition respectively. Optimal operating parameters in refolding of rhlFN-γy assisted by sht GroEL191-345 were as follows: refolding temperature 15 ℃, refolding time 4 h, pH 7.7, initial concentration of IFN- y 100-200μg/mL and the mol ratio of sht GroEL191-345 versus IFN- y 1:1-2:1. Furthermore, the immobilization of sht GroEL191-345 on NHS-activated sepharose fast flow made it possible to be recycled. The protein yield and the specific activity of rhlFN- y were 46.29% and 9.05 x 106 IU/mg even the initial protein concentration was up to 400μg/mL.
Finally, preliminary results were obtained in the refolding of rhlFN- y assisted by artificial chaperone system (CTAB and P-CD). Optimal operating parameters in refolding of rhlFN- y assisted by CTAB and P-CD were as follows: refolding temperature 15℃, refolding time 4 h, pH=7.7, urea concentration 1.5mol/L, CTAB concentration 100μmol/L, the mol ratio of P-CD versus CTAB 18:1 and the point of adding beta-CD 30 min after the refolding process began. With the initial protein concentration of 100 μg/mL, the protein yield and specific activity of rhlFN- y assisted by CTAB and p-CD was 29.76% and 6.34 x106 IU/mg correspondingly.
引文
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