LZ8-Fc融合蛋白重组表达及其稳定性研究
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摘要
灵芝自古便是中国传统医学中的珍贵药材，被认为具有滋补强身、扶正固本之效。近代科学研究也证实灵芝的粗提物具有抗肿瘤和增强免疫力之功能，然而多数研究认为灵芝主要活性成分为多醣体及三萜类化合物，直至1989年Kino等科学家自灵芝的菌丝体纯化出具有免疫调节功能的小分子蛋白质—灵芝免疫调节蛋白（LZ-8），确立另一种活性物质免疫调节蛋白的存在。二十多年以来，国内外数十家课题组对其进行了深入研究，利用多种基因工程表达系统获得了重组LZ-8，揭示了LZ-8的晶体结构和主要理化性质，最为重要的是发现了LZ-8具有多种达到临床治疗价值的免疫调节和抗肿瘤活性。虽然LZ-8具有药用价值，但其被开发成为新药仍不被看好，主要原因是分子量小，仅13kDa，极容易被机体代谢，体内稳定性差，难以发挥其生物学作用。此外，LZ-8来源于灵芝，属于异源蛋白，易引起机体免疫系统产生严重的排异反应。考虑到以上问题，在本研究中将LZ-8与人IgG Fc段融合（LZ8-Fc）在毕赤酵母中进行重组表达，建立了中试发酵和纯化工艺，初步研究了LZ8-Fc的体内稳定性和LZ-8的融合表达策略，本论文的实验数据为融合蛋白表达共性问题中的不稳定性因素提供了理论基础，将对其长效剂型的开发具有一定的参考意义。
本研究中使用GGSS柔性肽段连接rLZ8和人IgG1中的Fc段，根据毕赤酵母密码子偏好性重新设计基因序列，两端设EcoRI（GAATTC）和KpnI（GGTACC）酶切位点，进行全基因合成。将目的基因片段连接至AOX1型启动子的甲醇诱导分泌型表达载体—pPICZɑ A，重组后的质粒命名为pPICZɑ A-LZ8-Fc。重组质粒电转化入毕赤酵母菌株X33，在摇瓶规模进行转化克隆的表达筛选，并初步优化了甲醇诱导表达条件。
在40升中试发酵规模下，通过摸索发酵过程工艺参数，包括pH值、DO值、罐压、温度、通气量，优化LZ8-Fc重组蛋白的甲醇诱导发酵工艺；发酵液上清SDS-PAGE电泳和Western Blot鉴定结果均显示，从甲醇诱导表达阶段，开始有目的蛋白条带，至发酵后期出现了降解的LZ-8蛋白条带。LZ8-Fc融合蛋白发酵上清经过两步切向流过滤处理，将样品经MabSelect Sure亲和层析和Superdex75凝胶过滤层析进行纯化，确定其稳定的纯化工艺，获得了纯度较高的LZ8-Fc融合蛋白，电泳显示为单一条带，质谱分析结果与预期一致。但是Western Blot结果表明LZ8-Fc融合蛋白在纯化过程中发生了降解。经检测，LZ8-Fc融合蛋白浓度为248g/mL，高效液相色谱的分子筛鉴定，LZ8-Fc融合蛋白的纯度为98.33%。
采用绵羊血红细胞凝集实验鉴定发现与相同摩尔质量的LZ-8蛋白比较，LZ8-Fc凝血活性降低。小鼠脾细胞的γ干扰素活性实验显示，LZ8-Fc未表现出LZ-8刺激脾细胞产生γ-干扰素的免疫调节活性。半衰期检测采用大鼠尾静脉给药的方法，按照5min，30min，1h，2h，4h，8h，24h，48h八个时间点取血，结果表明血清中LZ8在8h内明显下降，24h后超出检测范围，LZ8-Fc融合蛋白在4h后超出检测范围。
根据SCOP网站的数据库中的信息表明，LZ-8和IgG Fc段的晶体结构均为球蛋白，因而我们利用分子筛为原理的HPLC分析LZ8-Fc在自然状态下的保留时间，进而推断出LZ8-Fc的分子组装方式，实验结果显示LZ8-Fc仍以二聚体形式进行组装的。本研究中还发现一个重要的实验现象，即将LZ8-Fc浓缩至浓度（2.5mg/mL）时，LZ8-Fc分子出现了断裂。本研究中采用分子动力学计算分析LZ8-Fc分子的稳定性，并结合圆二色光谱结构分析，发现融合表达后分子的二级结构出现了很大变化，这可能是影响LZ-8分子稳定性的主要原因。
本研究为增强LZ-8的体内稳定性，降低其作为异源蛋白的免疫原性进行了初步探索。虽然LZ8-Fc分子仍不够稳定，但本论文的实验数据表明在融合蛋白表达后的应用中普遍存在的一个重要的问题，即位于N末端的蛋白可能会影响下游蛋白质的正确折叠。因此，融合表达中应尽量保留下游蛋白原有的N末端，为其他在融合蛋白表达的设计上提供了理论依据。
Ganoderma lucidum (G. lucidum) has been used in traditional Chinese medicineherbs for countries, and it is regarded as effective nutrients for human bodies. Modernmedical studies have confirmed that G. lucidum extracts have anti-tumor andimmune-regulatory functions. However, most of the studies focused on the mainactive components of G. lucidum such as polysaccharides and triterpenoids. In1989,Kino and colleagues extracted and purified a fraction of small molecule proteins fromthe mycelia of G. lucidum, and named it Ling Zhi immune-regulatory protein (LZ-8).This discovery confirmed the existence of immunomodulatory proteins. Ever sincethen, dozens of research groups investigated in-depth studies on LZ-8through variousgenetic expression systems to obtain the recombinant LZ-8. The crystal structure ofLZ-8has been revealed and the main physico-chemical properties are known. Mostimportantly, the therapeutic value of immunomodulation and antitumor activities ofLZ-8have been studied. Nevertheless, the pharmaceutical development based onLZ-8has not been favorable mainly due to the small molecular weight of LZ-8(13kDa) and its poor stability in vivo. In addition, LZ-8belongs to heterologousprotein family derived from G. lucidum, which leads to serious immune rejection. Inthis study we fused LZ-8with human IgG Fc fraction to produce the LZ8-Fc fusionprotein in a yeast (pichia pastoris) expressing system. The fermentation andpurification technology of LZ8-Fc was established, and its stability in vivo waspreliminary studied. This study will benefit the pharmaceutical development of LZ-8.
For technical approaches, the flexible peptide (GGSS) was used to connect LZ8and IgG1Fc fraction. The DNA sequence was optimized to yeast codons. The EcoRIand KpnI restrictive enzyme sites were inserted to both terminals of the LZ8-Fccoding sequence.
The the LZ8-Fc coding sequence was inserted to the plasmid (pPICZɑA). AOX1promoter was also inserted in the vectorfor methanol secretory induction. Therecombinant plasmid was named pPICZɑA-LZ8-Fc and transformed to the yeast strain (X33). The preliminary methanol-inducible expression condition of LZ8-Fc wasoptimized..
In the40-liter pilot-scale fermentation, the fermentation parameters, includingpH, DO, tank pressure, temperature, ventilation, were further optimized for methanolinduction of LZ8-Fc expression.
The results of SDS-page electrophoresis and Western Blot analysis on thesupernatant of fermentation indicated that LZ8-Fc protein bands were detectableduring methanol induction, and the degradation bands of LZ8-Fc were detectableafterwards. The fermentation supernatant was treated by a two-stage tangential flowfiltration. Afterwards, the samples were purified by MabSelect Sure affinity andSuperdex75gel filtration chromatography. It was determined that the purificationprocess was steady, and high purity LZ8-Fc fusion proteins were obtained.The resultof electrophoresis showed a single band, and the result of mass spectrometry analysiswas expected. However, the Western Blot analysis indicated that the LZ8-Fc wasdegraded during purification. The LZ8-Fc fusion protein concentration was248g/mL,and the purity was98.33%, detected by high-performance liquid chromatographicidentification. The hemagglutinating activity examination showed that LZ8-Fc hadlower activity than LZ-8in sheep red blood cell agglutination. γ interferon activityof splenocytes was carried out in mice. The result indicated that LZ8-Fc had nostimulation activity of γ-interferon on spleen cells compared with LZ-8. The half-lifedetection was performed by tail vein injection. The blood was collected at5min,30min,1h,2H,4H,8h,24h,48h time points, and the results indicated that rLZ8concentration in serum decreased significantly at8h and out of detection at24h, whileLZ8-Fc was out of detection at4h.
According to the information of SCOP online database, it showed that the crystalstructure of LZ-8and IgG Fc were both globulin. Thus, the retention time analysis ofLZ8-Fc was performed through molecular screen HPLC. The LZ8-Fc molecularassembly was a dimer. This study also found the phenomena that LZ8-Fc proteinstructure was broken in high concentration (2.5mg/mL). Molecular dynamics,combined with the structural analysis of circular dichroism spectra were utilized in thestability analysis of LZ8-Fc. It was discovered that considerable changes happened tothe secondary structure of LZ8-Fc after expression, which may explain the poor stability of LZ-8.
This study was carried out to enhancing the stability of LZ-8in vivo and toreduce its immunogenicity as a heterologous protein. Although the LZ8-Fc protein isnot very stable, the results suggested it is important to improve the expression offusion protein. The downstream of the N-terminal of the protein may interrupt thefolding of LZ8-Fc. Therefore, the downstream protein was suggested to retain, whichprovided a theoretical basis for further studies on fusion proteins.
本研究中使用GGSS柔性肽段连接rLZ8和人IgG1中的Fc段，根据毕赤酵母密码子偏好性重新设计基因序列，两端设EcoRI（GAATTC）和KpnI（GGTACC）酶切位点，进行全基因合成。将目的基因片段连接至AOX1型启动子的甲醇诱导分泌型表达载体—pPICZɑ A，重组后的质粒命名为pPICZɑ A-LZ8-Fc。重组质粒电转化入毕赤酵母菌株X33，在摇瓶规模进行转化克隆的表达筛选，并初步优化了甲醇诱导表达条件。
在40升中试发酵规模下，通过摸索发酵过程工艺参数，包括pH值、DO值、罐压、温度、通气量，优化LZ8-Fc重组蛋白的甲醇诱导发酵工艺；发酵液上清SDS-PAGE电泳和Western Blot鉴定结果均显示，从甲醇诱导表达阶段，开始有目的蛋白条带，至发酵后期出现了降解的LZ-8蛋白条带。LZ8-Fc融合蛋白发酵上清经过两步切向流过滤处理，将样品经MabSelect Sure亲和层析和Superdex75凝胶过滤层析进行纯化，确定其稳定的纯化工艺，获得了纯度较高的LZ8-Fc融合蛋白，电泳显示为单一条带，质谱分析结果与预期一致。但是Western Blot结果表明LZ8-Fc融合蛋白在纯化过程中发生了降解。经检测，LZ8-Fc融合蛋白浓度为248g/mL，高效液相色谱的分子筛鉴定，LZ8-Fc融合蛋白的纯度为98.33%。
采用绵羊血红细胞凝集实验鉴定发现与相同摩尔质量的LZ-8蛋白比较，LZ8-Fc凝血活性降低。小鼠脾细胞的γ干扰素活性实验显示，LZ8-Fc未表现出LZ-8刺激脾细胞产生γ-干扰素的免疫调节活性。半衰期检测采用大鼠尾静脉给药的方法，按照5min，30min，1h，2h，4h，8h，24h，48h八个时间点取血，结果表明血清中LZ8在8h内明显下降，24h后超出检测范围，LZ8-Fc融合蛋白在4h后超出检测范围。
根据SCOP网站的数据库中的信息表明，LZ-8和IgG Fc段的晶体结构均为球蛋白，因而我们利用分子筛为原理的HPLC分析LZ8-Fc在自然状态下的保留时间，进而推断出LZ8-Fc的分子组装方式，实验结果显示LZ8-Fc仍以二聚体形式进行组装的。本研究中还发现一个重要的实验现象，即将LZ8-Fc浓缩至浓度（2.5mg/mL）时，LZ8-Fc分子出现了断裂。本研究中采用分子动力学计算分析LZ8-Fc分子的稳定性，并结合圆二色光谱结构分析，发现融合表达后分子的二级结构出现了很大变化，这可能是影响LZ-8分子稳定性的主要原因。
本研究为增强LZ-8的体内稳定性，降低其作为异源蛋白的免疫原性进行了初步探索。虽然LZ8-Fc分子仍不够稳定，但本论文的实验数据表明在融合蛋白表达后的应用中普遍存在的一个重要的问题，即位于N末端的蛋白可能会影响下游蛋白质的正确折叠。因此，融合表达中应尽量保留下游蛋白原有的N末端，为其他在融合蛋白表达的设计上提供了理论依据。
Ganoderma lucidum (G. lucidum) has been used in traditional Chinese medicineherbs for countries, and it is regarded as effective nutrients for human bodies. Modernmedical studies have confirmed that G. lucidum extracts have anti-tumor andimmune-regulatory functions. However, most of the studies focused on the mainactive components of G. lucidum such as polysaccharides and triterpenoids. In1989,Kino and colleagues extracted and purified a fraction of small molecule proteins fromthe mycelia of G. lucidum, and named it Ling Zhi immune-regulatory protein (LZ-8).This discovery confirmed the existence of immunomodulatory proteins. Ever sincethen, dozens of research groups investigated in-depth studies on LZ-8through variousgenetic expression systems to obtain the recombinant LZ-8. The crystal structure ofLZ-8has been revealed and the main physico-chemical properties are known. Mostimportantly, the therapeutic value of immunomodulation and antitumor activities ofLZ-8have been studied. Nevertheless, the pharmaceutical development based onLZ-8has not been favorable mainly due to the small molecular weight of LZ-8(13kDa) and its poor stability in vivo. In addition, LZ-8belongs to heterologousprotein family derived from G. lucidum, which leads to serious immune rejection. Inthis study we fused LZ-8with human IgG Fc fraction to produce the LZ8-Fc fusionprotein in a yeast (pichia pastoris) expressing system. The fermentation andpurification technology of LZ8-Fc was established, and its stability in vivo waspreliminary studied. This study will benefit the pharmaceutical development of LZ-8.
For technical approaches, the flexible peptide (GGSS) was used to connect LZ8and IgG1Fc fraction. The DNA sequence was optimized to yeast codons. The EcoRIand KpnI restrictive enzyme sites were inserted to both terminals of the LZ8-Fccoding sequence.
The the LZ8-Fc coding sequence was inserted to the plasmid (pPICZɑA). AOX1promoter was also inserted in the vectorfor methanol secretory induction. Therecombinant plasmid was named pPICZɑA-LZ8-Fc and transformed to the yeast strain (X33). The preliminary methanol-inducible expression condition of LZ8-Fc wasoptimized..
In the40-liter pilot-scale fermentation, the fermentation parameters, includingpH, DO, tank pressure, temperature, ventilation, were further optimized for methanolinduction of LZ8-Fc expression.
The results of SDS-page electrophoresis and Western Blot analysis on thesupernatant of fermentation indicated that LZ8-Fc protein bands were detectableduring methanol induction, and the degradation bands of LZ8-Fc were detectableafterwards. The fermentation supernatant was treated by a two-stage tangential flowfiltration. Afterwards, the samples were purified by MabSelect Sure affinity andSuperdex75gel filtration chromatography. It was determined that the purificationprocess was steady, and high purity LZ8-Fc fusion proteins were obtained.The resultof electrophoresis showed a single band, and the result of mass spectrometry analysiswas expected. However, the Western Blot analysis indicated that the LZ8-Fc wasdegraded during purification. The LZ8-Fc fusion protein concentration was248g/mL,and the purity was98.33%, detected by high-performance liquid chromatographicidentification. The hemagglutinating activity examination showed that LZ8-Fc hadlower activity than LZ-8in sheep red blood cell agglutination. γ interferon activityof splenocytes was carried out in mice. The result indicated that LZ8-Fc had nostimulation activity of γ-interferon on spleen cells compared with LZ-8. The half-lifedetection was performed by tail vein injection. The blood was collected at5min,30min,1h,2H,4H,8h,24h,48h time points, and the results indicated that rLZ8concentration in serum decreased significantly at8h and out of detection at24h, whileLZ8-Fc was out of detection at4h.
According to the information of SCOP online database, it showed that the crystalstructure of LZ-8and IgG Fc were both globulin. Thus, the retention time analysis ofLZ8-Fc was performed through molecular screen HPLC. The LZ8-Fc molecularassembly was a dimer. This study also found the phenomena that LZ8-Fc proteinstructure was broken in high concentration (2.5mg/mL). Molecular dynamics,combined with the structural analysis of circular dichroism spectra were utilized in thestability analysis of LZ8-Fc. It was discovered that considerable changes happened tothe secondary structure of LZ8-Fc after expression, which may explain the poor stability of LZ-8.
This study was carried out to enhancing the stability of LZ-8in vivo and toreduce its immunogenicity as a heterologous protein. Although the LZ8-Fc protein isnot very stable, the results suggested it is important to improve the expression offusion protein. The downstream of the N-terminal of the protein may interrupt thefolding of LZ8-Fc. Therefore, the downstream protein was suggested to retain, whichprovided a theoretical basis for further studies on fusion proteins.
引文
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