GPC3蛋白结构的分析及其表达纯化

Analysis of GPC3 protein structure and its expression purification

目的了解GPC3蛋白结构,构建GPC3蛋白的原核表达纯化系统,高效表达和纯化出GPC3蛋白。方法从GenBank中获取基因序列,通过Protparam、SOPMA、SWISS-MODEL等在线软件对GPC3蛋白(25-554 aa)的结构进行分析。从不同载体和菌株中筛选出相对优势的组合,优化其表达条件,并诱导表达TrxA-GPC3融合蛋白,通过包涵体的溶解和复性、亲和层析、标签切割和分子筛纯化等方法分离纯化出无TrxA标签的GPC3蛋白。结果GPC3蛋白的相对分子质量为60.26 kDa,等电点为5.76,不稳定系数为41.27,脂肪系数为83.66,平均亲水系数为-0.312,是不稳定的亲水性蛋白,其主要为α-螺旋和无规则卷曲结构,引入标签的TrxA-GPC3融合蛋白比GPC3蛋白更稳定。通过对比筛选出表达效率较高的BL21(DE3)-pET32a-GPC3工程菌,其在0.5%的葡萄糖浓度和0.1 mmol/L的IPTG浓度条件下,结合20℃的诱导温度及16 h的诱导时间,可显著提高蛋白表达量,通过亲和层析得到纯度90%以上的TrxA-GPC3融合蛋白,利用凝血酶将TrxA标签切除,最后通过分子筛纯化得到单一的GPC3蛋白。结论利用生物信息学分析了GPC3蛋白的理化性质和结构,并在原核系统中成功表达和纯化了GPC3蛋白,为高特异性抗体的制备或筛选以及结构-功能研究奠定了基础,同时也为其它蛋白的表达纯化提供科学的指导意义。

Objective To explore the structure of GPC3 protein and construct a prokaryotic sys⁃tem for its efficiently expression and purification.Methods Gene sequences were obtained from GenBank,and the structure of GPC3 protein(25⁃554 aa)was analyzed using online software Protparam,SOPMA and SWISS⁃MODEL.Select relatively advantageous combinations from different vectors and strains,optimize its expression conditions,and induce the expression of TrxA⁃GPC3 fusion protein.Separate and purify GPC3 pro⁃tein without TrxA label through dissolution and renaturation of inclusion body,affinity chromatography,label cleavage and molecular sieve purification.Results GPC3 is an unstable hydrophilic protein with the molecu⁃lar weight about 60.26 kDa,theoretical pI of 5.76,instability index of 41.27,Aliphatic index of 83.66 and grand average hydrophilicity of-0.312.It mainly consists of alpha⁃helices and random coil structures.The TrxA⁃GPC3 fusion protein with an introduced tag is more stable than the GPC3 protein alone.By comparison,the BL21(DE3)⁃pET32a⁃GPC3 engineered bacteria,which exhibit higher expression efficiency,were select⁃ed.Under the conditions of 0.5%glucose concentration and 0.1 mmol/L IPTG concentration,combined with an induction temperature of 20℃and an induction time of 16 hours,the protein expression level can be signifi⁃cantly increased.Through affinity chromatography,TrxA⁃GPC3 fusion protein with a purity of over 90%was obtained.The TrxA tag was then removed using thrombin,and finally,a single GPC3 protein was obtained through molecular sieve purification.Conclusions In this study,we analyzed the physicochemical properties and structure of GPC3 protein using bioinformatics,and successfully expressed and purified GPC3 protein in prokaryotic systems.This laid a foundation for the preparation or screening of highly specific antibodies and structural⁃functional studies,and provided scientific guidance for the expression and purification of other pro⁃teins.