Effect of Freeze-Thaw and Urea in Solubility of GPC3-Csub Protein Expressed in Escherichia coli  

作　　者：Xuan-Truc Chu-Dao Kim-Tuyen Huynh-Dam Dang-Thuc Ngo-Luong Quang-Luan Le Thanh-Thao Vo-Nguyen Xuan-Truc Chu-Dao;Kim-Tuyen Huynh-Dam;Dang-Thuc Ngo-Luong;Quang-Luan Le;Thanh-Thao Vo-Nguyen(Department of Medical Biotechnology, Biotechnology Center of Ho Chi Minh City, Ho Chi Minh City, Vietnam)

机构地区：[1]Department of Medical Biotechnology, Biotechnology Center of Ho Chi Minh City, Ho Chi Minh City, Vietnam

出　　处：《Journal of Biosciences and Medicines》2024年第4期288-297,共10页生物科学与医学（英文）

摘　　要：Glypican-3 is a protein encoded by the Glypican-3 gene located on human X chromosome (Xq26), composed of two subunits, a 40 kDa N-terminal subunit, and a 30 kDa C-terminal subunit. Glypican-3 is a currently potential target molecule for liver cancer treatments because of its over-expression and growth effects on hepatocellular carcinoma (HCC). This study examined the expression and purification of a C-terminal subunit of Glypican-3 protein (GPC3-Csub) due to its application in both diagnosis and therapy for hepatocellular carcinoma. The gene encoding for GPC3-Csub was successfully cloned into plasmid pET28a fused with an affinity tag composed of six consecutive histidine residues (His-tag). Recombinant protein GPC3-Csub was expressed in Escherichia coli BL21 (DE3) in the condition of adding 3% ethanol with IPTG induction. GPC3-Csub was extracted using repeated freeze-thaw cycles with lysozyme, and inclusion bodies were solubilized by 8M Urea, SDS 10% in pH 12. His-tag fused GPC3-Csub proteins allowed it to be purified by affinity chromatography method using the Nickel-nitrilotriacetic acid (Ni-NTA) column. High expression of GPC3-Csub was confirmed by Coomassie staining and western-blot. GPC3-Csub could be isolated with a Ni-NTA column and have a purity of about 90%.Glypican-3 is a protein encoded by the Glypican-3 gene located on human X chromosome (Xq26), composed of two subunits, a 40 kDa N-terminal subunit, and a 30 kDa C-terminal subunit. Glypican-3 is a currently potential target molecule for liver cancer treatments because of its over-expression and growth effects on hepatocellular carcinoma (HCC). This study examined the expression and purification of a C-terminal subunit of Glypican-3 protein (GPC3-Csub) due to its application in both diagnosis and therapy for hepatocellular carcinoma. The gene encoding for GPC3-Csub was successfully cloned into plasmid pET28a fused with an affinity tag composed of six consecutive histidine residues (His-tag). Recombinant protein GPC3-Csub was expressed in Escherichia coli BL21 (DE3) in the condition of adding 3% ethanol with IPTG induction. GPC3-Csub was extracted using repeated freeze-thaw cycles with lysozyme, and inclusion bodies were solubilized by 8M Urea, SDS 10% in pH 12. His-tag fused GPC3-Csub proteins allowed it to be purified by affinity chromatography method using the Nickel-nitrilotriacetic acid (Ni-NTA) column. High expression of GPC3-Csub was confirmed by Coomassie staining and western-blot. GPC3-Csub could be isolated with a Ni-NTA column and have a purity of about 90%.

关 键 词：GLYPICAN-3 Affinity Chromatography Inclusion Body Liver Cancer 

分 类 号：R73[医药卫生—肿瘤]