Construction of high level prokaryotic expression and purification system of PD-L1 extracellular domain by using Escherichia coli host cell machinery.
Programmed cell death 1 ligand 1 (PD-L1) is a trans-membrane protein highly expressed on the membrane of cancer cell, which binds inhibitory receptor of PD-1 on the T cells and attenuates anti-tumor immune response.The strategy of blocking PD1 and PD-L1 interaction has been widely used for anti-cancer drug development. The DNA encoding extracellular domain of PD-L1 was cloned and expressed with the pET30(+) and Escherichia coli BL21(DE3) system. Cloning of PD-L1 extracellular domain was confirmed by PCR and enzymatic digestion. Sequence analysis of cloned targeted genes showed 100% homology of original sequence. The recombinant protein was expressed using 1mM/mL IPTG and purified by affinity chromatography on a column of Ni-NTA and confirmed by SDS-PAGE and western blot analysis. Results showed that our constructed pET30(+)/PDL1-ECD system efficiently produces desired recombinant protein with molecular weight of 38.1kDa. The prokaryotic expression system provides an easy method to express PD-L1 extracellular domain that further facilitate the role of PD-1/PD-L1 binding inhibition and helps in valuable drug and antibodies production.
['Kalim|Muhammad|M|', 'Chen|Jie|J|', 'Wang|Shenghao|S|', 'Lin|Caiyao|C|', 'Ullah|Saif|S|', 'Liang|Keying|K|', 'Ding|Qian|Q|', 'Chen|Shuqing|S|', 'Zhan|Jinbiao|J|']
D000911:Antibodies, Monoclonal D060890:B7-H1 Antigen D015153:Blotting, Western D003001:Cloning, Molecular D004926:Escherichia coli D015870:Gene Expression D005822:Genetic Vectors D006801:Humans D007167:Immunotherapy D009369:Neoplasms D016133:Polymerase Chain Reaction D000072417:Protein Domains D011994:Recombinant Proteins D017421:Sequence Analysis
Expression PD-L1 PD-1 Antibody CD274 Immunotherapy