Establishment of a human cell line with a surface display system for screening and optimizing Na+-taurocholate cotransporting polypeptide-binding peptides

作者全名："Wang, Pei-yun; Yang, Xue; Guo, Lin; Wang, Yu-wei; Zhang, Wen-lu; Sun, Yu-xue; Li, Jie; Gan, Chun-yang; Long, Shao-yuan; Liu, Jia-jun; Fan, Shu-ying; Huang, Ai-long; Hu, Jie-Li"

作者地址："[Wang, Pei-yun; Yang, Xue; Guo, Lin; Zhang, Wen-lu; Sun, Yu-xue; Li, Jie; Gan, Chun-yang; Long, Shao-yuan; Liu, Jia-jun; Fan, Shu-ying; Huang, Ai-long; Hu, Jie-Li] Chongqing Med Univ, Key Lab Mol Biol Infect Dis, Minist Educ, Chongqing, Peoples R China; [Wang, Yu-wei] Chongqing Hosp Tradit Chinese Med, Dept Lab Med, Chongqing, Peoples R China; [Wang, Yu-wei] Chongqing Hosp Tradit Chinese Med, Lab Diag & Treatment Infect Dis Integrated Tradit, Chongqing, Peoples R China"

通信作者："Huang, AL; Hu, JL (通讯作者)，Chongqing Med Univ, Key Lab Mol Biol Infect Dis, Minist Educ, Chongqing, Peoples R China."

来源：FRONTIERS IN MICROBIOLOGY

ESI学科分类：MICROBIOLOGY

WOS号：WOS:000848530200001

JCR分区：Q2

影响因子：5.2

年份：2022

卷号：13

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：mammalian cell surface display; FACS; peptides; NTCP; screening and optimization

摘要："One of the most desirable targets for HBV medications is the sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for the hepatitis B virus (HBV). N-myristoylated preS1 2-48 (Myrcludex B or Hepcludex), an NTCP-binding peptide from the large surface protein of HBV, has been developed as the first-in-class entry inhibitor. However, its relatively large molecular weight contributes to increased immunogenicity and antibody production. As a result, it is preferable to look for an NTCP-binding peptide with a smaller size. To do this, we developed a human cell surface display strategy and screened peptides based on preS1-21. PreS1-21 (genotype D) was extended by 7 random amino acids and fused with mCherry and FasL transmembrane domain. The pooled constructs were transfected into HEK293 cells by using the transposon/transposase system to create a library displaying various peptides on the cell surface with red fluorescence. On the other hand, we expressed NTCP protein fused with EGFP on HEK293 and used the membrane lysate containing NTCP-GFP as the bait protein to select peptides with increased NTCP affinity. After 7 cycles of selection, the deep sequencing results revealed that some polypeptides were more than 1,000 times enriched. Further screening of the mostly enriched 10 peptides yields the peptide preS1-21-pep3. Replacing the preS1-21 sequence of preS1-21-pep3 with those from different genotypes demonstrated that the consensus sequence of genotype A-F had the best performance. The peptide (Myr-preS1-21-pep3) was synthesized and tested on the HepG2-NTCP cell model. The results showed that Myr-preS1-21-pep3 is approximately 10 times more potent than the initial peptide Myr-preS1-21 in preventing HBV infection. In conclusion, we developed a new strategy for screening peptides binding to membrane proteins and identified a new NTCP-binding peptide with a much smaller size than Hepcludex."

基金机构："National Natural Science Foundation of China [81871635, 81671997, U20A20392, 81902060]; Natural Science Foundation of Chongqing [cstc2021jcyj-msxmX0298, cstc2020jcyj-msxmX0764, cstc2020jscx-dxwtBX0022]; Science and Technology Commission of Yuzhong District, Chongqing [20180141]; 111 Project [D20028]; Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University [202104]; CQMU Program for Youth Innovation in Future Medicine [W0049]"

基金资助正文："This work was supported by the National Natural Science Foundation of China (Nos. 81871635, 81671997, U20A20392, and 81902060), the Natural Science Foundation of Chongqing (Nos. cstc2021jcyj-msxmX0298, cstc2020jcyj-msxmX0764, and cstc2020jscx-dxwtBX0022), the Science and Technology Commission of Yuzhong District, Chongqing (No. 20180141), the 111 Project (No. D20028), the Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University (No. 202104), and CQMU Program for Youth Innovation in Future Medicine (No. W0049)."