Establishment of a human cell line with a surface display system for screening and optimizing Na(+)-taurocholate cotransporting polypeptide-binding peptides.
Establishment of a human cell line with a surface display system for screening and optimizing Na(+)-taurocholate cotransporting polypeptide-binding peptides.
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建立具有表面展示系统的人类细胞系，用于筛选和优化牛磺胆酸钠共转运多肽结合肽
DOI：
10.3389/fmicb.2022.920280
复制
发表时间：
2022
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
--
作者：
HBV药物最理想的靶点之一是牛磺胆酸钠共转运多肽（NTCP），它是B肝炎病毒（HBV）的进入受体。N-肉豆蔻酰化preS 1 2-48（Myrcludex B或Hepcludex）是一种来自HBV大表面蛋白的NTCP结合肽，已被开发为第一类进入抑制剂。然而，其相对大的分子量有助于增加免疫原性和抗体产生。因此，优选寻找具有较小尺寸的NTCP结合肽。为此，我们开发了一种人细胞表面展示策略，并筛选了基于preS 1 -21的肽。PreS 1 -21（基因型D）通过7个随机氨基酸的延伸与mCherry和FasL跨膜结构域融合。通过使用转座子/转座酶系统将合并的构建体转染到HEK 293细胞中，以产生在细胞表面上展示具有红色荧光的各种肽的文库。另一方面，我们在HEK 293上表达了与EGFP融合的NTCP蛋白，并使用含有NTCP-GFP的膜裂解物作为诱饵蛋白来选择具有增加的NTCP亲和力的肽。经过7轮筛选，深度测序结果显示，部分多肽富集超过1,000倍。进一步筛选主要富集的10种肽产生肽preS 1 -21-pep 3。将preS 1 -21-pep 3的preS 1 -21序列替换为来自不同基因型的序列，结果表明基因型A-F的共有序列具有最好的性能。合成肽（Myr-preS 1 -21-pep 3）并在HepG 2-NTCP细胞模型上进行测试。结果表明，Myr-preS 1 -21-pep 3在预防HBV感染方面比初始肽Myr-preS 1 -21强约10倍。总之，我们开发了一种新的策略，筛选肽结合膜蛋白，并确定了一个新的NTCP结合肽的大小比Hepcludex小得多。
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.
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DOI：
10.1016/j.omtm.2021.11.002
发表时间：
2021-12-10
期刊：
Molecular therapy. Methods & clinical development
影响因子：
--
作者：
Uchida T;Park SB;Inuzuka T;Zhang M;Allen JN;Chayama K;Liang TJ
通讯作者：
Liang TJ
影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
影响因子：
16.6
作者：
Crook ZR;Sevilla GP;Friend D;Brusniak MY;Bandaranayake AD;Clarke M;Gewe M;Mhyre AJ;Baker D;Strong RK;Bradley P;Olson JM
通讯作者：
Olson JM
影响因子：
5.4
作者：
NASSAL, M
通讯作者：
NASSAL, M
影响因子：
4.9
作者：
Ladner, SK;Otto, MJ;King, RW
通讯作者：
King, RW