Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C.
Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C.
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重组破伤风类毒素重链C段半胱氨酸修饰的研究
DOI：
10.1021/jasms.1c00075
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发表时间：
2021-07-07
影响因子：
3.2
通讯作者：
Lei, Q. Paula
中科院分区：
文献类型：
--
作者：
Cai, Cindy X.;Schneck, Nicole A.;Cozine, Taryn;Ivleva, Vera B.;Ragheb, Daniel;Gollapudi, Deepika;Patel, Aakash;Barefoot, Nathan;Gowetski, Daniel B.;Lei, Q. Paula
为了开发缀合的HIV-1融合肽疫苗，应用重组破伤风类毒素重链片段C（rTTHC）作为载体蛋白以增强肽的免疫原性。了解rTTHC的特性是肽缀合之前的第一步。对E.在大肠杆菌中表达rTTHC。完整质量沿着肽图谱分析发现存在三种半胱氨酸修饰形式：谷胱甘肽化、三硫键修饰和二硫键改组，与疏水作用色谱（HIC）纯化步骤期间的三峰图谱相关。这些多种氧化形式的共存表明活性硫醇在rTTHC材料中进行氧化还原反应。通过MS分析对rTTHC载体蛋白的鉴别确认为评估纯化步骤提供了关键指导，并有助于确保疫苗开发可以进行。
For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.
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