Single-step conjugation of bioactive peptides to proteins via a self-contained succinimidyl bis-arylhydrazone.
Single-step conjugation of bioactive peptides to proteins via a self-contained succinimidyl bis-arylhydrazone.
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DOI：
10.1021/bc9002794
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发表时间：
2009-10-21
影响因子：
4.7
通讯作者：
Sanderson SD
中科院分区：
文献类型：
--
作者：
Phillips JA;Morgan EL;Dong Y;Cole GT;McMahan C;Hung CY;Sanderson SD
本文介绍了一种方法，一个单一的步骤，位点特异性的共轭生物活性肽的蛋白质，利用独特的UV签名吸光度的双芳基腙提供的监测优势。通过将十肽分子佐剂YSF κ B（MeL）aR（EP 67）缀合至两种测试蛋白，卵清蛋白（OVA）和牛血清白蛋白（BSA），以及缀合至在完整流感病毒和球孢子菌属的真菌节孢子（孢子）上表达的蛋白，证明了该方法的实用性。用EP 67的一种形式完成缀合，其中其N-末端用琥珀酰亚胺基-4-苯甲酰肼基-烟酰胺（S4 BHyNic）（肽7）修饰，从而能够通过与表面暴露的氨基形成酰胺键与这些大实体缀合。强吸收的双芳基腙S4 BHyNic（ε354 nm = 29，000 Lmol−1cm−1）的存在允许测定EP 67与蛋白质的摩尔取代比（MSR），其与氨基酸分析测定的MSR非常一致。与OVA缀合不损害EP 67接合携带C5 a受体的抗原呈递细胞（APC）的能力，如通过EP 67介导的白细胞介素-6（IL-6）从APC的释放所测量的。相对于单独的OVA，用所得OVA-EP 67疫苗缀合物免疫的小鼠产生高血清效价的OVA特异性IgG抗体。此外，EP 67的缀合不影响流感病毒粒子的表面完整性或球孢子的生物活性。这种将生物活性肽缀合至蛋白质和其它大生物实体的方法可以代表产生用于机理研究或新型治疗实体（诸如含EP 67的疫苗）的各种生物缀合物的方便且有效的方式。
This paper describes a method for a single-step, site-specific conjugation of bioactive peptides to proteins that exploits the monitoring advantages provided by the unique UV signature absorbance of a bis-arylhydrazone. The utility of this method is demonstrated by the conjugation of a decapeptide molecular adjuvant, YSFKDMP(MeL)aR (EP67), to two test proteins, ovalbumin (OVA) and bovine serum albumin (BSA), and to proteins expressed on intact influenza virons and fungal arthroconidia (spores) of Coccidioides. Conjugation is accomplished with a version of EP67 in which its N-terminus is modified with succinimidyl-4-benzoylhydrazino-nicotinamide (S4BHyNic) (peptide 7), thus enabling conjugation to these large entities via formation of amide bonds with surface-exposed amino groups. The presence of the strongly absorbing bis-arylhydrazone S4BHyNic (ε354 nm = 29,000 Lmol−1cm−1) allows for determination of EP67-to-protein molar substitution ratios (MSR), which are in good agreement with the MSRs determined by amino acid analysis. Conjugation to OVA does not compromise the ability of EP67 to engage C5a receptor bearing antigen presenting cells (APC) as measured by the EP67-mediated release of interleukin-6 (IL-6) from APCs. Mice immunized with the resulting OVA-EP67 vaccine conjugate produce high serum titers of OVA-specific IgG antibodies relative to OVA alone. Also, the conjugation of EP67 does not affect the surface integrity of influenza virons or the biological viability of Coccidioides spores. This method of conjugating bioactive peptides to proteins and other large biological entities may represent a convenient and effective way generating various bio-conjugates for use in mechanistic studies or novel therapeutic entities such as EP67-containing vaccines.
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