Kjeldsen Thomas, Hogendorf Wouter F J, Tornøe Christian W, Anderson Jonathan, Hubalek Frantisek, Stidsen Carsten E, Sorensen Jan L, Hoeg-Jensen Thomas
Novo Nordisk A/S, Novo Nordisk Park H5.S.51, DK-2760 Måløv, Denmark.
ACS Omega. 2020 Jul 31;5(31):19827-19833. doi: 10.1021/acsomega.0c02712. eCollection 2020 Aug 11.
Covalent cross-linking of biomolecules can be useful in pursuit of tissue targeting or dual targeting of two receptors on cell surfaces for avidity effects. Long linkers (>10 kDa) can be advantageous for such purposes, and poly(ethylene glycol) (PEG) linkers are most commonly used due to the high aqueous solubility of PEG and its relative inertness toward biological targets. However, PEG is non-biodegradable, and available PEG linkers longer than 5 kDa are heterogeneous (polydisperse), which means that conjugates based on such materials will be mixtures. We describe here recombinant linkers of distinct lengths, which can be expressed in yeast, which are polar, and which carry orthogonal reactivity at each end of the linker, thus allowing chemoselective cross-linking of proteins. A conjugate between insulin and either of the two trypsin inhibitor peptides/proteins exemplifies the technology, using a GQAP-based linker of molecular weight of 17 848, having one amine at the N-terminal, and one Cys, at the C-terminal. Notably, yeast-based expression systems typically give products with mixed disulfides when expressing proteins that are equipped with one unpaired Cys, namely, mixed disulfides with glutathione, free Cys amino acid, and/or a protein homodimer. To obtain a homogeneous linker, we worked out conditions for transforming the linker with mixed disulfides into a linker with a homogeneous disulfide, using excess 4-mercaptophenylacetic acid. Subsequently, the N-terminal amine of the linker was transformed into an azide, and the C-terminal Cys disulfide was reduced to a free thiol and reacted with halo-acetyl insulin. The N-terminal azide was finally conjugated to either of the two types of alkyne-containing trypsin inhibitor peptides/proteins. This reaction sequence allowed the cross-linked proteins to carry internal disulfides, as no reduction step was needed after protein conjugations. The insulin-trypsin inhibitor conjugates were shown to be stabilized toward enzymatic digestions and to have partially retained binding to the insulin receptor.
生物分子的共价交联在实现组织靶向或细胞表面两种受体的双靶向以产生亲和力效应方面可能是有用的。长连接子（>10 kDa）对于此类目的可能具有优势，聚乙二醇（PEG）连接子由于PEG的高水溶性及其对生物靶点的相对惰性而最为常用。然而，PEG是不可生物降解的，且现有的长度超过5 kDa的PEG连接子是异质的（多分散的），这意味着基于此类材料的缀合物将是混合物。我们在此描述了不同长度的重组连接子，其可在酵母中表达，具有极性，且在连接子的每一端都具有正交反应性，从而允许蛋白质进行化学选择性交联。胰岛素与两种胰蛋白酶抑制剂肽/蛋白中的任何一种之间的缀合物例证了该技术，使用基于GQAP的分子量为17848的连接子，其N端有一个胺基，C端有一个半胱氨酸。值得注意的是，基于酵母的表达系统在表达配备有一个未配对半胱氨酸的蛋白质时，通常会产生带有混合二硫键的产物，即与谷胱甘肽、游离半胱氨酸氨基酸和/或蛋白质同二聚体的混合二硫键。为了获得均一的连接子，我们制定了条件，使用过量的4-巯基苯乙酸将带有混合二硫键的连接子转化为具有均一二硫键的连接子。随后，连接子的N端胺基被转化为叠氮化物，C端半胱氨酸二硫键被还原为游离硫醇并与卤代乙酰胰岛素反应。N端叠氮化物最终与两种含炔基的胰蛋白酶抑制剂肽/蛋白中的任何一种缀合。该反应序列使交联蛋白带有内部二硫键，因为在蛋白质缀合后不需要还原步骤。胰岛素-胰蛋白酶抑制剂缀合物显示出对酶消化稳定，并且部分保留了与胰岛素受体的结合。
