Peptidyl Linkers for Protein Heterodimerization Catalyzed by Microbial Transglutaminase

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• 作者：Tsutomu Tanaka ; Noriho Kamiya ; and Teruyuki Nagamune
• 刊名：Bioconjugate Chemistry
• 出版年：2004
• 出版时间：May 2004
• 年：2004
• 卷：15
• 期：3
• 页码：491 - 497
• 全文大小：177K
• 年卷期：v.15,no.3(May 2004)
• ISSN：1520-4812

文摘

Specific peptidyl linkers that result in the heterodimerization of functional proteins, which is catalyzedby microbial transglutaminase from Streptomyces mobaraensis (MTG), were generated based on aribonuclease S-peptide using site-directed mutagenesis. The peptidyl linkers designated as Lys-tagand Gln-tag were designed to possess sole reactive Lys or Gln residue that was amenable for selectiveLys-Gln cross-linkage of different proteins. Green fluorescent protein variants, ECFP and EYFP, wereemployed as model proteins, and those Lys- and Gln-tags were fused to the N-termini of ECFP andEYFP, respectively. As a result, we succeeded in solely obtaining the ECFP-EYFP heterodimer withoutforming multiply cross-linked byproducts. It was found that the reactivity of peptidyl linkers variedaccording to the type of amino acid to be replaced. Peptidyl linkers with a basic amino acid (Arg)exhibited the highest reactivity in the cross-linking reaction, suggesting the cationic residue substratepreference of MTG. Kinetic analysis utilizing fluorescent resonance energy transfer (FRET), that isonly observed upon the heterodimeric ECFP-EYFP conjugation, revealed that the amino acidreplacement contributed to the acceleration of cross-linking reactions by increasing catalytic turnover(k_cat), rather than substrate binding affinity (K_m). Finally, using a ribonuclease S-protein, themanipulation of enzymatic protein cross-linking based on specific S-peptide:S-protein interactionswas explored. Since newly designed Lys- and Gln-tags retained binding affinities to the S-protein,the heterodimerization was perfectly restrained by wrapping them with the S-protein. The resultssuggest the possibility of limited protein conjugation by tuning steric hindrance against the MTG.Tailoring enzymatic posttranslational modifications with either engineering peptidyl substrates orby taking specific peptide-protein interactions into consideration may facilitate the development ofa new sequential protein conjugation method for the preparation of multifunctional protein.