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Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds.

ABSTRACT: To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC) and oxime ligation. This method was applied to the conjugation of biotin and ?-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

SUBMITTER: Muhlberg M

PROVIDER: S-EPMC4464295 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds.

Mühlberg Michaela M Hoesl Michael G MG Kuehne Christian C Dernedde Jens J Budisa Nediljko N Hackenberger Christian P R CPR

Beilstein journal of organic chemistry 20150513

To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC) and oxime ligation. This method was a ...[more]

PMID: 26124880

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Project description:Background: Protein theranostics integrate both diagnostic and treatment functions on a single disease-targeting protein. However, the preparation of these multimodal agents remains a major challenge. Ideally, conventional recombinant proteins should be used as starting materials for modification with the desired detection and therapeutic functionalities, but simple chemical strategies that allow the introduction of two different modifications into a protein in a site-specific manner are not currently available. We recently discovered two highly efficient peptide ligases, namely butelase-1 and VyPAL2. Although both ligate at asparaginyl peptide bonds, these two enzymes are bio-orthogonal with distinguishable substrate specificities, which can be exploited to introduce distinct modifications onto a protein. Methods: We quantified substrate specificity differences between butelase-1 and VyPAL2, which provide orthogonality for a tandem ligation method for protein dual modifications. Recombinant proteins or synthetic peptides engineered with the preferred recognition motifs of butelase-1 and VyPAL2 at their respective C- and N-terminal ends could be modified consecutively by the action of the two ligases. Results: Using this method, we modified an EGFR-targeting affibody with a fluorescein tag and a mitochondrion-lytic peptide at its respective N- and C-terminal ends. The dual-labeled protein was found to be a selective bioimaging and cytotoxic agent for EGFR-positive A431 cancer cells. In addition, the method was used to prepare a cyclic form of the affibody conjugated with doxorubicin. Both modified affibodies showed increased cytotoxicity to A431 cells by 10- and 100-fold compared to unconjugated doxorubicin and the free peptide, respectively. Conclusion: Bio-orthogonal tandem ligation using two asparaginyl peptide ligases with differential substrate specificities is a straightforward approach for the preparation of multifunctional protein biologics as potential theranostics.

Project description:The specificity of the kinase-regulator interaction is driven by a limited set of interfacial residues in each protein that strongly. To identify combinations of these interface residues that are functional and potentially insulated from existing two-component signaling pathways in E. coli, we constructed a dual library of mutants in which the key, coevolving interface residues of a canonical two-component system, PhoQ and PhoP, were randomized. We used NNS codons to randomize six residues in PhoQ and five residues in PhoP, all of which lie at the interface formed by the two proteins in complex and that are critical to determining partner specificity in all two-component signaling pathways. To identify functional combinations of residues, we first grew the library of PhoQ-PhoP variants overnight in medium with low Mg2+, which activates PhoQ. Because cells must phosphorylate PhoP to grow when extracellular Mg2+ is limiting, this step enriches for functional PhoQ-PhoP variants. Variants that survived selection in limiting Mg2+ were then subjected to Sort-Seq, using fluorescence-activated cell sorting (FACS) and deep sequencing to quantify the signal responsiveness of variants in the library. To gauge the phosphorylation of PhoP in vivo, we used a fluorescent transcriptional reporter, PmgrB-yfp. In the presence of low extracellular Mg2+, functional PhoQ promotes the phosphorylation of PhoP and the production of YFP, whereas in the presence of high concentrations of Mg2+, PhoQ drives the dephosphorylation of PhoP, limiting the accumulation of YFP). The library was grown in each condition for 6 hours before sorting and sequencing. To identify variants that are signal responsive and drive YFP production specifically in low Mg2+, we sorted cells from each condition into 8 separate bins and deep sequenced the randomized regions of variants collected in each bin. We then calculated the frequency of each variant in each bin to yield the distributions of individual variants in low and high Mg2+, which were fit to Gaussians. From these fits, we assessed the mean level of YFP in each condition and the fold-induction, or signal responsiveness, of each variant detected in the library. The 11 codons / amino acids listed in this dataset refer to codons 12, 14, 15, 18, and 19 in PhoP and codons 284, 288, 289, 292, 302, and 303 in PhoQ, in that order.

Dataset Information

Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds.

Publications

Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds.

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