Abstract
Head-to-tail cyclization of genetically encoded peptides and proteins can be achieved with the split intein circular ligation of peptides and proteins (SICLOPPS) method by inserting the desired polypeptide between the C- and N-terminal fragments of a split intein. To prevent the intramolecular protein splicing reaction from spontaneously occurring upon folding of the intein domain, we have previously rendered this process light-dependent in a photo-controllable variant of the M86 intein, using genetically encoded ortho-nitrobenzyltyrosine at a structurally important position. Here, we report improvements on this photo-intein with regard to expression yields and rate of cyclic peptide formation. The temporally defined photo-activation of the purified stable intein precursor enabled a kinetic analysis that identified the final resolution of the branched intermediate as the rate-determining individual reaction of the three steps catalyzed by the intein. With this knowledge, we prepared an R143H mutant with a block F histidine residue. This histidine is conserved in most inteins and helps catalyze the third step of succinimide formation. The engineered intein formed the cyclic peptide product up to 3-fold faster within the first 15 min after irradiation, underlining the potential of protein splicing pathway engineering. The broader utility of the intein was also shown by formation of the 14-mer sunflower trypsin inhibitor 1.
Funding source: Deutsche Forschungsgemeinschaft
Award Identifier / Grant number: MO 1073/7-1
Funding statement: We thank Stephanie Wulff for technical assistance with MS measurements. This work was supported by the Deutsche Forschungsgemeinschaft, funder id: 10.13039/501100001659, (MO 1073/7-1).
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©2019 Walter de Gruyter GmbH, Berlin/Boston
Articles in the same Issue
- Frontmatter
- Protein engineering comes of age
- Microbial transglutaminase for biotechnological and biomedical engineering
- Computational design of structured loops for new protein functions
- Formylglycine-generating enzymes for site-specific bioconjugation
- Chemical modification of neuropeptide Y for human Y1 receptor targeting in health and disease
- Manipulating the stereoselectivity of a thermostable alcohol dehydrogenase by directed evolution for efficient asymmetric synthesis of arylpropanols
- Radiometal-labeled anti-VCAM-1 nanobodies as molecular tracers for atherosclerosis – impact of radiochemistry on pharmacokinetics
- Hit evaluation of an α-helical peptide: Ala-scan, truncation and sidechain-to-sidechain macrocyclization of an RNA polymerase Inhibitor
- Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing
- An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications
- Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase
- Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning
- Peptide binding affinity redistributes preassembled repeat protein fragments
- Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
- Rational design of an improved photo-activatable intein for the production of head-to-tail cyclized peptides
- Characterization and engineering of photoactivated adenylyl cyclases
Articles in the same Issue
- Frontmatter
- Protein engineering comes of age
- Microbial transglutaminase for biotechnological and biomedical engineering
- Computational design of structured loops for new protein functions
- Formylglycine-generating enzymes for site-specific bioconjugation
- Chemical modification of neuropeptide Y for human Y1 receptor targeting in health and disease
- Manipulating the stereoselectivity of a thermostable alcohol dehydrogenase by directed evolution for efficient asymmetric synthesis of arylpropanols
- Radiometal-labeled anti-VCAM-1 nanobodies as molecular tracers for atherosclerosis – impact of radiochemistry on pharmacokinetics
- Hit evaluation of an α-helical peptide: Ala-scan, truncation and sidechain-to-sidechain macrocyclization of an RNA polymerase Inhibitor
- Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing
- An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications
- Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase
- Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning
- Peptide binding affinity redistributes preassembled repeat protein fragments
- Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
- Rational design of an improved photo-activatable intein for the production of head-to-tail cyclized peptides
- Characterization and engineering of photoactivated adenylyl cyclases