Posttranslational Chemical Mutagenesis Methods to Insert Posttranslational Modifications into Recombinant Proteins

Harel, Omer; Jbara, Muhammad

doi:10.3390/molecules27144389

Cited by 13 publications

(9 citation statements)

References 90 publications

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“…Chemical and enzymatic approaches have been implemented to generate proteins with O -glycosylation mimetics. The site-directed mutated cysteine residue is transformed into Dha by alkylating reagents and reacted with active glycosylation derivatives. , Furthermore, thioglycoligase engineered from Streptomyces plicatus hexosaminidase is able to transfer N -acetylglucosamine (GlcNAc) to a site-directed mutated cysteine residue to form a protein with S -GlcNAc, which is a physiological mimetic of O -glycosylation . However, both methods only apply to in vitro studies with purified proteins.…”

Section: Serine/threonine Modificationsmentioning

confidence: 99%

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

Gan,

Fan

2024

Chem. Rev.

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Post-translational modifications (PTMs) endow proteins with new properties to respond to environmental changes or growth needs. With the development of advanced proteomics techniques, hundreds of distinct types of PTMs have been observed in a wide range of proteins from bacteria, archaea, and eukarya. To identify the roles of these PTMs, scientists have applied various approaches. However, high dynamics, low stoichiometry, and crosstalk between PTMs make it almost impossible to obtain homogeneously modified proteins for characterization of the site-specific effect of individual PTM on target proteins. To solve this problem, the genetic code expansion (GCE) strategy has been introduced into the field of PTM studies. Instead of modifying proteins after translation, GCE incorporates modified amino acids into proteins during translation, thus generating site-specifically modified proteins at target positions. In this review, we summarize the development of GCE systems for orthogonal translation for site-specific installation of PTMs.

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Section: Serine/threonine Modificationsmentioning

confidence: 99%

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

Gan,

Fan

2024

Chem. Rev.

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“…The elevated temperature, coupled with a continuous flow process, extends the length of peptides routinely accessible via standard SPPS to leverage protein production. Chemical protein synthesis and semisynthetic strategies have enabled the preparation of numerous modified protein targets, including glycosylated, phosphorylated, sulfonated, and ubiquitinated analogs for several structural and functional studies [1, 10a, 29] . Such control of the covalent insertion of desired modification has provided a unique opportunity to systematically correlate molecular structure with function, and it paves the way to access complex protein targets toward the development of advanced bioactive analogs [4]…”

Section: Synthetic Strategies To Prepare Modified and Artificial Prot...mentioning

confidence: 99%

Chemical Synthesis of Bioactive Proteins

Harel

Jbara

2023

Angew Chem Int Ed

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Nature has developed a plethora of protein machinery to operate and maintain nearly every task of cellular life. These processes are tightly regulated via post-expression modifications-transformations that modulate intracellular protein synthesis, folding, and activation. Methods to prepare homogeneously and precisely modified proteins are essential to probe their function and design new bioactive modalities. Synthetic chemistry has contributed remarkably to protein science by allowing the preparation of novel biomacromolecules that are often challenging or impractical to prepare via common biological means. The ability to chemically build and precisely modify proteins has enabled the production of new molecules with novel physicochemical properties and programmed activity for biomedical research, diagnostic, and therapeutic applications. This minireview summarizes recent developments in chemical protein synthesis to produce bioactive proteins, with emphasis on novel analogs with promising in vitro and in vivo activity.

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“…Sulfonyl Chlorides, Epoxides, Maleimides, 2,4-Dinitrofluorobenzene, Disulfides, α-Bromoacids and α-Bromoamides Reagents that target cysteine residues are important, and, due to the low abundance of exposed cysteine surface residues in the majority of proteins [87], site-directed mutagenesis has been a very successful strategy to introduce a surface-exposed cysteine for labeling or for crosslinking studies [13,130,[149][150][151]. The reaction of thiols with epoxides is important in the thiol-epoxy "Click" reaction, allowing for the rapid formation of covalent bonds under mild conditions and having importance in the preparation of hydrogels used in tissue engineering, for example [152,153].…”

Section: Cysteine Bioconjugation Reactionsmentioning

confidence: 99%

Thermodynamic Overview of Bioconjugation Reactions Pertinent to Lysine and Cysteine Peptide and Protein Residues

Lopandic,

Merza,

Honek

2023

Compounds

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Bioconjugation reactions are critical to the modification of peptides and proteins, permitting the introduction of biophysical probes onto proteins as well as drugs for use in antibody-targeted medicines. A diverse set of chemical reagents can be employed in these circumstances to covalently label protein side chains, such as the amine moiety in the side chain of lysine and the thiol functionality in cysteine residues, two of the more frequently employed sites for modification. To provide researchers with a thermodynamic survey of the reaction of these residues with frequently employed chemical modification reagents as well as reactive cellular intermediates also known to modify proteins non-enzymatically, a theoretical investigation of the overall thermodynamics of models of these reactions was undertaken at the T1 and G3(MP2) thermochemical recipe levels (gas phase), the M06-2X/6-311+G(2df,2p)/B3LYP/6-31G(d) (gas and water phase), and the M06-2X/cc-PVTZ(-f)++ density functional levels of theory (water phase). Discussions of the relationship between the reagent structure and the overall thermodynamics of amine or thiol modification are presented. Of additional interest are the observations that routine cellular intermediates such as certain thioesters, acyl phosphates, and acetyl-L-carnitine can contribute to non-enzymatic protein modifications. These reactions and representative click chemistry reactions were also investigated. The computational survey presented herein (>320 reaction computations were undertaken) should serve as a valuable resource for researchers undertaking protein bioconjugation. A concluding section addresses the ability of computation to provide predictions as to the potential for protein modification by new chemical entities, with a cautionary note on protein modification side reactions that may occur when employing synthetic substrates to measure enzyme kinetic activities.

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Posttranslational Chemical Mutagenesis Methods to Insert Posttranslational Modifications into Recombinant Proteins

Cited by 13 publications

References 90 publications

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

Chemical Synthesis of Bioactive Proteins

Thermodynamic Overview of Bioconjugation Reactions Pertinent to Lysine and Cysteine Peptide and Protein Residues

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