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Modified Labeling For Polyderm Wbc (tm)

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Last Updated: 02 July 2021

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General | Latest Info

Site-specific modification of proteins is a challenging problem in modern chemical biology. 1-4 With applications in fields including biology, chemistry, and medicine, 5 developing methods to site-specifically modify proteins is of great importance. Site-specific Protein modifications have been used to create antibody-drug conjugates, 6 to study natural post-translational modifications, to introduce fluorophores and other small molecules for biophysical studies, orient Protein immobilization, 7 to prepare Protein-polymer conjugates, 8 and to examine properties of proteins such as their folding, their dynamics and their interactions With other proteins. 9 Protein immobilization is an important first step for many biotechnology applications, including construction of protein microarrays, biosensors, immunoassays and protein conjugates which are used for medical therapies. 10 11 One of the main advantages of site-specific protein modification is that it can be used to create structurally defined covalent linkages between proteins and surfaces, materials or biomolecules. 12 this ensure homogeneous coverage and accessibility to active site of protein. These are important factors in studying protein expression and localization, to improve bioavailability and pharmacokinetics of Protein-base drugs, in structure-function studies, and in development of biosensors. 13 early methods of Protein functionalization exploit reactivity of either cysteine or lysine residues by reacting protein with excess of thiol-or amine-reactive reagents, such as maleimides or N-hydroxysuccinimidyl esters, respectively. In addition to lysine and cysteine modifications, methods have also been developed to chemically modify tyrosine, N-or C-terminus, and aspartate or glutamate residues. 14 Transimination 15-17 and periodate oxidation 4 18 are also among common chemical methods for protein modifications which introduce aldehyde or ketone moiety at the N-terminus. Although all of these methods are currently in use and have widespread utility, variety of alternative approaches have been developed to achieve greater reaction yield, higher selectivity and site-specificity, compatibility with complex biological systems, and improved reaction rates. 12 One approach for protein labeling is to use reactions that are chemoselective, whose functional groups are both inert in biological media and which do not natively occur in proteins or other biological macromolecules. In many cases, combine strategy of described modification methods are used, such as taking advantage of selectivity of reactive functional groups on proteins which is used to introduce new reactive group that then provide superior reaction kinetics for second, more complex conjugation reaction. Using this strategy, large excess of initial reagent can be used to introduce reactive handle into protein, followed by a second reaction using stoichiometric quantities of biomolecule or probe of interest, which can be more costly to obtain. The discovery and application of green fluorescent protein and its derivatives into chemical biology has had great impact on exploring protein behavior in living cells and has dramatically increased our knowledge of biological processes. 19-21 However, use of GFP to tag proteins comes with disadvantages; many proteins do not tolerate fusion with GFP without compromising function or intracellular distribution.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Sortagging

Sortases catalize transpeptidation reaction, where enzyme cleave amide bonds between Threonine and Glycine of Sortase recognition sequence, generating thioacyl intermediate. Subsequently, this intermediate is resolved by the N-terminus of oligoglycine nucleophile, creating a new peptide bond that links substrate to the incoming nucleophile. Sortagging Kit takes advantage of this reaction to label virtually any Recombinant antibody or protein containing Sortase recognition sequence. Labeling efficiency of greater than 90 % is achieved by taking advantage of the highly Active Sortase 5 Pentamutant. Simply combine your protein of interest with Sortase 5 and polyglycine label, and incubate for 1 hour at 37C. Purification columns are included to remove Sortase 5 and excess label. The Kit also includes Ubiquitin-LPETGH6 and GGG-Clover as positive controls.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

PFTase post-translational modification

In nature, numerous proteins are post-translationally modified on sulfur atom present in cysteine residue near C-termini of proteins by protein farnesyltransferase. PFTase employs farnesyl pyrophosphate as its natural substrate to transfer farnesyl group to cysteine of CaaX-box tetrapeptide sequence. Reaction can also be performed in vitro. PFTase is promiscuous in nature of its substrates, in that it can tolerate a variety of CaaX sequences as well as some modifications to the FPP structure. Recently, number of groups have used this feature to site-specifically modify proteins using analogues of FPP. Various isoprenoid surrogates containing azide, 96 97 alkyne, 98 aldehyde, 42 99 biotin, 100 or aryl groups 101 have been synthesize and shown to be successfully processed by PFTase. PFTase-catalyze transfer of synthetic alkyne-or azide-functionalized farnesyl analogues in combination with Huisgen cycloaddition or Staudinger ligation has been used for site-specific fluorophore labeling and oriented immobilization of proteins. 96 Importantly, CaaX-box sequences such as CVIA, or CVIM can be genetically encoded into C-termini of many proteins, making them efficient substrates for PFTase. In early work, Poulter and coworkers use this strategy to immobilize protein on solid surfaces. 7 They synthesize alkyne-functionalized FPP and incorporated alkyne moiety into proteins at their C-termini using PFTase. The Glass-slide was functionalized with azide group and covalent immobilization of proteins to slide was subsequently achieved using copper-catalyze cycloaddition. In subsequent work, they synthesize collection of FPP analogues containing either alkyne or azide moieties and analyze their kinetic parameters as substrates for PFTase. 102 Out of eleven synthetic analogues, two of them give steady-state kinetic parameters very similar to those of natural substrate FPP. Waldmann and coworkers use PFTase strategy to modify proteins including H-, N-, and K-Ras GTPases, which are important in cellular signaling and are among human oncogene products, at their C-termini with farnesyl group. Subsequently, they use thiol-functionalized surfaces to react photochemically with alkenes present in farnesyl groups introduced into proteins as noted above. In this way, proteins were covalently immobilize in a site-specific manner under mild conditions in about 10 minutes. 103 in other work, They develop several azide-, biotin-and diene-functionalized analogues of FPP. After kinetic analysis of enzymatic reactions with PFTase, they conclude that all of these new substrates, except biotin-containing analogue, can be transferred onto proteins that possess CaaX box. 104 Subsequently, proteins functionalized with azide or diene groups were successfully modified by Staudinger ligation or Diels-Alder cycloaddition. In related work, Alexandrov and coworkers describe structure-guided design of engineer farnesyl transferase and geranylgeranyl transferase-I that could both efficiently use biotin-geranyl pyrophosphate as substrate. 100 Using these mutant enzymes and biotin-geranyl pyrophosphate as substrate, they could detect femtomolar levels of prenylatable proteins in cells and organs and identify their cognate protein prenyltransferases. Distefano and coworkers have exploited PFTase-catalyze reaction for a number of applications.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Biotin ligase

Identification of protein-protein associations is a fundamental approach to the study of protein function, subcellular proteomes, and biological mechanisms. The Proximity-dependent bio tin ID entification method was developed to overcome barriers imposed by conventional screening methods for PPAs. The BioID method is based on proximity-dependent cellular biotinylation by promiscuous bacterial biotin ligase fuse to bait protein to generate history of PPAs over time in living cells. These biotinylated proteins can be selectively isolated by conventional biotin capture methods and identified using mass spectrometry analysis. These candidates identified by the BioID method can represent direct interactors, indirect interactors, and / or vicinal proteins that do not physically interact with fusion protein. Owing to its applicability to insoluble proteins and weak / transient PPAs, BioID method has rapidly become widely used to study PPAs in nuclear envelope, centrosomes, cell-cell junctions, spatiotemporal dynamics of epigenetic factors, cancer development, cross-talk between cytoskeletons, mRNA decay, signaling pathways regulating various essential cellular mechanisms, and ubiquitin metabolism. BioID has also been applied to elucidate interplay mechanisms between host and virus or microorganisms for HIV, chlamydia trachomatis, chlamydia psittaci, toxoplasma gondii, trypanosoma brucei, and herpes viruses. One challenge for any method that relies on expression of fusion proteins is proper subcellular targeting. Although BioID is slightly larger than green fluorescent protein, we have observed that fusion to BioID occasionally prevents efficient targeting of certain fusion proteins. In an attempt to ameliorate that limitation, we report our development and analysis of a new, smaller promiscuous biotin ligase that, in addition to its smaller size, is more efficient at labeling proximate proteins.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Lipoic acid ligase

Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediate peptide labeling utilizing lipoic acid ligase and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In the first step, W37V mutant of lipoic acid ligase from Escherichia coli is used to ligate synthetic chemical handle site-specifically to lysine residue in 13 amino acid peptide motifa short sequence that can be genetically express as fusion with any protein of interest. In the second step, molecular probe can be attached to chemical handle in bioorthogonal Diels-Alder reaction with inverse electron demand. This method is a complementary approach to protein labeling using genetic code expansion and circumvents larger protein tags while maintaining label specificity, providing experimental flexibility and straightforwardness.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Sources

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

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