Information about Native Chemical Ligation
Native chemical ligation is a common form of chemical ligation, a technique for constructing a large peptide from two or more smaller peptides. In native chemical ligation a peptide containing a C-terminal thioester reacts with another peptide containing an N-terminal cysteine, in the presence of an exogenous thiol catalyst. In a thermodynamically-controlled, freely reversible first step a transthioesterification occurs. The product rearranges irreversibly under the usual reaction conditions to form the desired amide bond. The process was developed in the laboratory of Stephen Kent at The Scripps Research Institute in 1994.
Peptide-thioesters to be used in native chemical ligation are usually prepared by Boc chemistry SPPS; the thioester piece cannot be synthesized with a nucleophilic base, thus disfavoring Fmoc chemistry. Fmoc techniques for generating thioesters involving the modifications of the Kenner 'safety catch' linker are known. Protecting groups on the N-terminal piece cannot have constituents which release as aldehydes or ketones since these will cap the N-terminal cysteine. For the same reason, acetone should be avoided in general use, particularly prior to lyophilization and in washing glassware.
The payoff is that coupling long peptides by this technique is in many cases nearly quantitative and provides synthetic access to proteins otherwise impossible to make, due to length or decoration by posttranslational modification.
A limitation of this technique is that cysteine has to be part of the produced protein at a suitable place. For some proteins homocysteine can be used and methylated after coupling to form methionine. Cysteine can also be desulfurized to alanine. Alternately, ligation auxiliaries can be used that mimic an N-terminal cysteine for the ligation reaction, but which can be removed after synthesis.
Polypeptide C-terminal thioesters produced by recombinant DNA techniques can be reacted with an N-terminal Cys containing polypeptide by the same native ligation chemistry to provide very large semisynthesized proteins. Thus, by exploiting nature's inteins to prepare a recombinant C-terminal thioester, the size restriction of the reacting peptide segment is removed. Similarly, a recombinant protein containing an N-terminal Cys can be reacted with a synthetic polypeptide thioester. Native Chemical Ligation of this kind using a recombinant polypeptide segment is known as Expressed Protein Ligation.
Muir TW, Sondhi D, Cole PA. 1998. Expressed Protein Ligation: A General Method for Protein Engineering. Proc. Natl. Acad. Sci. USA 95:6705-6710.
Nilsson BL, Soellner MB, Raines RT. 2005. Chemical Synthesis of Proteins. Annu. Rev. Biophys. Biomol. Struct. 34:91-118
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Native chemical ligation
Peptide-thioesters to be used in native chemical ligation are usually prepared by Boc chemistry SPPS; the thioester piece cannot be synthesized with a nucleophilic base, thus disfavoring Fmoc chemistry. Fmoc techniques for generating thioesters involving the modifications of the Kenner 'safety catch' linker are known. Protecting groups on the N-terminal piece cannot have constituents which release as aldehydes or ketones since these will cap the N-terminal cysteine. For the same reason, acetone should be avoided in general use, particularly prior to lyophilization and in washing glassware.
The payoff is that coupling long peptides by this technique is in many cases nearly quantitative and provides synthetic access to proteins otherwise impossible to make, due to length or decoration by posttranslational modification.
A limitation of this technique is that cysteine has to be part of the produced protein at a suitable place. For some proteins homocysteine can be used and methylated after coupling to form methionine. Cysteine can also be desulfurized to alanine. Alternately, ligation auxiliaries can be used that mimic an N-terminal cysteine for the ligation reaction, but which can be removed after synthesis.
Polypeptide C-terminal thioesters produced by recombinant DNA techniques can be reacted with an N-terminal Cys containing polypeptide by the same native ligation chemistry to provide very large semisynthesized proteins. Thus, by exploiting nature's inteins to prepare a recombinant C-terminal thioester, the size restriction of the reacting peptide segment is removed. Similarly, a recombinant protein containing an N-terminal Cys can be reacted with a synthetic polypeptide thioester. Native Chemical Ligation of this kind using a recombinant polypeptide segment is known as Expressed Protein Ligation.
See also
References
Dawson PE, Muir TW, Clark-Lewis I, Kent, SBH. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779.Muir TW, Sondhi D, Cole PA. 1998. Expressed Protein Ligation: A General Method for Protein Engineering. Proc. Natl. Acad. Sci. USA 95:6705-6710.
Nilsson BL, Soellner MB, Raines RT. 2005. Chemical Synthesis of Proteins. Annu. Rev. Biophys. Biomol. Struct. 34:91-118
Chemical ligation is a set of techniques used for creating long peptide or protein chains. It is the second step of a convergent approach. First smaller peptides containing 30-50 amino acids are prepared by conventional chemical peptide synthesis.
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Peptides (from the Greek πεπτίδια, "small digestibles") are short polymers formed from the linking, in a defined order, of α-amino acids.
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Thioesters are compounds resulting from the bonding of sulfur with an acyl group with the general formula R-S-CO-R'. They are the product of esterification between a carboxylic acid and a thiol (as opposed to an alcohol in regular esters).
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Cysteine (abbreviated as Cys or C)[1] is an α-amino acid with the chemical formula HO2CCH(NH2)CH2SH. It is not an essential amino acid, which means that humans can synthesize it. Its codons are UGU and UGC.
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Stephen B. H. Kent is a chemist at the University of Chicago who developed native chemical ligation and also demonstrated the principle that mirror-image amino acids put together to form a protein create a mirror-image protein which, if an enzyme, can catalyze the mirror-image
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The Scripps Research Institute (TSRI) is a medical research facility that focuses on research in the basic biomedical sciences. Primarily located in La Jolla, California, with a sister facility in Jupiter, Florida, the institute is home to 3,000 scientists, technicians, graduate
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aldehyde is an organic compound containing a terminal carbonyl group. This functional group, which consists of a carbon atom which is bonded to a hydrogen atom and double-bonded to an oxygen atom (chemical formula O=CH-), is called the aldehyde group.
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A ketone (pronounced as key tone) is either the functional group characterized by a carbonyl group (O=C) linked to two other carbon atoms or a chemical compound that contains this functional group.
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The chemical compound acetone (also known as propanone, dimethyl ketone, 2-propanone, propan-2-one and β-ketopropane) is the simplest representative of the ketones.
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Freeze drying (also known as lyophilization) is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport.
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Homocysteine is a chemical compound with the formula HSCH2CH2CH(NH2)CO2H. It is a homologue of the naturally-occurring amino acid cysteine, differing in that its side-chain contains an additional methylene (-CH2
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Methionine (abbreviated as Met or M)[1] is an α-amino acid with the chemical formula HO2CCH(NH2)CH2CH2SCH3. This essential amino acid is classified as nonpolar.
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Alanine (abbreviated as Ala or A)[1] is an α-amino acid with the chemical formula HO2CCH(NH2)CH3. The L-isomer is one of the 20 proteinogenic amino acids, i.e. the building blocks of proteins.
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An intein is a segment of a protein that is able to excise itself and rejoin the remaining portions (the exteins) with a peptide bond. Inteins have also been called "protein introns".
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Recombinant may refer to :
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- Recombinant DNA - a form of articifial DNA
- VRLA (also referred to as Recombinant) - valve regulated lead acid, a certain kind of lead-acid battery
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Thioesters are compounds resulting from the bonding of sulfur with an acyl group with the general formula R-S-CO-R'. They are the product of esterification between a carboxylic acid and a thiol (as opposed to an alcohol in regular esters).
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In organic chemistry, peptide synthesis is the creation of peptides, which are organic compounds in which multiple amino acids bind via peptide bonds which are also known as amide bonds.
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Protein synthesis is the creation of proteins using DNA and RNA. Biological and artificial methods for creation of proteins differ significantly.
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- For biological protein synthesis, see protein biosynthesis.
- For artificial protein synthesis, see peptide synthesis.
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