native chemical ligation of polypeptides

UNIT 18.4Native Chemical Ligation of Polypeptides

The total synthesis and semisynthesis of proteins allows the site-specific incorporation

of unnatural amino acids, post-translational modifications, and biophysical/biochemical

probes into the target molecule. Among the various chemical and enzymatic approaches

available for the synthesis/semisynthesis of proteins, the native chemical ligation tech-

nique has proven especially useful and will be the exclusive focus of this unit. Native

chemical ligation allows native backbone proteins to be assembled from fully unprotected

polypeptide building blocks. To facilitate the ligation reaction, the α-carboxylate group

of the N-terminal polypeptide fragment must be mildly activated as an aryl thioester, while

the C-terminal polypeptide fragment must contain an amino-terminal cysteine residue.

The reaction is normally carried out in aqueous buffers at around neutral pH and can be

performed with complete regioselectivity in the presence of all the functionalities com-

monly found in proteins. Even the presence of additional cysteine sulfhydryl groups in

one or both peptide fragments does not affect the reaction regioselectivity. The unit first

discusses how to choose the ligation site(s) in the target protein and then outlines how to

obtain the necessary polypeptide building blocks using either chemical synthesis or

recombinant DNA expression (see Strategic Planning). Next, the synthesis of a protein

by native chemical ligation of two polypeptide fragments is described (see Basic Protocol

1). The synthesis of a protein from three polypeptide fragments using a sequential native

chemical ligation strategy is also described (see Basic Protocol 2). The support protocols

describe how to obtain the necessary polypeptide fragments using either chemical

synthesis or recombinant DNA expression.

NOTE: All the operations involving volatile chemicals should be performed in a well

ventilated fume hood. See APPENDIX 2A for general safety guidelines.

STRATEGIC PLANNING

The first step in synthesizing a protein by native chemical ligation is to choose the ligation

site(s), thereby defining the polypeptide fragments to be used in the ligation reactions.

The position of the ligation site depends upon several factors: the primary sequence of

the protein; the secondary and tertiary structures of the protein (if known); and the type

of strategy being used to generate the fragments, i.e., chemical synthesis or recombinant

DNA expression. Ideally the target protein should be divided up into synthetically

accessible fragments the size of which depends upon whether chemical synthesis or

biosynthesis is being used for their generation; optimized solid-phase peptide synthesis

(SPPS; also see UNIT 18.1) allows polypeptides of up to ∼50 residues to be efficiently

prepared, whereas recombinant DNA expression permits the preparation of much larger

fragments. For example, a hypothetical 100-amino-acid protein could be assembled either

from two ∼50-residue synthetic peptides or from a 20-residue synthetic fragment and an

80-residue recombinant fragment. Note that in the latter strategy (i.e., semisynthesis) it

is important to design the synthesis in such a way that the chemically synthesized fragment

encompasses the region of the protein to be engineered. Where possible, naturally

occurring X-Cys motifs in the native sequence should be chosen as the ligation sites,

where X can be any residue except Asp, Asn, Glu, Gln, and Pro. In the absence of an

endogenous Cys at the appropriate position in the primary sequence, it will be necessary

to mutate a native residue in order to facilitate ligation. The effect of this Cys mutation

on the structure and function of the protein can usually be minimized by following a few

simple rules: (1) If the tertiary structure of the protein is known, choose a region remote

from the active site of the protein, preferably in a flexible surface loop; (2) choose the

mutation to be as conservative as possible, e.g., Ser→Cys or Ala→Cys; (3) try to avoid

Supplement 15

Contributed by Julio A. Camarero and Tom W. MuirCurrent Protocols in Protein Science (1999) 18.4.1-18.4.21

Copyright © 1999 by John Wiley & Sons, Inc.

18.4.1

Preparation andHandling ofPeptides

mutating residues that are conserved across a gene family; and (4) take advantage of any

known mutational data on the system since the effect on structure/function of mutating a

particular residue may already be known.

Chemical synthesis and recombinant DNA expression each allow the generation of

polypeptides containing an amino-terminal cysteine residue (C-terminal fragment) and

the generation of polypeptides containing an α-thioester functionality (N-terminal frag-

ment). For polypeptides of ∼50 residues or less, chemical synthesis is usually the method

of choice since it is relatively quick and efficient and allows the incorporation of unnatural

amino acids. Amino-terminal cysteine–containing peptides can be chemically synthesized

via established t-butyloxycarbonyl (Boc) or 9-fluorenyloxycarbonyl (Fmoc) SPPS pro-

tocols using commercially available resins (e.g., from NovaBiochem, Bachem, Peptides

International, Applied Biosystems, or Neosystems) and amino acid derivatives (see Table

18.4.1). The synthesis of peptide α-thioesters can currently only be achieved using Boc

chemistry, due to the sensitivity of the thioester functionality to the base-deprotection

conditions associated with the Fmoc-SPPS strategy. Note, the resin required for the

synthesis of peptide α-thioesters is not commercial available, but can be prepared in the

laboratory in a single day.

For polypeptide fragments larger than ∼50 amino acids in length, a biosynthetic strategy

should be employed. It must be noted that this assumes the gene has been cloned.

Polypeptides containing amino-terminal Cys residues can be obtained using a mutagene-

sis/proteolysis strategy. This involves constructing an expression vector in which a DNA

sequence encoding the peptide Ile-Glu-Gly-Arg-Cys is inserted between a upstream

affinity-purification handle (e.g., GST or His tag) and the appropriate gene fragment of

interest (for cloning strategies see UNIT 6.6). Following expression and affinity purification

(UNIT 6.6), the fusion protein is treated with Factor Xa to give the desired recombinant

Cys-polypeptide. Polypeptides containing α-thioester functionalities can be obtained by

expressing the corresponding polypeptide sequence using the IMPACT expression system

(New England Biolabs). This results in the generation of a fusion protein in which the

polypeptide of interest is attached through its C-terminus to an intein-CBD, where the

intein is an engineered protein-splicing element and CBD (chitin binding domain) is an

Table 18.4.1 Resins and Amino Acid Derivatives Usually Employed in the Synthesis of N-Terminal Cys and

α-Thioester Polypeptidesa

PolypeptideNα-protectinggroup strategy

Side-chain protecting groups Resin(s)C-terminal

functionality aftercleavage

N-terminal Cys Boc Arg(Tos), Asn(Xan), Asp/Glu(OcHx),

Cys(Meb), Gln(no protection), His(Dnp),

Lys(2-Cl-Z), Met(no protection),

Ser/Thr(Bzl), Trp(For), Tyr(2-Br-Z)

MBHA —CONH2

PAM —COOH

Fmoc Asn(Trt), Arg(Pmc), Asp/Glu(OtBu),

Cys(Trt), Gln(Trt), His(Trt), Lys(Boc),

Met(no protection), Ser/Thr/Tyr(tBu),

Trp(Boc)

Rink amide —CONH2

Wang —COOH

α-thioester Boc As in the Boc strategy for obtaining N-

terminal Cys polypeptides, except for

His(Bom) and Trp(no protection or Hoc)

3-mercapto-

propionamide

-MBHA

—COS-

CH2CH2CONH2

aAbbreviations: Boc, t-butyloxycarbonyl; Bom, benzyloxymethyl; 2-Br-Z, 2-bromobenzyloxycarbonyl; tBu, t-butyl; 2-Cl-Z, 2-chlo-

robenzyloxycarbonyl; Dnp, 2-4-dinitrophenyl; Fmoc, 9-fluorenyloxycarbonyl; For, formyl; Meb, p-methylbenzyl, OtBu, t-butyl ester;

OcHx, cyclohexyl ester; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Tos, tosyl (p-toluenesulfonyl); Trt, trityl; Hoc, cyclohexy-

loxycarbonyl; PAM, 4-hydroxymethylphenylacetamidomethyl; Xan, xanthyl.

Supplement 15 Current Protocols in Protein Science

18.4.2

Native ChemicalLigation of

Polypeptides

affinity handle. The desired recombinant α-thioester polypeptide can be obtained by

simply treating the immobilized fusion protein with an appropriate thiol; the intein-CBD

construct remains attached to the affinity matrix.

BASIC

PROTOCOL 1

NATIVE CHEMICAL LIGATION OF TWO POLYPEPTIDES

This protocol describes the chemical ligation of two polypeptide segments to afford a

target protein containing both fragments linked through a native peptide bond. The

reaction is initiated by dissolving both peptides in aqueous buffer at pH 7.5 in the presence

of thiol cofactors. The reaction usually takes between 1 and 3 days to go to completion,

at which point the product is purified using reversed-phase high-performance liquid

chromatography (RP-HPLC) and subsequently folded. A schematic of the chemistry

behind the procedure is presented in Figure 18.4.1.

Materials

6 M guanidine⋅HCl buffer (see recipe)

Nitrogen source (ultrapure)

Benzyl mercaptan (Aldrich)

Thiophenol (Aldrich)

Polypeptide with N-terminal Cys residue (see Support Protocols 1 and 2)

Polypeptide with α-thioester functionality (see Support Protocols 3 and 4)

1 M NaOH

Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 99% pure; Strem Chemicals)

OH

SHCO2–

NH

NH2H2N+

NH3

+

O

SRH3N

+H2N

HS

CO2–CONH

OH

SHCO2–

NH

NH2H2N+

NH3

+

Polypeptide 2 Polypeptide 1

OH

SHCO2–

NH

NH2H2N+

NH3

+

O

SH3N

+H2N

CONH

SHCO2–

NH

NH2H2N+

NH3

+

Polypeptide 1

reversible transthioesterification

spontaneous S to N acyl transfer

Polypeptide 2

OH

SHCO2–

NH

NH2H2N+

NH3

+

O

NHH3N

+SH

CO2–CONH

OH

SHCO2–

NH

NH2H2N+

NH3

+

Polypeptide 2Polypeptide 1

Polypeptide 2

OH

CO2–

Figure 18.4.1 The mechanism of native chemical ligation. The initial step is a reversible

transthioesterification reaction involving the thiol group of the N-terminal Cys-polypeptide (C-termi-

nal fragment) and the α-thioester moiety of the N-terminal polypeptide fragment. This intermediate

undergoes a spontaneous rearrangement to form a natural peptide bond at the ligation site.

continued

Current Protocols in Protein Science Supplement 15

18.4.3


Buffers for analytical C18 reversed-phase HPLC

Buffer A: H2O containing 0.1% trifluoroacetic acid (TFA)

Buffer B: 1 part H2O/9 parts acetonitrile/0.1% TFA

Appropriate final buffer in which target protein will be dissolved (e.g., forlong-term storage, activity studies, or structural studies), containing 6 Mguanidine⋅HCl and 5 mM DTT

Appropriate final buffer in which target protein will be dissolved, containing 4 M,2 M, and 0 M guanidine⋅HCl (APPENDIX 3A) and 1 mM DTT

Dialysis membrane with MWCO substantially smaller than target protein

Centricon filter (Amicon; optional)

Tabletop centrifuge (Sorvall RT-7 or equivalent)

Additional reagents and equipment for analytical and semipreparative orpreparative C18 reversed-phase HPLC, electrospray ionization (ESI) ormatrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) massspectrometry (MS; UNITS 16.1 & 16.2), dialysis (UNIT 4.4 & APPENDIX 3B), andspectrophotometric determination of protein concentration (UNIT 3.1)

Prepare the ligation buffer

1. Degas the 6 M guanidine⋅HCl buffer by purging with ultrapure nitrogen for at least

20 min.

2. Add benzyl mercaptan and thiophenol, each at a concentration of 0.5% (v/v), to the

degassed buffer. Mix by vortexing to produce the ligation buffer.

IMPORTANT NOTE: This ligation buffer should be prepared just prior to use and should

not be stored for long periods of time. Thiols are, in general, toxic and have an unpleasant

odor; therefore thiol-containing solutions should be treated with 5% (v/v) commercial

bleach in 1:1 (v/v) methanol/water before being disposed of.

Since benzyl mercaptan and thiophenol are barely soluble in aqueous buffers, vortexing is

required to form a homogeneous thiol suspension in the ligation buffer.

Ligate the N-terminal and C-terminal polypeptide fragments

3. Dissolve equimolar amounts of the polypeptides to be ligated (i.e., one with an

N-terminal Cys residue and the other with an α-thioester functionality) in the ligation

buffer to a final concentration of at least 0.5 mM with respect to each peptide.

Reactions can be run in volumes of 50 to 100 µl for analytical purposes and up to 0.5 to 5

ml for preparative purposes.

Native chemical ligation reactions are usually carried out in the presence of chaotropic

agents (guanidium⋅HCl or urea) in order to maximize the concentration of the two reactants

and to increase the availability of reacting moieties by alleviating any steric hindrance that

might be associated with folded polypeptides. The use of chemical denaturants, of course,

presumes that the final product can be properly folded in vitro. In cases where the fragments

being ligated must be kept in a folded state, the ligation reaction can be carried out in the

absence of chemical denaturants.

4. Check the pH of the ligation mixture by spotting a 2- to 3-µl aliquot onto a strip of

pH paper. If it is above or below 7.0, adjust the pH by titrating in small increments

with 1 M HCl or NaOH, respectively. Repeat this process until a pH of ∼7.5 is reached.

Dissolving lyophilized peptides at high concentrations can cause the pH of the ligation

mixture to drop; this is particularly so when HPLC-purified materials are being used (i.e.,

from acidic conditions). It is extremely important to ensure that the pH of the reaction

mixture is kept between 7 and 8, since the efficiency of the native chemical ligation is

strongly dependent on the pH. As the pH drops below pH 7.0, the reaction slows down, and

as it increases above pH 8.0 the chemoselectivity of the process is lost.


18.4.4


Polypeptides

5. Keep the reaction at room temperature with gentle stirring for 24 hr.

6. After 24 hr, take a 5-µl aliquot of the reaction mixture (allowing the rest of the mixture

to continue reacting as in step 5) and dilute the aliquot in a polypropylene microcen-

trifuge tube with 50 µl of 6 M guanidine⋅HCl buffer in the presence of one crystal of

tris-(2-carboxyethyl)phosphine hydrochloride (TCEP, used as a reducing agent).

Shake the sample until the TCEP is completely dissolved, wait 10 min, then

microcentrifuge 5 min at 10,000 rpm.

7. Analyze 25 µl of the supernatant by analytical C18 reversed-phase HPLC, initially

using a linear gradient from 0% buffer B to 73% buffer B over 60 min at a flow rate

of 1 ml/min and refining as necessary based on the elution times of the reactants and

product. Monitor the run via UV detection at 214 nm (and 280 nm if aromatic residues

are present in the target protein). Manually collect every peak (except the solvent

peak) into a separate polypropylene microcentrifuge tube and analyze each by

ESI-MS (UNITS 16.1 & 16.2), which will allow the retention times of the reactants and

the product to be determined

Occasionally the peak corresponding to the ligation product is buried beneath the thiophe-

nol or benzyl mercaptan peaks. This can usually be resolved by optimizing the HPLC

gradient or, alternatively, by lyophilizing the reduced sample a few times and then

reanalyzing it by HPLC.

8. After 36 hr take another 5-µl aliquot of the reaction mixture and analyze it by HPLC

as in step 7. If significant amounts of both reactants still remain and the product peak

area is still increasing, then leave the reaction for another 12 hr and repeat the analysis.

If, on the contrary, both reactants have been consumed or if the product peak area has

not increased appreciably, then terminate the reaction as in step 9.

Ligation reactions normally take 24 to 36 hr to go to completion, although on rare

occasions longer periods (3 to 4 days) or shorter periods (a few hours) may be required.

9. Dilute the crude reaction mixture into 5 to 10 vol of 6 M guanidine⋅HCl buffer and

reduce by adding 15 molar equivalents of TCEP (i.e., based on the quantities of

reactants used; 15 times the number of moles of either one of the polypeptides added).

Shake the solution until all the TCEP is dissolved and wait for 20 min.

10. Centrifuge the sample 20 min at 3000 rpm in a tabletop centrifuge (Sorvall RT-7 or

equivalent), room temperature. Retain the supernatant.

A white cloudiness often appears in the reaction medium during the course of the reaction.

This is due to the slow formation of insoluble benzyl mercaptan and thiophenol disulfides.

The reactants and ligation product will generally remain in solution, allowing the unwanted

disulfides to be removed by centrifugation.

The crude ligation mixture can be stored at −80°C for 2 to 3 days, although it is advisable

to purify it as soon as possible.

Purify the target protein

11. Purify the crude reaction mixture by semipreparative or preparative reversed-phase

HPLC using an optimized gradient system. Analyze each HPLC fraction by mass

spectrometry (UNITS 16.1 & 16.2) and analytical reversed-phase HPLC in order to identify

those fractions containing the target protein. Pool the fractions of highest purity

(>95%) and lyophilize.

Reversed-phase HPLC purification will denature the majority of proteins, meaning that a

subsequent refolding step is required. In cases where the ligation reaction has been carried

out under more physiological conditions (i.e., without chemical denaturants in the ligation

buffer), the final purification step can be performed using, e.g., gel-filtration chromatog-

raphy (UNIT 8.3) or ion-exchange chromatography (UNIT 8.4) under conditions compatible

with protein folding.


18.4.5


Fold the purified protein

12. Dissolve the lyophilized target protein to a final concentration of ≤100 µM in the

appropriate freshly degassed buffer (e.g., for long-term storage, activity studies, or

structural studies), containing 6 M guanidine⋅HCl and 5 mM DTT.

Although the present protocol involves the stepwise dialysis from 6 M guanidium⋅HCl, it

should be stressed that this approach may not work for all proteins and that a certain

amount of optimization will be required for every system. It should also be noted that for

disulfide-containing proteins, an additional oxidation step will have to be performed.

In general, the lower the final concentration of protein the better, since this will tend to

minimize aggregation during the refolding step.

13. Dialyze the solution at 4°C (UNIT 4.4 & APPENDIX 3B) against the same buffer as in step

12, this time containing 1 mM DTT and decreasing amounts of guanidine⋅HCl (i.e.,

first against 4 M guanidine⋅HCl, then against 2 M guanidine⋅HCl, then against final

storage buffer). In each step perform dialysis for at least 12 hr and change the buffer

2 to 3 times during this period.

The MWCO of the dialysis membrane should be chosen to be substantially smaller than

the purified protein.

14. Remove sample from dialysis bag, centrifuge to remove any precipitate, and quantify

the supernatant by UV spectrophotometry at 280 nm (UNIT 3.1). If necessary concen-

trate the protein solution using a Centricon filter (UNIT 4.4; Amicon), then divide into

aliquots and freeze at −70°C.

BASIC

PROTOCOL 2

SEQUENTIAL CHEMICAL LIGATION OF THREE POLYPEPTIDES

If the region of interest in the protein is >50 residues from the N- or C-termini, then its

chemical modification will be extremely difficult using a two-fragment ligation strategy,

since this would require the chemical synthesis of a peptide >50 residues in length. This

problem can be overcome by assembling the target protein from three polypeptide

fragments using a sequential native ligation strategy. The basic method for sequential

native ligation uses a N-terminal Cys polypeptide (C-terminal fragment), an α-thioester

polypeptide (N-terminal fragment), and an Nα(methylsulfony)ethyloxycarbonyl-Cys,

α-thioester polypeptide (central fragment). The procedure starts by ligating the middle

fragment and the C-terminal fragment at pH 7.5 in the presence of thiol cofactors. The

Nα(methylsulfony)ethyloxycarbonyl (Msc) group is then removed and the resulting

polypeptide (central plus C-terminal fragment) is ligated with the N-terminal fragment

to give the target protein, which is purified and refolded. The general approach is depicted

in Figure 18.4.2.

Materials

Three polypeptide fragments to be ligated:

Polypeptide with N-terminal Cys residue (see Support Protocols 1 and 2)

Polypeptide with Nα(Msc)-Cys, α-thioester functionality (see Support Protocol 5)

Polypeptide with α-thioester functionality (see Support Protocols 3 and 4)

1 M HCl

Additional reagents and equipment for native chemical ligation of twopolypeptides (see Basic Protocol 1)

1. Prepare the ligation buffer (see Basic Protocol 1, steps 1 and 2). Ligate the central

[i.e., Nα(Msc)-Cys, α-thioester] and C-terminal (N-terminal Cys) polypeptide frag-

ments [see Basic Protocol 1, steps 3 to 8, but use the Nα(Msc)-Cys, α-thioester

polypeptide in the reaction mix in place of the α-thioester polypeptide].


18.4.6


Polypeptides

2. When the ligation reaction is complete, remove the Nα-Msc protecting group by

raising the pH of the crude ligation mixture to 13 with 1 M NaOH solution (see Basic

Protocol 1, step 4). After 1 min, lower the pH to 5.0 to 7.0 with 1 M HCl (use ∼1.1

to 1.2 times the volume of 1 M NaOH required to raise the pH to 13).

Usually, for a 1-ml ligation mixture, the pH can be raised by adding 75 µl of 1 M NaOH,

then dropped by adding 95 µl of 1 M HCl. It is recommended that the pH be dropped by

adding the acid all at once, instead of by titration, for reasons of speed.

The presence of thiols improves the yield in the deprotection step, since they trap the still

reactive methyl ethylenyl sulfone side product.

3. Analyze the crude reaction mixture (see Basic Protocol 1, steps 6 and 7) and examine

to see if the Msc deprotection is complete. Purify the polypeptide fragment (see Basic

Protocol 1, steps 9 to 11), which will consist of the central fragment ligated to the

C-terminal fragment.

4. Starting with the lyophilized (central plus C-terminal) polypeptide fragment prepared

in steps 1 to 3 above and the N-terminal polypeptide fragment (containing an

α-thioester functionality), perform the ligation reaction to generate the target protein

(see Basic Protocol 1, steps 3 to 10).

5. Purify and refold the target protein (see Basic Protocol 1, steps 11 to 15).

peptide 2

HS

Msc-HN-Cys CO-SRMsc-HN-Cys

HS

H2N-Cys peptide 1+

peptide 3 CO-SR

CO2–

peptide 2Msc-HN-Cys CO-NH- Cys peptide 1 CO2–

HS

aqueous buffer, pH 7.5

0.5%thiophenol

benzyl mercaptan

HS

ligation 1

HS

peptide 2 CO-NH- CysH2N-HN-Cys peptide 1 CO2

–

HS

removal of Msc group

aqueous buffer, pH 7.5

0.5%thiophenol

benzyl mercaptan

peptide 3 CO-NH-CysH3N-Cys +

HS

peptide 2 CO-NH-Cys

HS

peptide 1 CO2–

H3N+

ligation 2

pH 13

peptide 3

peptide 2

Figure 18.4.2 The principle of sequential native chemical ligation. The key to this approach is the

reversible protection of the α-amino group of the central peptide fragment, thereby preventing

self-reaction with the α-thioester moiety present in the same molecule. This can be accomplished

with the base-labile Msc [Nα(methylsulfony)ethyloxycarbonyl] group, which can be easily removed

by brief treatment with base after the first ligation step. The newly deprotected fragment is now

ready for the next ligation step.


18.4.7


SUPPORT

PROTOCOL 1

CHEMICAL SYNTHESIS OF N-TERMINAL CYS-POLYPEPTIDES

The chemical synthesis of N-terminal Cys-polypeptides can be easily carried out using

standard SPPS with Boc or Fmoc Nα-protected amino acids and the appropriate resins

(see Table 18.4.1 and UNIT 18.1). In both cases, the synthesis can be performed on automated

solid-phase peptide synthesizers, which are available in the core facilities of many

institutions.

NOTE: Extreme care must be taken to avoid exposing N-terminal Cys-polypeptides to

even trace amounts of carbonyl-containing compounds (e.g., acetone or formaldehyde).

These chemicals react rapidly with the α-amino and thiol groups of the N-terminal Cys

to give a very stable thiazolidine derivative which prevents the peptide from participating

in subsequent native chemical ligation reactions. For the same reason, the groups

benzyloxymethyl (Bom) and t-butyloxymethyl (Bum), used to protect the side-chain of

His in Boc and Fmoc SPPS, respectively, should be avoided during the synthesis of these

polypeptides since they release formaldehyde during the cleavage/deprotection step.

SUPPORT

PROTOCOL 2

BIOSYNTHESIS OF N-TERMINAL CYS POLYPEPTIDES

Biosynthesis represents a complementary approach to chemical synthesis (see Support

Protocol 1) for obtaining N-terminal-Cys polypeptides, and is especially useful when the

target polypeptide fragment is somewhat greater than 50 residues in length (i.e., too large

to be chemically synthesized). Assuming that the cDNA for the gene is available,

N-terminal Cys polypeptides can be obtained using a mutagenesis/proteolysis strategy

(Erlandson et al., 1996). This involves constructing an expression vector in which a DNA

sequence encoding the peptide Ile-Glu-Gly-Arg-Cys is inserted between a upstream

affinity-purification handle (e.g., MBP, GST, or His tag) and the appropriate gene

fragment of interest (for cloning and mutagenesis see UNIT 6.6). Following expression and

affinity purification, the fusion protein is treated with Factor Xa to give the desired

recombinant Cys-polypeptide, which can then be further purified if necessary. These

recombinant Cys-polypeptides can be used in native chemical ligation reactions as per

their synthetic counterparts.

SUPPORT

PROTOCOL 3

CHEMICAL SYNTHESIS OF α-THIOESTER POLYPEPTIDES

This protocol describes the chemical synthesis of a 3-mercaptopropionamide α-thioester

polypeptide on a 3-mercaptopropionamide-MBHA resin. This resin can be easily pre-

pared from commercially available MBHA resin using a three-step solid-phase procedure

(Fig. 18.4.3). The solid-phase synthesis of the polypeptide is achieved using Boc amino

acid derivatives employing in situ neutralization/HBTU [2-(1H-benzotriazolyl)-1,1,3,3-

tetramethyluronium hexafluorophosphate] activation protocols for Boc-SPPS (Schnölzer

et al., 1992). The corresponding α-thioester polypeptide, suitable for native chemical

ligation, is obtained after cleavage with hydrogen fluoride and purification.

Materials

Methylbenzhydrylamine (MBHA) resin (Peptides International)

Dimethylformamide (DMF, spectrophotometric grade; Fisher)

5% (v/v) diisopropylethylamine (DIEA, peptide synthesis grade; Perkin-ElmerApplied Biosystems) in DMF (store up to 1 month at room temperature)

97% 3-bromopropionic acid

Dichloromethane (DCM, spectrophotometric grade; Fisher)

99% diisopropyl carbodiimide (DIPC, 99%; Aldrich)

Diisopropylethylamine (DIEA, peptide synthesis grade; Perkin-Elmer AppliedBiosystems)

continued


18.4.8


Polypeptides

Ac2O/DIEA/DMF solution (see recipe)

AcSH/DIEA/DMF solution (see recipe)

BME/DIEA/DMF solution (see recipe)

Boc–amino acyl–N-hydroxysuccinimide ester (Boc-AA-OSu; where AA is thefirst amino acid to be incorporated into the polypeptide; Bachem)

Trifluoroacetic acid (TFA, BioGrade)

Ninhydrin test reagents: monitor 1, monitor 2, and monitor 3 (Perkin-ElmerApplied Biosystems)

HF/p-cresol solution (see recipe)

Diethyl ether, cold

10% to 50% acetonitrile in H2O containing 0.1% TFA

15-ml manual peptide synthesis vessel (Peptides International)

Black rubber tubing (1/4 in. i.d. × 5/8 in. o.d. × 3/16 in. wall thickness; Fisher),resistant to acids and organic solvents

2-liter side-arm flasks with rubber stoppers and glass tubing to fit

Pasteur pipet containing glass wool for filtration

2-ml polypropylene column (Microcolumn X from Isolab) with Teflon stopcock

13 × 100–mm glass test tube

110°C heating block

60% ethanol

HF cleavage apparatus (Peptides International)

Additional material and equipment for solid-phase peptide synthesis (Schnölzer etal., 1992; UNIT 18.1), preparative C18 reversed-phase HPLC (see Basic Protocol1, step 11), and ESI-MS (Chapter 16)

Prepare the MBHA resin

1. Build a manual solid-phase peptide synthesis system (Fig. 18.4.4) consisting of a

15-ml manual peptide synthesis vessel attached by 12 in. of black rubber tubing

through a rubber stopper to a 2-liter side-arm flask, which is in turn connected to a

vacuum source.

MBHA NH2 NH BrCO2H, DIPC

DCM

OCH3-COSH, DIEA

DMFNH S

O

CH3

Br

NH SH

O

NH S-AA-Boc

OBoc-AA-OSu

NSOH,DIEA,DMF

DMF

O

MBHA MBHA

MBHA MBHA

Figure 18.4.3 Scheme showing the preparation of the Boc-aminoacyl-3-mercaptopropionamide-MBHA resin using solid-

phase transformations. Abbreviations: AA, aminoacyl; Boc, t-butyloxycarbonyl; CH3-COSH, thioacetic acid; DCM, dichlo-

romethane; DIEA, diisopropylethylamine; DIPC, diisopropyl carbodiimide; DMF, dimethylformamide; MBHA,

methylbenzhydrylamine; OSu, hydroxysuccinimide ester.


18.4.9


2. Place 1 g of MBHA resin (1.0 mmol/g) into the peptide synthesis vessel, add enough

DMF to cover the dry resin (∼5 ml), and wait 30 min.

This preswelling step is crucial for the success of any reaction carried out on a solid

support, since it renders the functional groups on the polymer available for reaction.

3. Drain the resin. Cover the resin with 5% DIEA/DMF, leave for 1 min, then drain the

resin. Repeat this process two more times, then perform three 20-sec flow washes

with DMF—i.e., wash the resin with a continuous flow of DMF for 20 sec while

keeping a constant volume of solvent above the resin bed (usually 3 to 4 mm), then

drain the resin.

A continuous flow wash is an extremely efficient way of exchanging solvent in a swollen

polymer—much more so than the commonly used bulk wash (i.e., by adding solvent, mixing,

and draining).

Prepare the symmetrical anhydride of 3-bromopropionic acid

4. Dissolve 1.222 g 3-bromopropionic acid (8 mmol) in a minimal volume of DCM (∼1

to 2 ml), then add 630 µl of 99% DIPC (4 mmol). Shake the solution vigorously and

wait 10 min.

A white precipitate corresponding to the diisopropyl urea will appear during the activation

reaction. If no such precipitate forms, the reaction has not taken place and should be

repeated.

It is best to prepare the symmetrical anhydride fresh for each coupling reaction.

5. Filter the solution through a Pasteur pipet containing glass wool.

It is very important to remove the diisopropyl urea by filtration before adding the symmet-

rical anhydride solution to the resin. The authors have found that if this step is not carried

out the acylation reaction does not go to completion.

Couple the symmetrical anhydride of 3-bromopropionic acid to the MBHA resin

6. Add the filtered solution from step 5 to the MBHA resin in the peptide synthesis

vessel. Without applying any suction, add 800 µl DIEA (4.5 mmol) and the minimal

amount of DMF (1 to 2 ml) required to give a good slurry.

7. Leave the coupling reaction for 30 min with occasional stirring using a glass stirring

rod.

rubber tubing resistantto acids and organic solvents

waste container

solid-phase peptidereaction vessel

vacuum

resin

stopcock

Figure 18.4.4 Suggested apparatus for the manual solid-phase synthesis of thioester peptides.


18.4.10


Polypeptides

8. Drain the resin and wash thoroughly with three 20-sec DMF flow washes as in step

3.

9. Repeat the coupling process (steps 4 to 8) two more times.

10. Add 5 ml Ac2O/DIEA/DMF solution to the resin and wait 10 min. Drain the resin

and wash three times with DMF as in step 3.

This acetylation step ensures that the small amount of unreacted amine groups remaining

on the MBHA resin cannot participate in any subsequent acylation steps.

Prepare the 3-mercaptopropionamide-MBHA resin

11. Add 5 ml AcSH/DIEA/DMF solution to the resin and wait 20 min. Drain the resin

and wash three times with DMF as in step 3. Repeat this entire process (treatment

with AcSH/DIEA/DMF solution, along with the washings) two more times.

12. Add 5 ml BME/DIEA/DMF solution to the resin and wait 20 min. Drain the resin

and wash three times with DMF as in step 3. Repeat this entire process (treatment

with BME/DIEA/DMF solution, along with the washings) two more times.

The 3-mercaptopropionamide-MBHA resin which is the product of this step is susceptible

to oxidation upon long-term storage. The authors therefore recommend that it be immedi-

ately acylated with the appropriate Boc amino acid derivative.

Couple the first Boc amino acid to the 3-mercaptopropionamide-MBHA resin

13. Dissolve 3 molar equivalents of the appropriate Boc-AA-OSu (where AA is the first

amino acid to be incorporated in the synthesis) in ∼6 ml DMF, then add this solution

to the 3-mercaptopropionamide-MBHA resin. Add 714 µl DIEA (4 molar equiva-

lents, 4 mmol) and leave the coupling reaction for 3 to 4 hr with occasional stirring.

Most of the Boc amino acid derivatives are commercially available as N-hydroxysuccin-

imide esters. If the required Boc amino acid derivative is not commercially available it can

be readily prepared manually (Bodanszky and Bodanszky, 1994).

14. Drain the resin and wash three times with DMF as in step 3. Add 5 ml

Ac2O/DIEA/DMF solution to the resin and wait 10 min, then wash the resin three

times with dichloromethane (DCM) using 20-sec flow washes as described in step

3. Dry the resin under vacuum and store at −20°C.

Determine the final substitution of the Boc-amino acyl-3-mercaptopropionamide-

MBHA resin

15. Place ∼5 mg of the dry Boc–amino acyl–3-mercaptopropionamide–MBHA resin

from step 14 in a 2-ml polypropylene Microcolumn X column with a Teflon stopcock,

connected to a vacuum source.

16. Add 1 ml TFA to the resin and wait 2 min. Drain the column, then flow wash first

with DMF and then with DCM. Drain again and dry under vacuum.

17. Weigh an exact amount of the dried resin (∼3 to 5 mg) and place in a 13 × 100–mm

glass test tube.

18. Add 2 drops of ninhydrin test monitor 1 reagent, 4 drops of ninhydrin test monitor 2

reagent, and 2 drops of ninhydrin test monitor 3 reagent, then incubate 5 min at 110°C

in a heating block.

19. Dilute the blue solution with 60% ethanol to 25 ml and mix well. Measure the

absorbance at 570 nm using 1-cm path-length cuvette. Calculate the substitution of

the thioester resin (in mmol/g) as 1.67 × (A570/amount of resin in mg).


18.4.11


For a resin with an initial loading of ∼1 mmol/g the final substitution for the corresponding

acylated thioester resin is usually ∼0.2 mmol/g.

Perform solid-phase synthesis of the α-thioester polypeptide

20. Synthesize the polypeptide sequence using the in situ neutralization/HBTU activation

protocols for Boc-SPPS (Schnölzer et al., 1992; UNIT 18.1).

Solid-phase deprotection of the 2,6-dinitrophenyl (Dnp) and formyl (For) protecting groups

(commonly used for the protection of the side chains of His and Trp residues, respectively)

results in premature cleavage of α-thioester polypeptides from the resin. Therefore, these

residues should either be deprotected after the ligation reaction is complete or incorporated

as Boc-Trp-OH (no side-chain protection) or Boc-Trp(Hoc)-OH and Boc-His(Bom)-OH

during SPPS .

21. Once the synthesis is complete, flow wash with DMF and DCM as in step 14. Dry

the resin under vacuum and store at −20°C.

Cleave and purify the α-thioester polypeptide

22. Using 200 mg of peptide α-thioester resin from step 21, cleave the peptide-resin by

treating with 5 ml of HF:p-cresol solution for 1 hr at 4°C in an HF cleavage apparatus.

CAUTION: Anhydrous HF is a highly toxic and corrosive gas and should only be

manipulated in a fume hood using the commercially available specialized apparatus.

23. Remove the HF under vacuum and resuspend both peptide and resin in ∼40 ml cold

diethyl ether with gentle stirring for 10 min. Filter the suspension on a glass Buchner

funnel under vacuum, without letting air pass through the filter, as this could oxidize

the cleaved peptide.

24. Wash the material in the filter (containing the cleaved peptide and the resin) three

times, each time with 10 ml cold diethyl ether. Wash an additional three times, each

time with 10 ml dichloromethane, then wash with 10 ml cold diethyl ether again.

Discard the wash liquid.

These washes will remove most of the scavenger byproducts.

25. Add 10 ml freshly degassed 50% acetonitrile in water containing 0.1% TFA to the

filter, wait 10 min (to dissolve the cleaved peptide), then filter. Repeat this process

three more times, then recover and lyophilize the filtrates.

26. Analyze a 50-µl aliquot by reversed-phase HPLC and ESI-MS (Chapter 16; also see

Basic Protocol 1, step 7).

27. Purify the lyophilized propionamide α-thioester peptide by preparative C18 RP-

HPLC (see Basic Protocol 1, step 11).

SUPPORT

PROTOCOL 4

BACTERIAL EXPRESSION OF α-THIOESTER POLYPEPTIDES

This protocol describes the preparation of α-thioester polypeptides using bacterial ex-

pression in E. coli. The desired polypeptide-encoding gene fragment is first cloned into

a commercially available pTYB vector (Fig. 18.4.5). Following E. coli transformation,

soluble expression of the polypeptide-intein-CBD (chitin binding domain) fusion protein

is induced and the cells are harvested and lysed. The lysate is then loaded onto a chitin

column and the fusion protein affinity purified. Finally, the target α-thioester polypeptide

is cleaved from the column and eluted using a buffer containing ethanethiol.

NOTE: Initial studies using an MBP-intein-CBD system indicate that the majority of

amino acid residues, when located immediately before the intein N-terminal cysteine,

allow both purification of fusion proteins and efficient cleavage with thiols. However, Pro,


18.4.12


Polypeptides

Figure 18.4.5 pTYB1, one of the series of pTYB vectors useful for the cloning, expression and

purification of recombinant α-thioesters polypeptides. (A) pTYB1 vector; (B) sequence of pTYB1

cloning/expression region. This vector uses a bacteriophage T7 promoter-driven system (see UNIT

5.1 and Ausubel et al., 1999). The target gene is cloned into the multiple cloning site (MCS) polylinker

region to create an in-frame fusion between the C-terminus of the target gene and the N-terminus

of the gene encoding a modified intein. The DNA encoding a small chitin binding domain (CBD)

has been also added to the C-terminus of the intein gene to allow the resulting fusion protein to be

purified by affinity chromatography. Following purification, the target α-thioester polypeptide is

obtained through a transthioesterification reaction by treating the fusion protein with ethanethiol.

The pTYB vectors are available from New England Biolabs.


18.4.13


Cys, and Asn residues were found to inhibit the in vitro cleavage with thiols. Note that

partial cleavage during bacterial expression can sometimes be observed in certain systems,

resulting in a decrease in the yield of fusion precursors. For example, in the MBP system,

Asp, Arg, His, and Glu residues gave rise to in vivo cleavage (>50%).

Materials

Gene construct encoding target polypeptide

pTYB1 vector (New England Biolabs)

E. coli BL21 (or any other suitable E. coli strain)

LB medium containing 100 µg/ml ampicillin (APPENDIX 4A)

1 M isopropyl-1-thio-β-D-galactopyranoside (IPTG), filter sterilized

Chitin beads slurry: 50% (w/v) suspension of chitin beads (New England Biolabs)in 40% ethanol

Column buffer (see recipe)

Lysis buffer (see recipe)

Cleavage buffer (see recipe)

Refrigerated centrifuge with Beckman JA-10 rotor and centrifuge buckets to hold1 liter of culture

1.5 × 10–cm glass or polypropylene column

Refrigerated centrifuge with Beckman JA-17 rotor and appropriate centrifuge tubes

Shaker

Additional reagents and equipment for introducing plasmid vectors into bacterialcells (APPENDIX 4D), growth of bacteria in liquid medium (APPENDIX 4A), lysis ofbacterial cells using a French press (UNIT 6.3), ESI-MS (UNITS 16.1 & 16.2)

Express the fusion protein

1. Insert the gene construct encoding the target polypeptide into the pTYB1 vector (see

manufacturer’s instruction for vector and Ausubel et al., 1999) so as to express the

target protein as an intein-CBD fusion. Introduce the vector into the E. coli BL21

cells (see APPENDIX 4D and Ausubel et al., 1999). Inoculate 15 ml LB/ampicillin with

E. coli BL21 containing a pTYB vector expressing the desired protein fusion. Grow

overnight with shaking at 37°C (APPENDIX 4A).

2. Inoculate 1000 ml LB/ampicillin with 10 ml of the overnight culture and grow with

shaking at 37°C to an OD600 of 0.5 to 0.6 (APPENDIX 4A).

3. Add 1 ml of 1 M IPTG (final concentration of 1 mM) to the culture and continue

incubation for 1 to 6 hr at 37°C.

The optimal induction conditions (i.e., incubation time, temperature, and final IPTG

concentration) for soluble expression will depend on the in vivo properties of the overex-

pressed protein, and should be optimized for every system.

4. Centrifuge the cell culture 10 min at 8700 × g (7000 rpm in Beckman JA-10 rotor),

4°C. Discard supernatant. Proceed to step 5 immediately or freeze pellet at −70°C

indefinitely.

If extract preparation is to be be carried out immediately, the chitin column may be prepared

during the centrifugation step.

Prepare the affinity column

5. Add ∼10 ml of chitin bead slurry to a 1.5 × 10–cm column and allow liquid to drain

just to the top of the packed resin bed (∼5 ml).

6. Wash the column with 100 ml column buffer at a flow rate of 1 ml/min.


18.4.14


Polypeptides

Prepare the cell extract

7. If cell pellet was frozen, (step 4), thaw on ice. Resuspend pellet in 30 ml ice-cold

lysis buffer. Lyse cells using a French press (UNIT 6.3).

IMPORTANT NOTE: From this step on, all procedures should be carried out at 4°C.

8. Centrifuge lysate for 30 min (or until supernatant is clear) at 25,000 × g (14,000 rpm

in Beckman JA-17 rotor), 4°C. Decant supernatant into a clean container on ice and

discard pellet.

The supernatant can be frozen and stored at −70°C indefinitely before continuing with the

procedure.

In order to monitor the expression, extraction, and purification steps, it is convenient to

take small aliquots at every step and analyze them by SDS-PAGE (UNIT 10.1).

Purify the fusion protein

9. If extract is frozen, thaw on ice. Load onto the chitin column (at a rate no faster than

0.5 ml/min). Collect flowthrough and reapply to column, then repeat this process one

more time.

Chitin beads have a capacity of ∼2 mg of CBD-tagged protein per ml packed beads.

Therefore, the amount of extract that can be loaded on the column will depend on the

amount of soluble fusion protein in the extract.

10. Wash the column with 100 ml column buffer at a flow rate of 1 ml/min. Discard

flowthrough. Be sure that all traces of crude extract have been washed off the sides

of the column.

Obtain the α-thioester polypeptide

11. Add 5 ml cleavage buffer to the column and gently shake the reaction slurry overnight

at room temperature.

12. Drain the beads, then wash with 15 ml cleavage buffer (three times, each time with

5 ml). Pool all of the fractions.

IMPORTANT NOTE: Thioesters are susceptible to hydrolysis under alkaline conditions;

consequently purification and storage of α-thioester polypeptides should be performed at

pH 6.0 or below.

Analyze and purify the α-thioester polypeptide

13. Analyze a 50-µl aliquot by reversed-phase analytical HPLC and ESI-MS (see UNITS

16.1 & 16.2; also see Basic Protocol 1, step 7).

14. Purify the target α-thioester polypeptide by reversed-phase preparative HPLC see

Basic Protocol 1, step 11).

SUPPORT

PROTOCOL 5

CHEMICAL SYNTHESIS OF Nα(Msc)-CYS, α-THIOESTER POLYPEPTIDES

This protocol describes how to introduce the Nα(methylsulfony)ethyloxycarbonyl (Msc)

protecting group onto the α-amino group of an N-terminal Cys polypeptide synthesized

on a 3-mercaptopropionamide-MBHA resin (see Support Protocol 3). The resulting

Nα(Msc)-Cys, α-thioester polypeptides are used in sequential native chemical ligation

reactions (see Basic Protocol 2).

NOTE: Boc-SPPS of α-thioester polypeptides requires the use of the Bom side-chain

protecting group for His. The Bom group releases fomaldehyde during its deprotection

with HF; however the formation of the thiazolidine adduct with the N-terminal Cys cannot

take place due to the presence of the NαMsc group.


18.4.15


Additional Materials (also see Support Protocol 3)

Fully protected Boc-polypeptide-3-mercaptopropionamide-MBHA resin (seeSupport Protocol 3, step 21), dried

(Methylsulfonyl)-ethyl 4-nitrophenyl carbonate (Msc-ONp; Fluka)

Prepare the Boc-polypeptide-3-mercaptopropionamide-MBHA resin

1. Place ∼0.5 g of fully protected Boc-polypeptide-3-mercaptopropionamide-MBHA

resin in a peptide synthesis vessel attached to a vacuum source and swell with DMF

(see Support Protocol 3, steps 1 and 2).

2. Deprotect the Boc group by adding ∼5 ml of TFA to the resin, waiting 1 min, then

draining the resin. Repeat this process one additional time, then drain the resin and

perform three 20-sec flow washes with DMF (see Support Protocol 3, step 3).

3. Neutralize the polypeptide-3-mercaptopropionamide-MBHA resin by treating with

5% DIAE/DMF and flow washing with DMF (see Support Protocol 3, step 3).

Introduce the Msc group on the α-amino group of the polypeptide-3-mercapto-

propionamide-MBHA resin

4. Dissolve 560 mg (2 mmol) of Msc-ONp in 4 to 5 ml DMF and add this mixture to

the peptide thioester resin, then add 340 µl (2 mmol) of DIEA. Leave the coupling

reaction for 3 hr, with occasional stirring using a glass stirring rod, then drain the

resin and perform three 20-sec flow washes with DMF.

5. Place ∼1 mg of resin from step 4, in a column (see Support Protocol 3, step 15). Flow

wash first with DMF and then with DCM, drain again, and dry under vacuum. Place

the aliquot in a glass test tube and perform the ninhydrin test (see Support Protocol

3, step 18). If the color of the solution is strongly blue, repeat the coupling reaction

(step 4) and test again; if not, continue with step 6, below.

Sometimes the ninhydrin test can give false positives (i.e., strong blue coloration) even

although the acylation reaction is complete. This is especially true when the α-amino group

is protected with a base-labile group (i.e., Fmoc or Msc). Therefore, if the ninhydrin test

is still positive after the third coupling, proceed to step 6.

6. Perform three 20-sec flow washes with DMF and then with DCM. Dry the resin under

vacuum and store at −20°C until further use.

7. Cleave and purify 200 mg of peptide-thioester resin (see Support Protocol 3, steps

22 to 26).

REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent for the preparation of all buffers. For common stock solutions,see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.

Ac2O/DIEA/DMF solution

15 parts (v/v) 99% acetic anhydride (Ac2O)

15 parts (v/v) diisopropylethylamine (DIEA, peptide synthesis grade; Perkin-El-

mer Applied Biosystems)

70 parts (v/v) dimethylformamide (DMF, spectrophotometric grade; Fisher)

Prepare fresh

AcSH/DIEA/DMF solution

1 part (v/v) 96% thiolacetic acid (AcSH; Fluka)

1 part (v/v) diisopropylethylamine (DIEA, peptide synthesis grade; Perkin-Elmer

Applied Biosystems)


Prepare fresh


18.4.16


Polypeptides

BME/DIEA/DMF solution

1 part (v/v) 98% 2-mercaptoethanol (BME)

1 part (v/v) diisopropylethylamine (DIEA, peptide synthesis grade; Perkin-Elmer

Applied Biosystems)


Prepare fresh

Cleavage buffer

0.1 mM EDTA

200 mM sodium phosphate, pH 6.0

250 mM NaCl

3% (v/v) ethanethiol

0.1% (v/v) Triton X-100

Adjust pH to 6.0 with 1 M NaOH or HCl

Store up to 6 months at 4°C

Column buffer

0.1 mM EDTA

20 mM sodium phosphate, pH 7.2

250 mM NaCl

0.1% (v/v) Triton X-100

Adjust pH to 7.2 with 1 M NaOH or HCl


Guanidine⋅HCl buffer, 6 M

6 M guanidine⋅HCl (APPENDIX 3A)

0.1 M sodium phosphate

1 mM EDTA

Adjust pH to 7.5 with 1 M NaOH

Store up to 6 months at room temperature

HF/p-cresol solution

96 parts (v/v) hydrogen fluoride (HF; anhydrous)

4 parts (v/v) p-cresol

Prepare fresh

CAUTION: Anhydrous HF is a highly toxic and corrosive gas and should be manipulated

only in a fume hood, using the commercially available specialized apparatus (HF cleavage

apparatus from Peptides International).

Lysis buffer

0.1 mM EDTA

1 mM PMSF

25 mM HEPES, pH 8.0

250 mM NaCl

5% (v/v) glycerol

Adjust pH to 8.0 with 1 M NaOH


COMMENTARY

Background InformationThe introduction of solid-phase peptide-

synthesis (SPPS) by Bruce Merrifield revolu-

tionized the chemical synthesis of peptides

(Merrifield, 1963; also see UNIT 18.1). Despite

the enormous impact of SPPS in the generation

and study of small bioactive peptides, it is now

clear that the combination of incomplete acy-

lation/deprotection reactions and other well

documented side reactions places an intrinsic

limit on the size of peptides accessible by effi-

cient stepwise SPPS. Thus, polypeptides of up


18.4.17


to ∼50 residues in length can be prepared by

stepwise SPPS with reasonable confidence, but

beyond this, the chances of success fall off

precipitously. In order to overcome this size

limitation, recent years have seen renewed in-

terest in the use of convergent synthetic strate-

gies—i.e., the synthesis of large polypeptides

from smaller peptide building blocks which are

themselves accessible via the SPPS approach.

It should be noted that fragment condensation

has a long and illustrious history in the peptide

chemistry field; however, there have always

been serious problems associated with the ma-

nipulation of the fully protected peptides in

these classical convergent strategies. Although

the use of minimal protection strategies repre-

sented a step in the right direction (see Lloyd-

Williams et al., 1993 for an extensive review),

it was not until the early 1990s that a truly

practical way of performing fragment conden-

sations was developed—i.e., chemical ligation

(for reviews see Muir, 1995; Wallace, 1995).

The original chemical ligation strategies were

all based on the premise that an unnatural moi-

ety could be used to covalently join two fully

unprotected (and hence water-soluble)

polypeptides, each bearing unique and mutu-

ally reactive groups. A number of different

chemistries have been developed for this pur-

pose, all of which give rise to an unnatural

covalent structure at the ligation site (Muir,

1995; Wallace, 1995).

In 1994, a second-generation ligation chem-

istry was introduced, known as “native chemi-

cal ligation,” which allows the preparation of

proteins with native backbone structures from

fully unprotected peptide building blocks

(Dawson et al., 1994). This important extension

of the chemical ligation concept makes use of

the mild acylating power of the α-thioester

functionality. The principle of native chemical

ligation is depicted in Figure 18.4.1. The first

step involves the chemoselective reaction

which occurs between the free thiol group of

an unprotected N-terminal Cys-polypeptide

and a second, unprotected polypeptide contain-

ing an α-thioester group. This transthioesteri-

fication reaction gives rise to a thioester-linked

intermediate which spontaneously rearranges

to form a native peptide bond at the ligation

site. The target full-length polypeptide is thus

obtained without any further manipulation. Na-

tive chemical ligation reactions are performed

in aqueous buffers at pH 7 to 7.5 in the presence

of thiol cofactors. At this pH, the regioselectiv-

ity of the reaction is such that the reaction can

to be performed in the presence of all the

functionalities commonly found in proteins.

Even the presence of additional Cys residues in

one or both fragments does not affect the re-

gioselectivity of the ligation (Hackeng et al.,

1997). Small proteins or protein domains ∼100

to 120 amino acids in length can be reliably

generated from two peptide building blocks in

a single chemical ligation step (for a few exam-

ples see Dawson et al., 1994, 1997; Lu et al.,

1996; Hackeng et al., 1997; Camarero et al.,

1998).

The total chemical synthesis of larger pro-

tein targets (>100 residues) via the ligation of

just two fragments becomes more and more

problematic as the size increases. This is due to

the difficulties associated with the direct step-

wise SPPS of polypeptide segments bigger than

50 residues. This difficulty can be overcome by

performing multiple ligation reactions using

three or more synthetic peptides (e.g., Canne et

al., 1995). Native chemical ligation has been

extended to allow multiple ligation steps to be

performed sequentially in a controlled and di-

rected way (Muir et al., 1997; Camarero et al.,

1998). The general approach is depicted in

Figure 18.4.2. Key to this strategy is the tem-

porary protection of the α-amino group of the

central peptide with the base-labile 2-(methyl-

sulfonyl)ethyloxycarbonyl (Msc) group

(Tesser and Balvert-Geers, 1975). The presence

of this protecting group prevents the N-terminal

Cys from reacting in an intramolecular or in-

termolecular fashion with the α-thioester func-

tionality present in the same polypeptide frag-

ment. Once the first ligation reaction is fin-

ished, the Nα-Msc group can be efficiently

removed, allowing the next ligation step to be

performed (Fig. 18.4.2).

The native chemical ligation approach can

also be used in the semisynthesis of proteins

(Muir et al., 1998; Severinov and Muir, 1998;

Erlandson et al., 1996; Evans et al., 1998) from

recombinant and synthetic polypeptide frag-

ments. As described above, native chemical

ligation of two polypeptides requires that one

of the fragments possess an N-terminal Cys and

that the other contain an α-thioester moiety.

Polypeptides containing an N-terminal cyste-

ine residue for use in litigation can be obtained

using standard recombinant DNA expression

methods (see Chapter 5). Importantly, biosyn-

thetic methods are also now available for the

generation of α-thioester polypeptides (Muir et

al., 1998; Severinov and Muir, 1998; Evans et

al., 1998). This is made possible using the

IMPACT expression system, commercially

available from New England Biolabs (Chong


18.4.18


Polypeptides

et al., 1997). This system utilizes a protein

splicing element, an intein from the Sac-

charomyces cerevisiae VMA1 gene, in con-

junction with a chitin binding domain (CBD)

which allows purification by affinity chroma-

tography (Fig. 18.4.6). The natural intein has

been modified in the expression system

(Asn454→Ala) in order to block the normal

protein splicing reaction in midstream. This

results in the formation of a thioester linkage

between the polypeptide of interest and the

intein (Fig. 18.4.6). Cleavage of the thioester

can thus be induced by treatment of the ex-

pressed fusion protein with the appropriate

thiol to give the target α-thioester polypeptide

(Muir et al., 1998; Severinov and Muir, 1998;

Evans et al., 1998).

Critical Parameters andTroubleshooting

As mentioned in Basic Protocol 1, pH is the

most critical parameter in the native chemical

ligation of two fully unprotected peptides in

aqueous buffers. The efficiency of the ligation

reaction is strongly dependent on the pH; above

pH 8.0 the reaction loses its regioselectivity and

below pH 6.0 the reaction is usually very slow.

N-extein

A HS

NH intein

HS

NH2

NHC-extein

O

O

N→S acyl transfer

N-exteinHS

NH2

NH

O

Ointein C-exteinH2N

S

transthioesterification

S

NH2

NH

O

Ointein C-exteinH2N

HS

O

S→N acyl transfer

and succinimide formation

intein

SH

HN

O

N-extein

intein

NH

O

OinteinH2N

HS +

O

O

recombinant protein intein*CO-NH-Cys

clone protein gene in IMPACT system

express in E. coli

HS

recombinant protein intein*CO-NH-Cys

HS

recombinant protein CO-S

recombinant protein CO-SCH2CH3

intein*H2N-Cys

i) 3% ethanethiol at pH 6

overnight

ii) filtration

transthioesterification

N→S Acyl transfer

Affinity purification

on chitin beads

B

CBD

CBD

N-extein

CBD

C-extein

Figure 18.4.6 Principles of the biosynthetic preparation of α-thioester polypeptides by recombinant techniques. (A)

Scheme representing the proposed mechanism of protein splicing involving the intein from Saccharomyces cerevisiae VMA1

gene (Xu and Perler, 1996). (B) Expression, purification, and cleavage of polypeptide-intein*-CBD fusion protein (where the

asterisk refers to the mutation Asn454→Ala in the intein element and CBD refers to the chitin binding domain) with an

appropriate thiol to give the α-thioester polypeptide.


18.4.19


Another crucial factor is the concentration

of the two reactants. The bimolecular nature of

native chemical ligation (and ligations in gen-

eral) means that the concentration of both reac-

tants should be as high as possible for efficient

reaction. Generally the use of chemical dena-

turants (GdmCl or urea) will allow high con-

centrations of both reactants to be achieved.

Furthermore, the use of denaturing conditions

helps to alleviate potential steric problems that

may be associated with the use of folded

polypeptides.

Another important parameter for the success

of the ligation reaction is the availability of the

thiol and/or α-amino groups of the N-terminal

Cys polypeptide during the ligation reaction. If

one or both groups are chemically blocked, the

ligation will not take place. It is well known

that N-terminal Cys peptides can react rapidly

with carbonyl-containing compounds (e.g.,

acetone and formaldehyde) to give the corre-

sponding N-terminal thiazolidine adducts,

which are totally unreactive in the native liga-

tion process. It is thus crucial to avoid the use

of these substances while handling all peptides

and buffers. Note that the authors have found

acetone, a commonly used solvent for washing

glassware in many laboratories, to be particu-

larly problematic in this regard.

Anticipated ResultsNative chemical ligation has been applied to

the synthesis and semisynthesis of a large num-

ber of proteins and protein domains. These

studies indicate that very high yields (80% or

better) are typically obtained for the ligation

step. Following purification, the total yield usu-

ally drops to 50% to 60%. In sequential liga-

tions where the Nα-Msc group has to be depro-

tected in situ after the first ligation step, the

typical total yield after purification is ∼30% to

40%. It is also important to note that the pro-

tein-folding step may further decrease the final

yield of product.

Time ConsiderationsIn Basic Protocols 1 and 2, the chemical

ligation step typically requires 2 days, although

in some cases the ligation reaction can proceed

very rapidly (in a few hours) or somewhat more

slowly (in 4 days). The purification step (RP-

HPLC or other liquid chromatographies) can

be performed in half a day. Processing of the

purified samples (lyophilization or concentra-

tion) can take 1 to 2 days (depending on the

volume). Finally, folding of the protein (if nec-

essary) can be achieved in 3 days.

The thioester resin can be prepared in 1 day.

The time required for the chemical synthesis of

an α-thioester polypeptide (see Support Proto-

col 3) will depend on its size. Typically, for

peptides ∼50 residues in length, the solid-phase

chain assembly can be carried out in 3 days

manually or in 1 day using an automated syn-

thesizer. The deprotection-cleavage and purifi-

cation can be performed in 1 day, and lyophili-

zation of peptide fractions can take 1 to 2 days

(depending on the volume).

Bacterial expression of a peptide-intein-

CBD fusion protein and preparation of the

crude cell extract requires 2 days for the system

described here (see Support Protocol 4). Col-

umn preparation requires ∼2 to 3 hr. Loading

and washing the column can be done in 3 hr.

The cleavage of the α-thioester polypeptide

from the affinity column requires 10 to 15 hr

(overnight). Purification and processing of the

polypeptide fractions requires 1 to 2 days.

Literature CitedAusubel, F.A., Brent, R., Kingston, R.E., Moore,

D.D., Seidman, J.G., Smith, J.A., and Struhl, K.(eds.). 1999. Current Protocols in Molecular Bi-ology. John Wiley & Sons, New York.

Bodanszky, M. and Bodanszky, A. 1994. The Prac-tice of Peptide Synthesis, 2nd ed. Springer-Ver-lag, Berlin.

Camarero, J.A., Cotton, G.J., Adeva, A., and Muir,T.W. 1998. Chemical ligation of unprotectedpeptides directly from a solid support. J. Pept.

Res. 51:303-316.

Canne, L.E., Ferre-D’Amare, A.R., Burley, S.K.,and Kent, S.B.H. 1995. Total chemical synthesisof a unique transcription factor-related protein:cMyc-Max. J. Am. Chem. Soc. 117:2998-3007.

Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E.,Landry, D., Vence, L.M., Perler, F.B., Benner, J.,Kucera, R.B., Hirvonen, C.A., Pelletier, J.J.,Paulus, H., and Xu, M.Q. 1997. Single-columnpurification of free recombinant proteins using aself-cleavable affinity tag derived from a proteinsplicing element. Gene 192:271-281.

Dawson, P.E., Muir, T.W., Clark-Lewis, I., and Kent,S.B.H. 1994. Synthesis of proteins by nativechemical ligation. Science 266:776-779.

Dawson, P.E., Churchill, M.J., Ghadiri, M.R., andKent, S.B.H. 1997. Modulation in native chemi-cal ligation through the use of thiol additives. J.

Am. Chem. Soc. 119:4325-4329.

Erlandson, D.A., Chytill, M., and Verdine, G.L.1996. The leucine zipper domain controls theorientation of AP-1 in the NFAT AP-1 DNAcomplex. Chem. Biol. 3(12):981-991.

Evans, T.C., Benner, J.J., and Xu, M.-Q. 1998. Semi-synthesis of cytotoxic proteins using a modifiedprotein splicing element. Protein Sci. 7:2256-2264.


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Polypeptides

Hackeng, T.M., Mounier, C.M., Bon, C., Dawson,P.E., Griffin, J.H., and Kent, S.B. 1997. Totalchemical synthesis of enzymatically active hu-man type II secretory phospholipase A2. Proc.Natl. Acad. Sci. U.S.A. 94:7845-7850.

LLoyd-Williams, P., Albericio, F., and Giralt, E.1993. Convergent solid-phase peptide synthesis.Tetrahedron 49:11065-11133.

Lu, W., Qasim, M.A., and Kent, S.B.H. 1996. Com-parative total synthesis of turkey ovomucoidthird domain by both stepwise solid phase syn-thesis and native chemical ligation. J. Am. Chem.Soc. 118:8518-8523.

Merrifield, R.B. 1963. Solid phase peptide synthe-sis. I. The synthesis of a tetrapeptide. J. Am.

Chem. Soc. 85:2149-2154.

Muir, T.W. 1995. A chemical approach to the con-struction of multimeric protein assemblies.Structure 3:649-652.

Muir, T.W., Dawson, P.E., and Kent, S.B.H. 1997.Protein synthesis by chemical ligation of unpro-tected peptides in aqueous solution. MethodsEnzymol. 289:266-298.

Muir, T.W., Sondhi, D., and Cole, P.A. 1998. Ex-pressed protein ligation: A general method forprotein engineering. Proc. Natl. Acad. Sci.U.S.A. 95:6705-6710.

Schnölzer, M., Alewood, P., Jones, A., Alewood, D.,and Kent, S.B.H. 1992. In situ neutralization inBoc-chemistry solid phase peptide synthesis:Rapid, high yield assembly of difficult se-quences. Int. J. Pept. Protein Res. 40:180-193.

Severinov, K. and Muir, T.W. 1998. Expressed pro-tein ligation: A novel method for studying pro-tein-protein interactions in transcription. J. Biol.Chem. 273:16205-16209.

Tesser, G.I. and Balvert-Geers, I.C. 1975. Themethylsulfonylethyloxycarbonyl group: A newand versatile amino protective function. Int. J.Pept. Prot. Res. 7:295-305.

Wallace, C.J.A. 1995. Peptide ligation and semisyn-thesis. Curr. Opin. Biotechnol. 6:403-410.

Xu, M.-Q. and Perler, F.B. 1996. The mechanism ofprotein splicing and its modulation by mutation.EMBO J. 15:5146-5153.

Contributed by Julio A. Camarero and Tom W. MuirThe Rockefeller UniversityNew York, New York


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