the chemical modification of proteins 
 is an established tool for 
 studying the structure , function , and regulation of this class of 
 biopolymer .
moreover , in recent years , 
 a great deal of effort has been directed toward the modification of 
 proteins for therapeutic applications .
traditionally , protein conjugation chemistries have exploited the 
 reactivity of surface - exposed nucleophilic amino acids , such as cysteine 
 or lysine , however , these methods typically result in heterogeneous 
 mixtures of products , which can complicate biological studies or efficacious 
 medicinal applications . to address this concern ,
several strategies 
 have been developed for the site - specific modification of proteins , 
 ranging from total chemical synthesis ( usually via native chemical 
 ligation , ncl ) to the genetic incorporation 
 of unnatural amino acids or bio - orthogonal 
 functional groups . in between these two 
 extremes
the most 
 widely used protein semisynthesis technique is an extension 
 of ncl termed expressed protein ligation ( epl ) in which a recombinant 
 protein -thioester building block is ligated to a synthetic 
 molecule equipped with a 1,2-aminothiol moiety ( most commonly an n - terminal 
 cys - containing peptide ) through the formation of a native peptide 
 bond ( scheme 1 ) . since 
 its inception
, epl has been applied to a wide variety of proteins , 
 including enzymes , ion channels , transcription factors , transmembrane receptors , and antibodies ( for reviews see refs ( 15 and 16 ) ) .
one of the basic requirements 
 of epl is a thioester group at the c - terminus of a recombinant protein . 

this reactive handle is introduced by exploiting a process known as 
 protein splicing ( scheme 1 ) , which is mediated 
 by an autoprocessing domain called an intein .
protein splicing typically takes place through the formation of 
 one or more protein thioester intermediates , which ultimately resolve 
 to form a native peptide bond between the sequences flanking the intein 
 ( referred to as n- and c - exteins ) . by using appropriate intein mutants , 

it is possible to intercept these intermediates with exogenous thiols , 
 resulting in an n - extein of choice being cleaved from the mutant intein 
 as a reactive -thioester derivative suitable for chemical ligation .
int and int represent n- and c - intein fragments , respectively . despite the many successes of epl , the approach 
 often suffers from 
 low overall efficiency due to complications associated with the generation 
 of protein -thioesters .
in particular , fusions to inteins are , 
 to varying extents , susceptible to premature extein cleavage , both in vivo and during initial purification from cell lysates , 
 which reduces the isolated yield of the intein fusion needed for the 
 subsequent thiolysis step .
importantly , 
 the cleaved extein side - product is unreactive toward epl , and its 
 separation from the desired -thioester or the ligation product 
 is often difficult for large proteins , such as antibodies . to compound matters ,
the thiolysis reaction 
 itself can be slow and inefficient , further strengthening the need 
 to develop customized purification regimes , involving multiple chromatographic 
 steps , to isolate the desired product from complex mixtures .
collectively , these technical issues mean that a considerable investment 
 in time and resources is usually required before a semisynthetic protein 
 is obtained in useful quantities .
( a ) 
 schematic of the procedure 
 for the isolation of an -thioester derivative of a protein 
 of interest ( poi ) using engineered split intein fragments ( int and int ) .
epl can be performed in a one - pot fashion 
 during thiolysis from the split intein or immediately after elution , 
 without need of any further purification .
( b ) sequences of npu ( wt ) and the npuaa mutant used on the split intein 
 column .
catalytic residues mutated in npuaa are shown 
 in bold , and the linker sequence added for immobilization onto the 
 solid support is underlined .
sequences are numbered according to the 
 intein sequence alignment shown in figure s2 . to overcome the various drawbacks
associated with 
 the intein thiolysis 
 process central to epl , we envisioned an alternative strategy based 
 on naturally occurring split inteins . unlike inteins used in standard 
 epl , which are contiguous polypeptides that catalyze protein splicing 
 in cis , split
inteins consist of two discrete polypeptides , 
 herein termed int and int , which , upon association , 
 catalyze protein splicing in trans ( protein trans - splicing , pts , scheme 1 ) .
split inteins have two important properties 
 that make them attractive for an improved epl strategy .
first , cognate 
 int and int pairs often bind tightly to one 
 another ; dissociation constants in the low nanomolar range , reflecting 
 extremely fast on - rates , have been reported for members of the naturally 
 split dnae inteins from cyanobacteria .
this ability 
 of split inteins to self - associate has recently been exploited by 
 lu et al . as part of a traceless protein purification system , in this 
 case using an artificially split intein pair .
the potential utility of split inteins in epl is further enhanced 
 by the remarkable splicing efficiency of some members of the family .
for instance , many of the split dnae inteins have half - lives for 
 the splicing reaction of less than a minute , as compared to several 
 hours in the case of the cis - splicing inteins commonly 
 used in epl .
moreover , recent mechanistic 
 investigations indicate that these  ultrafast  dnae 
 inteins have a highly activated n - terminal splice junction , making 
 them superior reagents for protein -thioester generation . given the unique properties of split inteins , 
 in particular , the ultrafast split dnae inteins , we conceived the 
 integrated protein modification system shown in figure 1 in which the split intein association is employed both to 
 purify the desired protein from complex biological mixtures and to 
 trigger the generation of a desired protein -thioester for 
 epl . in principle
, this complementation system should address the 
 major issues attendant to the standard epl protocol , including premature 
 cleavage of the intein , which can not occur in the case of a split 
 intein fragment absent its cognate partner .
to implement 
 our system , we designed a mutant of the ultrafast nostoc 
 punctiforme ( npu ) split dnae intein suitable for efficient 
 -thioester generation . specifically , we mutated the catalytic 
 c - terminal residue in the int fragment ( asn137 ) and the 
 first residue in the c - extein ( cys+1 ) to ala , to allow for efficient 
 build up of the desired splicing intermediates upon exposure to an 
 n - extein - npu fusion ( figure 1 ) . 

preliminary studies showed that mixing n - extein - npu fusions 
 ( where n - extein corresponded to various model proteins ) with the mutant 
 npu ( npuaa ) led to highly efficient n - extein 
 -thioester formation in a thiol - dependent manner ( figures s3 and s4 ) .
importantly , only very low 
 levels of intein cleavage ( i.e. , unwanted hydrolysis ) were observed 
 in the absence of thiols , thereby fulfilling a requirement of our 
 integrated strategy . encouraged by these results , we adapted 
 the system for the one - pot purification and generation of c - terminally 
 modified proteins by taking full advantage of the strong and specific 
 interaction between the split intein fragments .
accordingly , the npuaa peptide was immobilized on a solid support through a unique 
 cys residue engineered within its c - extein region ( figure s5 ) . the resulting npuaa column ( hereafter 
 referred to as int column ) was then evaluated as an affinity - modification 
 resin .
three test proteins maltose binding protein ( mbp ) , ubiquitin 
 ( ub ) , and protein histidine phosphatase type 1 ( phpt1 ) were genetically 
 fused to npu and expressed in escherichia 
 coli . in each case , cells were lysed , and the soluble 
 fraction was loaded onto the int column to allow binding 
 of the npu tagged protein to the immobilized npuaa .
after a brief incubation (  5 min at rt ) the column was 
 extensively washed to remove contaminants , and thiolysis was triggered 
 by addition of a buffer containing the thiol , mercaptoethansulfonate 
 ( mes ) . in all three cases , the desired -thioester protein eluted 
 from the int column with high recovery ( 7595% ) 
 and high purity ( 95% as determined by rp - hplc and mass spectrometry ) 
 ( figure 2 ) .
total isolated yields of purified 
 protein -thioesters varied from one protein to another and 
 ranged from 2.5 mg ( per l of bacterial culture ) for ub - mes to 40 mg 
 for mbp - mes .
the calculated loading capacity of the int column used in these experiments was 36 mg of protein per 
 ml of beads ( 0.12 mols / ml ) , but higher or lower loadings could 
 easily be achieved by modifying the amount of npu - aa immobilized 
 on the solid support .
furthermore , we showed that the int column could be regenerated and reused at least 5 times with only 
 minimal loss of capacity ( figures s11 and s12 ) .
the utility of the -thioester derivatives of ub , mbp , and 
 phpt1 obtained from the column was demonstrated by ligating each of 
 them to an n - terminal cys - containing fluorescent peptide ( cgk(fl ) ) 
 to give the corresponding semisynthetic products in excellent yield 
 ( figure s10 ) .
importantly , one - pot thiolysis / ligation 
 reactions could be carried out , which allowed us to obtain a site - specifically 
 modified protein directly from cell lysates without isolating the 
 intermediate thioester ( figure 3 ) .
( a ) mbp , ( b ) phpt1 , and ( c ) ub mercaptoethansulfonate 
 ( mes ) -thioesters were purified in one step from e. coli cell lysates using the int column . 

the purifications were monitored by coomassie stained sds - page analysis 
 ( top ) ( inp : input , ft : column flow - through , w1 - 3 : washes , e1 - 4 : elutions , 
 and bds : resin beads ) .
rp - hplc ( detection at 214 nm ) and esi - tof ms 
 analysis of the eluted fractions ( bottom left and right , respectively ) 
 confirmed the identity of all protein -thioesters and indicated 
 high purity .
one - pot purification / ligation of ubiquitin to the h - cgk(fluorescein)-nh2 peptide ( cgk(fl ) ) .
ub - npu from e. coli cell lysates was bound to the int column , as shown in figure 2 , and after removal 
 of contaminants through extensive washes , intein cleavage and ligation 
 were triggered by addition of 200 mm mes and 1 mm cgk(fl ) peptide . 

coomassie stained sds - page analysis and in gel fluorescence of the 
 purification / ligation ( left ) .
rp - hplc ( detection at 214 and 440 nm ) 
 and esi - tof ms ( right ) of the eluted fractions confirm the desired 
 ligated protein was obtained in one step directly from cell lysates 
 with a ligation yield close to 95% ( quantified by rp - hplc ) .
an attractive feature of epl is that it can allow for the preparation 
 of site - specifically modified proteins in a virtually traceless manner . 

this is contingent on the ability to efficiently generate recombinant 
 protein -thioesters when any of the 20 proteinogenic amino 
 acids are present at the c - terminus of the protein .
the activity of 
 split inteins is known to be sensitive to the identity of the amino 
 acids immediately flanking the splice junction .
thus , we were eager to test the generality 
 of our strategy and asked whether we could generate -thioesters 
 of all 20 amino acids , using ubiquitin as the n - extein template .
twenty 
 ub - npu fusion proteins were individually expressed in e. coli and purified over the int column 
 as before .
thiol - induced cleavage yields from the solid support were 
 determined by sds - page analysis of the eluted and resin beads fractions , 
 and levels of competing side reactions ( mainly hydrolysis ) were measured 
 by rp - hplc and esi - tof ms .
the results clearly show that for most 
 amino acids > 60% of the bound proteins were recovered after mes 
 treatment . 
 furthermore , 8095% of the eluted material was the desired 
 -thioester product .
the only exceptions were pro and glu , for 
 which recovery were 49 and 50% , respectively ( figure 4 ) .
the asn mutant displayed high levels of cleavage from the 
 split intein , but almost no -thioester could be isolated due 
 to side - chain cyclization to form a c - terminal succinimide .
a second 
 problematic residue was asp , for which we observed some premature 
 cleavage during initial binding to the int resin .
moreover , 
 rp - hplc analysis of the eluted fractions upon thiolysis revealed two 
 species with the molecular weight of the desired -thioester . 

these results were not wholly unexpected , as asp is known to cleave 
 prematurely from contiguous inteins through side - chain cyclization , and its -thioesters have been reported to migrate to the 
 side - chain carboxylate yielding mixtures of - and -isomers .
these minor constraints aside , it is clear from 
 these studies that our streamlined epl system is compatible when the 
 majority of amino acids are present as the last residue in the protein 
 of interest .
the 20 mutants of the protein ub - x - npu were expressed 
 in e. coli varying the identity of 
 the c - terminal amino acid of ub ( x ) from the wt gly to all other proteinogenic 
 amino acids .
thiol - induced cleavage yields from the int column were calculated from the sds - page analysis of the eluted 
 fractions and left over resin beads .
ratios of -thioester vs 
 side products were determined from rp - hplc and esi - tof ms analysis 
 of the eluted fractions .
the major competing reaction for all amino 
 acids was hydrolysis with the exception of asn for which its succinimide 
 form was isolated instead . *
protein semisynthesis frequently requires the preparation of protein 
 fragments , which are often poorly behaved and need to be purified 
 in the presence of strong chaotropic agents .
we first confirmed that 
 the model protein ub - npu could bind the int column in the presence of 2 and 4 m urea and that the corresponding 
 ub -thioester could be generated with similar yields as obtained 
 under native conditions ( figure s14 ) .
this 
 is consistent with a previous study , which demonstrated npu dnae could 
 splice in the presence of high concentration of denaturants .
we then turned to a more challenging target , 
 namely a fragment of histone h2b ( residues 1116 ) , a polypeptide 
 that is prone to aggregation and difficult to generate as an -thioester 
 derivative using standard epl procedures .
we expressed human histone h2b(1116 ) fused to npu in e. coli , extracted it from inclusion 
 bodies in 6 m urea , and diluted it to 2 m urea prior to loading on 
 the int - intein column .
the incubation was performed for 
 3 h at ph 6.0 to maximize binding while avoiding premature cleavage 
 through hydrolysis .
the ph was then raised to 7.2 , and thiolysis was 
 carried out for 36 h at rt ( note that the presence of the denaturant 
 slows down the thiolysis rate ) . using these conditions ,
hh2b(1116)-mes 
 was obtained in excellent purity ( > 90% by rp - hplc ) and isolated 
 yield 
 ( 20 mg per l of culture ) .
this represents a significant improvement 
 over previous protocols which afford less protein ( 4 mg per l of culture ) 
 and require the use of multiple chromatographic purification steps 
 including rp - hplc .
importantly , the hh2b(1116)-mes 
 thioester obtained from the int column could be directly 
 used in epl reactions without further purification .
accordingly , we 
 successfully ligated the protein to a hh2b(117125 ) peptide 
 containing an acetylated lys at position 120 to yield semisynthetic hh2b - k120ac ( figure 5 ) .
coomassie 
 stained sds - page analysis of hh2b(1116 ) -thioester 
 generation in the presence of 2 m urea ( sup : cell lysate supernatant , 
 trit : 1% triton wash of the inclusion bodies , inp : solubilized inclusion 
 bodies used as input for the int column ) .
e1e3 
 were collected after 18 h of incubation with mes and e4e6 
 after an additional 18 h. e1e6 were pooled , concentrated to 
 150 m , and ligated to the peptide h - cvtk(ac)ytsak - oh at 1 mm 
 for 3 h at rt .
( b ) rp - hplc ( left ) of the ligation reaction mixture 
 and ms ( right ) of the ligated hh2b - k120ac product .
finally , we tested our streamlined epl methodology for the modification 
 of a monoclonal antibody .
the site - specific modification of antibodies 
 has become highly desirable in the area of biopharmaceuticals and 
 diagnostics .
currently , most commercially 
 utilized methods to conjugate cargo to antibodies are relatively nonspecific 
 and result in polydisperse mixtures that may vary from batch - to - batch . 
 since this heterogeneity can adversely affect both efficacy and safety 
 of the conjugate
indeed , protein semisynthesis via standard epl and pts has recently 
 been used to generate monoclonal antibody conjugates with full activity . given this , we were eager to see whether our 
 streamlined epl process could be used in the facile generation of 
 antibody conjugates .
as a model immunoglobulin ( igg ) for our studies , 
 we used an antibody against the dec205 receptor , a c - type lectin found 
 predominantly on dendritic cells .
accordingly , 
 we designed a construct in which npu was fused to the 
 c - terminus of the heavy chain of the antibody ( dec205-npu ) .
initial expression tests of dec205-npu in 293 t cells resulted in very low levels of the antibody being 
 secreted ( figure 6a ) .
we have observed previously 
 that the identity of the int can have an effect on expression 
 levels of its fusions .
consequently , 
 we asked whether we could obtain higher levels of secreted dec205-int by varying the identity of the intein n - fragment .
several 
 new dec205-int constructs were generated in which 
 int corresponded to the n - fragment of a series of ultrafast 
 split dnae inteins , namely , ava , csp , cra , cwa , mcht , oli , and ter . 
 we also tested an npu mutant ( c - s ) where the noncatalytic 
 cysteines , cys28 and cys59 , were mutated to ser , to determine whether 
 these residues influence secretion and maturation of the igg tetramer 
 ( figure 6a ) .
importantly , each of the int fragments in this set can cross - react with the c - fragment 
 of npu without significant loss of splicing efficiency .
thus , the int column already in 
 hand is compatible with all of these dec205-int fusions .
the contiguous mxe gyra intein was also fused to dec205 
 to test whether the use of n - intein fragments negatively affected 
 expression levels compared to a full - length intein .
an expression 
 screen of this dec205-int library was performed 
 in 293 t cells , revealing that the ava and csp fusions reproducibly exhibited higher expression levels than the 
 other n - inteins , with the former being the best .
indeed , the expression 
 levels of the dec - ava construct were at least as 
 good as the dec - gyra construct . based on this ,
the dec205-ava fusion was chosen and purified over the int column 
 in an analogous manner to that of the soluble proteins described above 
 ( figure 6b ) .
elutions from the column contained 
 the thiolyzed dec205 , which was subsequently ligated to the 
 cgk(fl ) peptide ( figure 6c ) .
size exclusion 
 chromatography ( sec ) coupled to multiple angle light scattering ( mals ) 
 analysis confirmed that the antibody retained its tetrameric folded 
 state after thiolysis and ligation ( figure 6d ) .
we also performed ms analysis of the deglycosylated and fully 
 reduced antibody , which confirmed the formation of a stable , nonreducible 
 amide bond between the dec205 heavy chain and the fluorescent 
 peptide with a 75% yield ( figure 6e ) . importantly
, 
 we demonstrated that the semisynthetic dec - cgk(fl ) retains 
 its ability to bind the dec205 receptor to the same extent as a control 
 dec205 , previously shown to be fully functional in 
 vivo(46 ) ( figure 7 ) .
binding of dec - cgk(fl ) to the dec205 receptor could 
 be monitored not only by flow cytometry using an antimouse igg secondary 
 antibody ( figure 7 ) but also through the site - specifically 
 incorporated fluorescein ( figure s20 ) .
purification 
 of a monoclonal antibody -thioester using a 
 split intein and its site - specific modification .
( a ) test expression 
 of dec205 genetically fused to the contiguous mxe gyra intein 
 and different split dnae inteins through the c - terminus of its heavy 
 chain ( hc ) .
western blot of 293 t cell supernatants of several dec205-int 
 fusions using an antibody against mouse igg .
( c ) elution fractions containing dec205-mes 
 were concentrated to 20 m and ligated to the cgk(fl ) fluorescent 
 peptide at 1 mm for 48 h at rt .
( d ) sec - mals analysis of the ligated 
 antibody showing that it retains its tetrameric structure after thiolysis 
 and ligation ( mw = 151 kda , mw calcd = 148 kda ) .
( e ) esi - tof ms analysis of degycosylated 
 and fully reduced hc after ligation , showing 75% of the hc is labeled . 

( a ) dose 
 dependent binding of dec205-cgk(fl ) to cho cells expressing 
 the mouse dec205 receptor monitored by flow cytometry using a pe - labeled 
 -mouse igg .
binding to control cho / neo cells , which do not 
 express the receptor is shown in gray .
( b ) as in ( a ) but using a control 
 -dec205 antibody .
we have shown that split dnae inteins can 
 be engineered for the 
 efficient generation and isolation of protein -thioesters . 

furthermore , the strategy can be seamlessly integrated with epl , and 
 one pot purification / ligations can be performed without isolation 
 of the -thioester intermediates .
we note , however , that the 
 ligation step ( i.e. , ncl ) still requires the use of high concentrations 
 ( high m or above ) of n - terminal cysteine peptide for efficient 
 reactions . the strong and specific interaction between the two intein 
 fragments facilitates the purification of protein -thioesters 
 under a variety of conditions , including strong denaturants , and the 
 isolation of these products directly from cell lysates proceeds significantly 
 faster than via many mainstream epl strategies , which often take multiple 
 days and several intermediate enrichment / purification steps .
additionally , 
 while split intein fusions are usually associated with low levels 
 of expression , we show here that certain dnae int fragments 
 express to the same levels as commonly used contiguous inteins when 
 fused to a monoclonal ab .
moreover , the absence of premature cleavage 
 events allowed us to generate semisynthetic proteins , such as h2b - k120ac 
 in far superior yields than those of previously established protocols . 

importantly , we have shown the utility of this methodology for the 
 modification of complex biomolecules such as an igg .
thus , streamlined 
 epl via our split dnae intein column should provide an efficient route 
 to site - specifically modified proteins for basic biochemical research 
 as well as translational applications .
npu - aa - cys - ome peptide ( 0.5 mols per ml of resin ) was dissolved 
 in 2 column volumes ( cv ) of coupling buffer ( 50 mm trishcl at 
 ph 8.5 , 250 l for 125 l resin ) and treated with 25 mm 
 tcep from a 1 m stock for 15 min .
the peptide solution was then added 
 to 1 cv of agarose sulfolink resin ( from pierce , loading : 18.4 mol 
 iodoacetyl groups / ml of resin ) in a small fritted column and incubated 
 for 15 min on a nutator , followed by 30 min standing at rt .
the column 
 flow - through was collected , and the column was washed twice with 2 
 cv of coupling buffer .
unreacted iodoacetyl groups on the resin were 
 blocked by a treatment with 50 mm h - cys - ome in coupling buffer for 
 15 min on a nutator , followed by 30 min standing at rt .
column was 
 washed twice with 1 cv of coupling buffer , 2 cv of 1 m nacl , and finally 
 2 cv of water .
int columns were stored in storage buffer 
 ( 100 mm phosphate , 150 mm nacl , 1 mm edta , 0.05% nan3 , 
 ph 7.2 ) , at 4 c for up to 2 weeks .
e. coli bl21(de3 ) 
 cells transformed with the desired poi - npu plasmid were 
 grown in 1 l of lb containing 100 g / ml of ampicillin at 37 
 c until od600 = 0.6 .
after harvesting the cells by centrifugation 
 ( 10  500 rcf , 30 min ) , the cell pellets were transferred to 
 50 ml conical tubes with 20 ml of high - salt binding buffer ( 100 mm 
 phosphate , 500 mm nacl , 1 mm edta , 1 mm tcep , ph 7.2 ) with complete 
 protease inhibitor cocktail ( roche ) and stored at 80 c . 

resuspended cells were lysed by sonication , and the soluble fraction 
 recovered by centrifugation ( 17  000 rcf , 10 min ) . onto 62.5 
 l of int column , 300 l of the soluble fraction 
 were loaded and incubated at rt for 5 min .
after incubation , the flow - through 
 was collected , and the column was washed with 300 l of high - salt 
 binding buffer , 300 l of wash buffer ( 100 mm phosphate , 300 
 mm nacl , 1 mm edta , 1 mm tcep , ph 7.2 ) , and 300 l of binding 
 buffer ( 100 mm phosphate , 150 mm nacl , 1 mm edta , 1 mm tcep , ph 7.2 ) . 
 the column was capped and incubated with 150 l of elution buffer 
 ( 100 mm phosphate , 150 mm nacl , 200 mm mes , 10 mm tcep , 1 mm edta , 
 ph 7.2 ) for 18 h. flow - through was collected , and the column was washed 
 three times with 150 l elution buffer . onto 62.5 
 l of int column ,
300 l cell lysate containing 
 ub - npu was loaded and incubated as described before .
the column was 
 washed with 300 l high - salt binding buffer , 300 l wash 
 buffer , and 300 l binding buffer .
the column was capped and 
 incubated with 75 l epl buffer ( 100 mm phosphate , 150 mm nacl , 
 200 mm mes , 50 mm mpaa , 1 mm edta , 10 mm tcep , ph 7.9 ) containing 
 1 mm cgk(fl ) peptide for 18 h. the flow - through was collected , and 
 the column was washed three times with 75 l elution buffer . 

gels were first imaged using 
 the green fluorescence channel on a ge imagequant las 4010 imager 
 and then coomassie stained .
t cells were transiently 
 cotransfected with antimouse - dec205-lc and antimouse - dec205-hc - ava using lipofectamine 2000 ( invitrogen ) , according to the manufacturer s 
 instructions .
after 4 days incubation at 37 c with 5% co2 , cell 
 supernatants were harvested and spun down at 2000 rcf for 20 min at 
 4 c , filtered through a 0.22 m filter , and supplemented 
 with complete protease inhbitors .
for a typical purification , 50 ml 
 of dec205-ava transfected cell supernatants were 
 concentrated to a final volume of 5 ml and exchanged into binding 
 buffer . the resulting ab solution ( input )
was applied to an int column of 300 l beads ( loading : 1.8 mol npuc 
 peptide / ml ) and incubated at rt for 30 min .
column flow - through was 
 collected , and column washed three times with 3 cv of wash buffer 
 and once with 3 cv of binding buffer .
the column was capped and incubated 
 with 3 cv of ab elution buffer ( 100 mm phosphate , 150 mm nacl , 200 
 mm mes , 1 mm tcep , 1 mm edta , ph 7.2 ) for 20 h. the column flow - through 
 was collected , and the column washed three times with 3 cv of ab elution 
 buffer .
ligation was initiated by addition of 1 mm cgk(fl ) 
 peptide and 1 mm tcep and adjusting ph to 7.58.0 .
the reaction 
 was incubated in the dark at rt for 48 h and monitored by sds - page 
 imaged using a fluorescence scanner and coomassie staining .
once the 
 reaction was completed , the ligated ab was diluted to 200500 
 l and dialyzed into 100 mm phosphate , 150 mm nacl , 1 mm edta , 
 1 mm tcep , ph 7.2 .
 chemically modified proteins are invaluable tools for 
 studying 
 the molecular details of biological processes , and they also hold 
 great potential as new therapeutic agents . 
 several methods have been 
 developed for the site - specific modification of proteins , one of the 
 most widely used being expressed protein ligation ( epl ) in which a 
 recombinant -thioester is ligated to an n - terminal cys - containing 
 peptide . despite the widespread use of epl , the generation and isolation 
 of the required recombinant protein -thioesters remain challenging . 
 
 we describe here a new method for the preparation and purification 
 of recombinant protein -thioesters using engineered versions 
 of naturally split dnae inteins . 
 this family of autoprocessing enzymes 
 is closely related to the inteins currently used for protein -thioester 
 generation , but they feature faster kinetics and are split into two 
 inactive polypeptides that need to associate to become active . taking 
 advantage of the strong affinity between the two split intein fragments , 
 we devised a streamlined procedure for the purification and generation 
 of protein -thioesters from cell lysates and applied this strategy 
 for the semisynthesis of a variety of proteins including an acetylated 
 histone and a site - specifically modified monoclonal antibody . 
Introduction
Results and Discussion
Conclusion
Experimental Section