manual e 6901

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Iuci Maua

Impac Ki

ProteIn exPressIon & AnAlysIs

neB #e6901s

s a 20C

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1

tab C:

Kit Components 1

Introduction 2

Advantages o the IMPACT System 3

Description o System Components 3

Construction o the Fusion Plasmid 8

Primer Design 10

Cloning a PCR Fragment 12

Fusion Protein Expression 14

Anity Purication and On-Column Cleavage 16

Simplied Expression and Purication Protocol 18

Media and Solutions 20

Frequently Asked Questions 21

Reerences 24

IMPACT User Publications 25

Appendices

Appendix I: Cloning into pMXB10 26

Appendix II: Intein-mediated Protein Ligation (IPL) and Protein Labeling 27

Appendix III: Sequencing Primers 29

Appendix IV: Research Use Assurance Statement 29

Ordering Inormation 30

Ki Cmp:

Vector DNA pTXB1, pTYB21 10 g o each (50 l)

Control Vector pMXB10 10 g (50 l)

E. coliStrain ER2566 02 ml cells (not competent)

Anti-Chitin Binding Domain Serum (rabbit) 50 l

Chitin Beads (store at 4C) 20 ml

1, 4-Dithiothreitol (DTT), 1 M 5 ml

3X Sample Buer 15 ml

IMPACt Ki

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Iduci:The IMPACT (Intein Mediated Purication with an Anity Chitin-binding Tag)system is a novel protein purication system which utilizes the induciblesel-cleavage activity o protein splicing elements (termed inteins) to separatethe target protein rom the anity tag (1) It distinguishes itsel rom all other

purication systems by its ability to puriy, in a single chromatographic step,a native recombinant protein without the use o a protease Each intein tagcontains a chitin binding domain (CBD) or the anity purication o theusion protein on a chitin resin (2-4) Induction o on-column cleavage, usingthiol reagents such as dithiothreitol (DTT), releases the target protein rom theintein tag (Figures 1,2)The vectors included in this kit allow or the usion o the target protein at itsC-terminus (pTXB1) (3,5) or at its N-terminus (pTYB21) (4,6) to the intein tag

In addition, with the use o pTXB1, native recombinant proteins that possess areactive C-terminal thioester can be isolated or applications, including proteinsemisynthesis and site-specic labeling [3,7, Intein Mediated Protein Ligation(IPL, Appendix II)]

Figu 1: schmaic iuai h

IMPACt sm.

Intein

Tag

TargetProtein

Intein

Tag

TargetProtein

TargetProtein

TargetProtein

Target Gene

MCS

N-terminal C-terminal

Target Gene

MCS Intein Tag

N-terminal C-terminal

Intein Tag

Load &Wash

Inducible Cleavage+DTT at 4C

C-terminal Fusion N-terminal Fusion

T7 Promoter

T7 Promoter

ChitinChitin

Elute &Dialyze

Elute

pTXB1 pTYB21

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3

Advaag h IMPACt sm:

Single column purifcation without the use o a protease to remove the afnity tag

Ability to produce a native target protein without vector derived amino acids

Fusion to either the C-terminus or the N-terminus o the target protein

Isolation o proteins with or without an N-terminal methionine residue

Ligation and labeling o recombinant proteins

T7 promoter-driven system to achieve high levels o expression and tighttranscriptional control in E. coli.

Dcipi sm Cmp:VcpTXB1 (NEB #N6707) contains a mini-intein rom the Mycobacterium xenopigyrA gene (Mxe GyrA intein; 198 amino acid residues) that has been modiedto undergo thiol-induced cleavage at its N-terminus (3,5) The vector allowsor the purication o a target protein without any extra amino acids by cloninginto the NdeI and SapI sites The target protein is used at its C-terminus toa sel-cleavable intein tag (~28 kDa) that contains the chitin binding domain

(CBD, 6 kDa) allowing or anity purication o the usion precursor on achitin column

The pTYB21 (NEB #N6701) vector utilizes an intein rom the SaccharomycescerevisiaeVMA1 gene (Sce VMA1 intein; 454 amino acids)(4,6) The targetprotein is used at its N-terminus to a sel-cleavable VMA1 intein-CBD tag(56 kDa); the tag allows or the anity purication o the usion precursoron a chitin column The vector is designed to allow or purication o a targetprotein without any extra amino acids, or without an N-terminal methionine

residue, by cloning its 5 end into the SapI site The SapI site should be usedas the 3 cloning site in pTXB1 or the 5 cloning site in pTYB21 I anotherrestriction site is used an unavorable amino acid or cleavage will be encodedat the cleavage site, adjacent to the intein Please see 'Construction o the Fu-sion Plasmid' or details

The control vector, pMXB10 (NEB #N6903), derived rom pTXB1, carriesthe control target protein, maltose binding protein (MBP), already insertedupstream o the Mxe GyrA intein-CBD Induction yields the MBP-Mxe GyrAintein-CBD usion (~71 kDa) which, when cleaved, results in the elution oMBP (43 kDa) The polylinker regions fanking the coding region or MBP canconveniently be used to clone a gene o interest However, ater intein cleavagethe target protein will contain additional amino acids at its C-terminus, includ-ing (LEY), which has had a high rate o successul cleavage (see Appendix 1or cloning into pMXB10)

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The IMPACT vectors utilize a T7 promoter to provide stringent control o expres-sion o the usion gene in E. coli(8) The IMPACT vectors carry the Ampr genemarker (the bla gene), which conveys ampicillin resistance to the host strain;one vector sold separately, pKYB1 (NEB #N6706), is kanamycin resistant

oh vc ad appicaiAdditional IMPACT vectors with dierent multiple cloning sites or inteins(pTYB series, pTWIN series, pKYB1, pTXB3) are also available or the usion othe cleavable intein tag to either the C-terminus or the N-terminus o a targetprotein (or a complete list see wwwnebcom) The fexibility in usion proteinconstruction is designed to increase the probability o successul expression andpurication o a target protein, as well as to conduct sophisticated protein engi-neering projects such as protein ligation and cyclization [pTWIN1 (NEB #N6951)or pTWIN2 (NEB #N6952)] NEB also supplies peptides to specically label theC-terminus o a thioester tagged protein with either a fuorescein (NEB #P6606),a biotin (NEB #P6607), a 549 fuor (NEB #P6013) or a 649 fuor (NEB #6014) Iyou do not wish to use a thiol reagent or cleavage the Intein1 (SspDnaB intein)in the pTWIN vectors may be used; cleavage is induced by temperature (roomtemperature) and/or pH shit (rom pH 85 to 6)

Lane 1: Protein Marker, Broad Range (NEB #P7702, 15 l).

Lane 2: Crude extract rom uninduced cells (15 l).

Lane 3: Crude extract rom cell induced at 15C or 16 hours (15 l).

Lane 4: Load or claried lysate (4 l).

Lane 5: Flow through rom chitin column (4 l).

Lane 6: Wash (4 l).

Lane 7: DTT fush to distribute it evenly throughout the column (4 l).

Lanes 8-9: Elution o MBP ater stopping column fow and inducing a cleavage reaction at 4C or 16 hours.

Lane 10: Chitin beads aliquot ater elution (6 l).

Load F.T.Strip with

SDSEluteWash +DTT

+IPTG

Cleave

4C

o.n.

IPTG

1 2 3 4 5 6 7 8 9 10

972

664556

427

365

266

200

kDa

Intein-CBD

MBP

Precursor

Figu 2: epi ad Puifcai E. colima-bidig

pi (MBP) uig h ptxB vc (pMxB10).

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Figu 3A: Pik i h vc ptxB1 ad ptyB21.idica ii cavag i.

pTXB1

XbaI...CGC GAA ATT AAT ACG ACT CAC TAT AGG GGA ATT GTG AGC GGA TAA CAA TTC CCC TCT AGA

NdeI NheI NruI NotIAAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG GCT AGC TCG CGA GTC GAC GGC GGC CGC

M A S S R V D G G R

EcoRI XhoI SapI SpeIGAA TTC CTC GAG GGC TCT TCC TGC ATC ACG GGA GAT GCA CTA GTT... (89 bp)E F L E G S S C

*pTYB21

SapI NdeI NcoI NotI(117 bp) 5...GGA TCC CAG GTT GTT GTA CAG AAC GGA AGA GCT CAT ATG TCC ATG GGC GGC CGC

V Q N G R A H M S M G G R

EcoRV SalI BamHI EcoRI SbfI/PstIGAT ATC GTC GAC GGA TCC GAA TTC CCT GCA GGT AAT TAA...3 (58 bp)

D I V D G S E F P A G N *

InteinIntein Forward Primer

T7 Terminator Reverse Primer

T7 promoter lacoperator

Shine Dalgarno

T7 Universal primer

Intein

Mxe InteinReverse IIPrimer

*Note: NdeI and SapI should be used or cloning the target gene

See Frequently Asked Questions (page 21 and web site) and Companion Products(page 30)

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PvuI 555

FspI 702BsaI 855

SwaI 1096

PsiI 1194

PciI 2310

BstZ17I 2480

BstEII 3916

ApaI - PspOMI 3891

KasI - NarI - SfoI 3459

AfeI 3194

MluI 4098

EcoNI 4562

PmeI 5606XbaI 5683

Acc65I - KpnI 5782BaeI 5782

HindIII 6224

BmtI - NheI 6589

MscI 6750

StuI 7150

PmlI 7175

SacII 7185

BlpI 7430

SpeI 7372

AvaI - BsoBI 1213

DraIII 1319

BssHII 3687

BstBI 7122

MfeI 6646

BglII 6255

PstI 7355SbfI 7354

EcoRI 7348

BamHI 7342

SalI 7336

EcoRV 7330

NotI 7322

NcoI 7316

NdeI 7309

Eco53KI - SacI 7304

BspQI - SapI 7301BspEI 7508

ApR

M13ori

+

-

ori

rop

lacI

MCS

StuI 7150PmlI 7175

SacII 7185

Sce

in

tein

CBD

ptyB217,512 bp

AatII - ZraI 6019

AfeI 3194

AgeI 6379

AlwNI 1896

ApaI - PspOMI 3891

BamHI 6570

BclI 4084

BlpI 6622

BsaBI 5618

BsiWI 5959

BsmI 6100 BspEI 6700

BsrGI 5948

BssHII 3687

BstEII 3916

BstZ17I 2480

BtgI - SacII 6129

DraIII 1319

EcoO109I 6649

EcoRV 3650

FspI 702

HpaI 3594KasI - NarI - SfoI 3459

MfeI 6454

PciI 2310

PflFI - Tth111I 2504

PmeI 5606

PshAI 3253

PsiI 1194

PstI 6559

PvuI 555EcoRI 5755

NdeI 5722BmtI - NheI 5728

NotI 5747

NruI 5734

PaeR7I - TliI - XhoI 5761BspQI - SapI 5768

ScaI 444

SgrAI 6288

SpeI 5793

StuI 6393

SwaI 1096XbaI 5683

MCS ApR

M13ori

+

ori

rop

Mxein

tein

lacI

PspXI 5760

CBD

ptxB16,706 bp

Figu 3B:

ptxB1 vc.A uiqu i a

hw.

Figu 3C:

ptyB21 vc.

A uiqu i

a hw.

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E. coliai er2566ER2566 is provided as a host strain or the expression o a target gene clonedinto the IMPACT vectors ER2566 carries a chromosomal copy o the T7 RNApolymerase gene inserted into the lacZgene, and thus is under the controlo the lac promoter The strain is decient in both lonand ompT proteases

ER2566 is supplied as 50% glycerol stock; these cells are not competent.Recommended long term storage (>30 days) is at 70C. In addition, T7Express Competent E. colirom NEB (NEB #C2566), and other T7 expressionstrains and derivatives can also be used, including BL21(DE3), etc

ER2566 Genotype: fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) endA1 (mcrC-mrr)114::IS10.

Chii Bad (neB #s6651)A chitin anity matrix is used to isolate the usion precursor that contains thetarget protein, intein and a chitin binding domain (CBD) 20 ml o chitin beads(NEB #S6651) are supplied as a 40 ml slurry in 20% ethanol The bindingcapacity or the intein tag used to the target protein, maltose binding protein(MBP), is 2 mg o eluted MBP protein per ml o chitin beads Chitin beadsshould be stored at 4C.

Ai-Chii Bidig Dmai sum (ai-CBD)(neB #s6654)Rabbit serum raised against a peptide derived rom the Bacillus circulanschitin binding domain is provided or Western blot analysis (1:5000) Anti-CBDMonoclonal Antibody (NEB #E8034) is also available separately Store at20C

1, 4-Dihihi (Dtt)A 10 M DTT solution is provided in the IMPACT Kit Since DTT is not particu-larly stable ater dilution, the cleavage buer should be reshly prepared beoreuse DTT stock solutions should be aliquoted to minimize reeze/thaw cyclesStore at 20C

3x sDs samp Bu(Bu ladig Bu Pack, neB #B7703)

1875 mM Tris-HCl (pH 68 @ 25C), 6% (w/v) SDS, 30% glycerol and 003%(w/v) bromophenol blue (store at room temperature) DTT should be added tothe 3X SDS Sample Buer, to a nal concentration o 40 mM (see note in Table1A) Store at 20C

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Cuci h Fui Pamid:Cig cidaiThe identity o the amino acid residues adjacent to the intein has been shownto aect the cleavage reaction and should be taken into consideration (seeTables 1A and 1B) These tables are only a guide, since the olding o the entireusion protein, not just a single amino acid, aects cleavage

It is dicult to accurately predict the expression o a target protein-inteinusion, thus we recommend cloning the insert into dierent vectors andconducting small scale purications In our experience we have ound pTXB1to be the most consistent and reliable expression vector

I a target protein without vector-derived amino acids is required, the pTXB1 orthe pTYB21 vector can be used

I a C-terminal usion is required, where the C-terminus o the target proteinis used to the intein, then the pTXB1 vector should be used I a N-terminalusion is required the pTYB21 vector should be used

In the presence o thiols such as DTT, -mercaptoethanol or cysteine, theintein undergoes specic sel-cleavage which releases the target protein romthe chitin-bound intein tag resulting in a single-column purication o thetarget protein Furthermore, when pTXB1 is utilized, the use o thiol reagents,such as 2-mercaptoethanesulonic acid (MESNA), releases a protein with areactive thioester at the C-terminus o the target protein or use in intein medi-ated protein ligation (IPL)

Cig i ptxB1The mini-intein (MxeGyrA intein) in pTXB1 has been used to puriy a widerange o target protein usions Cloning into the NdeI and the SapI sites resultsin the usion o the intein to the C-terminus o the target protein, without anyextra amino acids on the target protein ater cleavage o the intein tag TheSapI site must be used to clone the 3 end o an insert. The SapI site canalso be used or the inclusion o extra amino acid residues avorable or con-trollable cleavage (by engineering the extra codons into the primers; Table 2)

I the insert sequence contains an internal SapI site the ollowing options exist:

1 Use the pMXB10 vector (See Appendix I)

2 I the insert does not contain a SpeI site, then the SpeI site present nearthe N-terminus o the MxeGyrA intein can be used by designing a primerthat contains the intein sequence; this can result in a usion without anyvector-derived residues ollowing cleavage

3 PCR with a prooreading polymerase to generate a blunt-end product orligation with pTXB1 that has been digested with SapI and lled in TheQuick Blunting Kit (NEB #E1201) can be used to generate a vector withblunt ends

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Eect o the C-terminal residue

o a target protein on DTT-

induced cleavage with pTXB1.

The C-terminal amino acid o the

target protein, paramyosin, was

mutated immediately upstream o

the intein cleavage site. Cleavage

was induced with 40 mM DTT in30 mM Tris, pH 8.5, 0.5M NaCl.

Percent cleavage was determined

by Coomassie stained SDS-

PAGE analysis o chitin beads

beore and ater DTT cleavage.

Note: Boiling in SDS Sample

Buer containing DTT can cause

partial or complete cleavage,

resulting in an overestimation o

in vivo cleavage. I substantialin vivo cleavage is observed, the

cell extract should be evaluated

in a SDS Sample Buer contain-

ing no DTT.

1 Leu showed ~50% in vivo

cleavagewhen induced at 15C;at 37C in vivo cleavage was less

than 5%.2 Asp showed ~50% in vivo cleav-

age when expression was induced

at 15C and 37C.

% cleavage

after 16 hours*

% cleavage

after 40 hours*

c-terminal

residue of the

target protein

4c

23c

4c

23c

t

PhGAthlAaHilu1

M

65-80 80-95 75-90 85-95

I

AgGutpC

30-55 660-85 50-70 70-95

Va 30 70 60 90

G 10 40 20 60

Ap2 10 20 20 30

s

P

5-15 5-15 5-15 5-20

Eect o the N-terminal

residue o a target protein

on DTT-induced cleavage

with pTYB21 or pTYB11. The

N-terminal amino acid o the

target protein,T4 DNA ligase,

was mutated immediately

downstream o the inteincleavage site. Cleavage was

induced with 40 mM DTT in

30 mM Hepes, pH 8.0, 0.5M

NaCl. Percent cleavage was

determined by Coomassie

stained SDS-PAGE analysis

o chitin beads beore and

ater DTT cleavage.

% cleavage

after 16 hours*

% cleavage

after 40 hours*

n-terminal

residue of the

target protein

4c

23c

4c

23c

M

AaG

40-60 > 95 60-90 > 95

GluAtpPht

10-40 75-95 40-60 > 90

Va

IApGulAgHi

< 10 50-80 10-20 70-95

P < 10 < 10 < 10 < 10

ths

C

< 10 dmid

dmid

80 dmid

dmid

20 dmid

dmid

> 90 dmid

dmid

tab 1A

tab 1B

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Cig i ptyB21Use o the SapI site results in the usion o the target gene adjacent to theintein tag so that the target protein can be puried without any extra non-nativeresiduesThe SapI site can be used or the inclusion o extra amino acid residues avorable

or cleavage (by engineering the extra codons into the primers; Table 2) I theNdeI site is used our extra amino acids (Gly-Arg-Ala-His) will be added to theN-terminus o the protein A stop codon should be included in the reverse primerwhen constructing a N-terminal usion

When pTYB21 is used, a small peptide (15 amino acid residues, 16 kDa) is alsocleaved rom the intein tag and co-eluted with the target protein It cannot bedetected on a regular SDS-PAGE and can be dialyzed out

Cig i pMxB10For cloning into pMXB10 NdeI, SapI or NcoI can be used as the 5 cloning siteThe 3 cloning site can be SacI, HindIII, NotI, EcoRI or XhoI (See Appendix I)

Note: BspQI (NEB #R0712) is an isoschizomer of SapI (NEB #R0569) and can beused instead of SapI.

Pim Dig:Appropriate restriction sites, absent in the target gene, are incorporated in theorward and reverse primers when a target gene is generated by PCR The choiceo the restriction site in the primers determines whether any, or which, extraamino acid residues will be attached to the terminus o the target protein aterthe cleavage o the intein tag

Table 2 illustrates some examples o designing orward and reverse primersor pTXB1 and pTYB21 For cloning into pTXB1 one should clone a target gene

between the NdeI (orward primer) and the SapI (reverse primer) sites in pTXB1For the pTYB21 vector the SapI site can be used to clone the 5 end o thetarget gene (PstI as the 3 cloning site or pTYB21 is shown as an example or areverse primer in the table below)

When constructing a N-terminal usion (pTYB21) a stop codon should beencoded in the reverse primer The reverse primer or the C-terminal usion(pTXB1) should not include a stop codon

We recommend writing out your primers and cloning strategy in order to checkSapI (or BspQI) digestion, the reading rames etc For more inormation oncloning with SapI, please reer to our web site: http://wwwnebcom/nebecomm/tech_reerence/protein_expression/IMPACTFaqasp

In general, more than 15 bp o target gene sequence is required or PCR(represented by NNNNNN) In Table 2 the restriction site is underlined TheGGTGGT sequence at the 5 end o the primer is a random sequence o 6 bp toensure ecient DNA cleavage by the restriction enzyme

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tab 2: Pim dig ptxB1 ad ptyB21.

1 SapI digestion creates a 3-nt overhang (GCA) or ligation with the SapI-digested pTXB1 vector (containing a TGC over-

hang), resulting in an in-rame usion to the N-terminus o an intein. The SapI site can be used to add one or more extra

amino acid residue(s) to the target protein by including an appropriate sequence (e.g. add ACC in the reverse primer

corresponding to a GGT codon or a glycine residue). The SapI site is not regenerated ater cloning.2 SapI digestion creates a 3-nt overhang (AAC) compatible with the SapI digested pTYB21 (containing a GTT overhang).

The SapI site is not regenerated ater cloning.3 A stop codon should be included in the reverse primer when constructing a N-terminal usion.

The SapI site is not regenerated ater cloning.

restriction site sequence cloning vector

nd5- GGt GGt CAt AtG nnn nnn... -3

(wad pim)ptxB1

sap1

5- GGt GGt tGC tCt tCC GCA nnn nnn...-3

(v pim) ptxB1

sap25- GGt GGt tGC tCt tCC AAC nnn nnn... -3

(wad pim)ptyB21

P35- GGt GGt CtG CAG tCA nnn nnn... -3

(v pim)ptyB21

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Cig a PCr Fagm:The ollowing brie protocol describes the cloning o an amplied target generagment using restriction enzymes that create non-compatible sticky ends ordirect cloning into the IMPACT vectors Users can ollow standard techniquesto clone the PCR amplied gene For blunt-end or single site cloning, the vec-

tor may need to be treated with a phosphatase (Antarctic Phosphatase, NEB#M0289 or Cal Intestinal Phosphatase, NEB #M0290) I necessary, the PCRproduct can be sub-cloned into a PCR cloning vector beore cloning into anIMPACT vector

1 Restriction Digestion and Ligation: The amplied gene ragment iselectrophoresed on an agarose gel and the gene ragment is puriedThe puried ragment is double-digested with the appropriate restrictionenzymes; the vector (051 g) is also digested with the same enzymes

Note: SapI tends to settle in the tube so mix it with a pipette prior toremoving it from the vial.

Following a 1 to 2 hour digestion, both reaction mixtures are loaded ontoa 1% low melting TAE agarose gel Each o the gel slices (100200 l,the smaller the volume the better) containing the digested gene rag-ment and IMPACT vector are melted at 65C or 10 minutes and treatedwith -Agarase I (NEB #M0392; 1 l per 50 l) or one hour at 42C

Alternatively, a spin column can be used to isolate the DNA Ligation isconducted using the Quick Ligation Kit (NEB #M2200) or ve minutes atroom temperature by mixing the vector and the insert in an appropriateratio (about 1:3) A control ligation reaction containing digested vectoralone should be included

2 Transormation: To reduce the background rom the vectors sel-ligation,the ligation sample can be digested, prior to transormation, with arestriction enzyme whose recognition site is deleted rom the polylinkerduring cloning and is also absent rom the insert This linearizes any re-maining parental vector Fusion constructs should initially be establishedin a non-restricting (rm or rm+), non-expression E. colistrain [ie NEB10-beta (NEB #C3019) or NEB Turbo (NEB #C2984), not ER2566 or T7Express], especially when cloning a potentially toxic gene

For more inormation on transormation and competent cell selectionplease reer to our web site: http://wwwnebcom/nebecomm/tech_reer-

ence/competent/deaultasp3 Screening or the Presence o Inserts:

A Analysis o plasmid DNA: Restriction digests with the same restrictionenzymes that were used or cloning the target gene ragment can be usedto screen or correct clones, except when SapI is used because the SapIsite is lost ater ligation. It is also possible to use other restriction sitespresent in the plasmid

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Colony PCR or colony hybridization can be used to screen a large numbero transormants or the presence o the target gene

B Analysis o protein expression: Transorm the correct plasmid constructinto competent ER2566 or T7 Express Inoculate 510 reshly growncolonies, each in 4 ml LB+Amp media Grow the culture at 37C until it

reaches an OD600 o ~05 or slightly turbid Transer 2 ml to a sterile tubeas an uninduced control

Induce protein expression with 04 mM IPTG at 37C or 23 hours or at15C overnight Mix 40 l culture with 20 l 3X SDS-PAGE Sample BuerBoil or 5 minutes and load 15 l o both uninduced and induced sampleson SDS-PAGE and stain with Coomassie blue

Immunodetection with Anti-Chitin Binding Domain Serum can be usedto detect the intein-CBD usion proteins in total cell lysates Immunode-tection is not necessary i an induced band can be easily visualized byCoomassie blue staining o a SDS-PAGE

C Sequencing: Clones should be urther conrmed by DNA sequenc-ing beore proceeding to the cell culture and protein expression stepsSequencing primers are available or the sequencing o genes insertedinto the IMPACT vectors (See Figure 3A, Appendix III and CompanionProducts)

D Storage: The plasmid encoding the correct usion protein should bestored at 20C or a glycerol stock should be made o the cells contain-ing the expression plasmid and stored at 80C

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Fui Pi epi:The expression o the usion protein may be aected by a variety o actors suchas the (a) E. colistrain, (b) cell growth conditions (eg temperature, aeration,cell density, IPTG concentration, etc), (c) toxicity o the target protein, (d) codonusage and (e) structure and stability o mRNA E. coliER2566 cells are sup-

plied in the kit (not competent) as a host or usion protein expression rom anIMPACT vector High eciency competent cells o this strain, T7 Express (NEB#C2566) are available separately For expression o toxic proteins T7 Express IqCompetent E. coli(High Eciency) (NEB #C3016), T7 Express lysY/Iq CompetentE. coli(High Eciency) (NEB #C3013) and T7 Express lysYCompetent E. coli(High Eciency) (NEB #C3010) are also available Expression o a toxic proteinmay require lowering the culture temperature

Induction o protein expression at 1215C can oten help the olding and thesolubility o the usion protein and increase the cleavage eciency o the intein

For all protein samples remove a 40 l aliquot and mix with 20 l 3X SDS Sam-ple Buer (Figure 2) The samples are then boiled or ve minutes beore loadingonto a gel I necessary, the samples can be stored at 20C or a ew days

The ollowing protocol is provided as a general guideline (see Figure 4, page 19)

C Cuu1 Inoculate 1 liter o LB medium, containing 100 g/ml ampicillin, with a

reshly grown colony Using cells stored at 4C or an overnight culture maylower the protein yield

2 Incubate the culture in an orbital shaker at 37C until the OD600

reaches 05

Iduci Pi epi3 IPTG is added to a nal concentration o 04 mM or induction o protein

expression Beore the addition o IPTG, an aliquot o cell culture shouldbe removed and incubated separately as an uninduced control (sample1, uninduced) Initially induction at 37C or 24 hours can be tested orexpression and solubility

4 Remove a sample (40 l) and mix with 20 l 3X SDS Sample Buer or thetotal cell extract or induced protein sample (sample 2, crude cell extract orinduced) The cells rom the IPTG-induced culture are spun down at 5000 xg or 15 minutes at 4C and the supernatant is discarded The cell pellet can

be stored at 80C5 The cell pellet rom a one-liter culture is resuspended in 100 ml o the ap-

propriate ice-cold Column Buer (See Media and Solutions, page 26)

The inclusion o nonionic detergents in the cell lysis buer can reducenonspecic protein binding to the chitin resin during the anity columnstep Oxidation-sensitive proteins can be stabilized during purication byusing the reducing agents TCEP [tris-(2-carboxyethyl)phosphine] or TCCP[tris-(2-cyanoethyl)phosphine] (01 mM) in the cell lysis buer Egg white

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lysozyme is not recommended or cell lysis because it is known to bindand degrade the chitin beads Cell lysis reagents, such as B-PER (ThermoScientic, Rockord, IL), can be used

6 Lyse the cells by sonication on ice

Sonicate in short pulses, keep the cell culture cold during sonication and

do not allow the build up o bubbles/oam The release o protein can bemonitored by using Bradord Reagent (BioRad, Hercules, CA) or OD

280

Continue sonication until the level o the released protein level reaches amaximum

7 Centriuge the cells at 15,000 x g or 30 minutes at 4C; the supernatantis the claried extract to be loaded on the chitin column Remove 40 lrom the supernatant or the claried protein sample (sample 3, clariedextract) Save the pellet at 80C or uture analysis

opimizai epiI the target usion protein is present as an induced band in the crude cellextract but absent rom the claried cell extract, this may indicate a solubilityproblem Also i in vivocleavage is detected (the induced sample contains theintein-CBD and target protein) dierent induction conditions should be testedUsually induction at lower temperatures and/or with lower IPTG concentra-tions results in increased solubility and improved olding and subsequent thiol

induced cleavage In order to optimize expression conditions we recommendsplitting a one liter culture into several samples (100200 ml each) and testingor optimal expression conditions The optimal incubation temperature and timeor induction will vary depending on the target protein We recommend varyinginduction temperature and time to optimize expression (37C or 24 hours,30C or 46 hours, 2225C or 616 hours and 1215C overnight using04 mM IPTG) One sample with no IPTG should be incubated as a control oruninduced cells Varying IPTG concentrations (up to 1 mM) can also be tested

Lowering the IPTG concentration (00101 mM) may also reduce the usionprotein expression in inclusion bodies For low temperature induction (eg1215C) the culture can be incubated at 37C until the OD

600reaches 0607

Fusion protein expression can be examined by SDS-PAGE, ollowed byCoomassie staining or western blot analysis, or by an activity assay Analyzesamples rom both the total cell extract (soluble and insoluble proteins) andclaried extract (soluble) I the usion protein is not detected by Coomassiestaining, a Western blot with the Anti-Chitin Binding Domain Serum may beperormed

I the cell pellet needs to be tested, dissolve the pellet rom a 100200 mlculture in 1020 ml column buer and mix 40 l o the pellet suspension with20 l o the 3X SDS Sample Buer The pellet can also be dissolved in a buercontaining urea This sample can be saved or analysis (by loading 510 l on aSDS-PAGE) i the usion protein is not detected in the claried (soluble) extract

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Afi Puifcai ad o-cum Cavag:This should be perormed with cold solutions at 4C, unless otherwise stated

Ppaai Chii CumThe chitin column should be washed with 10 column volumes o the Column

Buer prior to the loading o the crude cell extract The chitin-binding domain(CBD) present in the intein-tag, allows or the anity purication o the usionprotein using chitin beads Generally, a column packed with 10 ml o chitinbeads (10 ml bed volume or 20 ml chitin beads slurry) should be used or aone liter culture (adjust the amount o beads according to expression level)

ladig h Caifd C eacLoad the claried extract onto the chitin column at a fow rate no aster than

051 ml/min Take a sample o the fow through (sample 4) and compare it tothe claried cell extract sample to indicate the binding eciency o the usionprecursor to the chitin column I some o the usion precursor is present inthe fow through you may need to increase the amount o resin or load moreslowly

Wahig h Chii CumAt least 20 bed volumes o the Column Buer should be used to wash the

column (sample 5) Due to the high anity o the CBD or the chitin beads, ahigher fow rate (eg, 2 ml/min) and stringent wash conditions can be usedto reduce nonspecic binding o other E. coliproteins [high salt concentration(051 M NaCl) and/or nonionic detergents]

Iduci o-cum CavagTo release the target protein, on-column cleavage is induced by a thiol reagentInduction o the on-column cleavage is conducted by quickly fushing the

column with 3 bed volumes o the Cleavage Buer, containing 50 mM DTT,to evenly distribute thiols throughout the column (sample 7) Ater the quickfush, stop the column fow and leave at 423C or 1640 hours (see Tables1A and 1B) Beore adding the thiol reagent, check cleavage eciency byremoving 100 l o resin and mixing with 50 l 3X SDS Sample Buer Aterboiling or 5 minutes, spin the resin down The supernatant (310 l) isdirectly used or SDS-PAGE analyses (sample 6)

I intein mediated protein ligation (IPL) or expressed protein ligation (EPL) is

to be conducted, typically 2-mercaptoethanesulonic acid (MESNA) is used asthe thiol reagent to induce cleavage (Appendix II)

Several actors aect the cleavage eciency and thus the nal yield: (i) aminoacid residue(s) at the cleavage site; (ii) temperature o the cleavage reaction;(iii) duration o the cleavage reaction; (iv) pH o the Cleavage Buer

Since cleavage is dependent on the protein structure, a single amino acidresidue at the cleavage site is not the only determinant or ecient cleavage,

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and only serves as a guideline In most cases (see Tables 1A & 1B), incubationo the column at 1623C or 16 hours (overnight) results in more than 50%cleavage o the usion precursor When the C-terminal usion vector (pTXB1)is used, the on-column cleavage reaction can be conducted at 4C overnightWhen the N-terminal usion vector (pTYB21) is used, higher temperatures(1623C) and longer cleavage reaction times (40 hours) are normallyrequired The data in Tables 1A & 1B provide a guideline or selecting an ap-propriate temperature and duration or the cleavage reaction The cleavage e-ciency can be determined by a SDS-PAGE by analyzing samples o the chitinresin ater thiol cleavage (sample 9)

I most o the precursor is not cleaved, longer incubation time and highertemperature or the cleavage reaction are recommended

eui h tag PiFollowing on-column cleavage the target protein is eluted rom the columnusing the Column Buer The intein-CBD tag remains bound to the resin Frac-tion sizes o about one third o the column bed volume typically result in theelution o the target protein within the rst ew ractions (sample 8)

The protein concentration in each raction can be determined by the BradordAssay and the eluted ractions should be analyzed by SDS-PAGE To checkcleavage eciency, remove 100 l o resin and mix with 50 l 3X SDS SampleBuer Boil or 5 minutes and spin the resin down Analyze the supernatant(310 l, sample 9) by SDS-PAGE I a large amount o the precursor stillremains uncleaved, continue incubation o the column or an additional 1224hours beore conducting a second elution

When pTYB21 is used, a small peptide (16 kDa) is also cleaved rom theintein tag and co-eluted with the target protein Due to its small molecularweight, the cleaved peptide cannot be detected on a regular SDS-PAGE gel and

can be removed by dialysis

sippig h Chii CumUncleaved usion precursor protein and the intein-tag remain bound to the chi-tin resin during elution and can be stripped rom the resin by 1% SDS or 03 NNaOH in column buer The elution should be conducted at room temperatureto prevent the precipitation o the SDS Since protein concentrations cannotbe determined by the Bradord dye binding assay when SDS is present, the

samples should be analyzed by SDS-PAGE

rgai h Chii riThe chitin resin can be regenerated 45 times using the ollowing protocolWash with 3 bed volumes o 03 M NaOH (stripping solution) Allow the resinto soak or 30 minutes and then wash with an additional 7 bed volumes oStripping Solution Rinse with 20 bed volumes o water ollowed by 5 bed vol-umes o Column Buer The resin can be stored at 4C For long term storage

002% sodium azide should be added to the Column Buer

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simpifd epi ad Puifcai Pc:1 Transormation: Transorm the plasmid bearing the target gene into

competent T7 Express or competent cells prepared rom ER2566

2 Cell Culture: Inoculate a reshly grown colony in LB medium containing

100 g/ml ampicillin and grow the cells at 37C When the OD600 reaches05, induce protein expression by adding IPTG to a nal concentration o04 mM, and incubate at 3037C

3 Column Preparation: Equilibrate a chitin column (20 ml slurry or 1 literculture) with 10 column volumes o Column Buer [20 Tris-HCl (pH 85),500 mM NaCl]

4 Cell Harvest: Centriuge cell culture at 5,000 x g or 15 minutes at 4CDiscard supernatant Resuspend cell pellet in column buer

5 Loading: Break cells by sonication in Column Buer, and centriuge at15,000 g or 30 minutes at 4C Slowly load the claried lysate onto thechitin column (0510 ml/minute)

6 Washing: Wash the column with at least 20 bed volumes o ColumnBuer to thoroughly remove the unbound proteins (up to 20 ml/minute)

7 Adding Thiols: Quickly wash the column with 3 column volumes oCleavage Buer [Column buer containing 50 mM DTT (or purication)

or 50 mM MESNA (or IPL)]

8 On-column Cleavage: Stop the fow and incubate the column at 4C23Cor 1640 hours The temperature and duration o the cleavage reactionare dependent on the on-column cleavage eciency which can bechecked by analyzing samples o chitin resin beore and ater cleavage

9 Elution: Elute the target protein with Column Buer by continuing thecolumn fow

10 Dialysis: Dialyze the target protein in to an appropriate storage buer; thiswill also remove the excess thiol reagent used in the Cleavage Buer andthe co-eluted small peptide (when using pTYB21)

11 Cleavage: To examine cleavage eciency remove 100 l o chitin resinand mix with 50 l o 3X SDS Sample Buer Ater boiling or 5 minutes,analyze the supernatant on a Coomassie stained SDS-PAGE gel to deter-mine the cleavage eciency

12 Regeneration o Chitin Resin: Wash the column with 3 bed volumeso the 03 M NaOH (Stripping Solution) Allow the resin to soak or 30minutes and wash the resin with an additional 7 bed volumes o 03 MNaOH Wash with 20 bed volumes o water, ollowed by 5 bed volumes ocolumn buer

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Flush columnwith Cleavage Buffer, stop flow and

leave at 4C overnight

Harvest and lyse cells. Load clarifiedcell extract onto chitin column

Wash column with Column Buffer

Elute column with additionalcolumn buffer

Analyze protein samples onSDS polyacrylamide gel

Determine cleavage efficiency

Induce with IPTG

Grow Cells

Sample 1: uninduced sample

Sample 2: crude cell extract

Sample 3: clarified extract

Sample 4: flow through (to determine binding efficiency)

Sample 7: determine cleavage during DTT wash

Sample 8: target protein

Sample 9: chitin beads

Sample 5: wash

Sample 6: chitin beads: before cleavage

Figu 4: Fw cha Pi epi ad Puifcai uig

h IMPACt sm. samp cci aai b sDs-PAGe i

idicad.

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Mdia ad sui:The ollowing are suggested media or cell culture, cell lysis and proteinpurication They can be modied according to the specic properties o thetarget protein

LB broth (per liter)

10 g tryptone

5 g yeast extract

10 g NaCl

Adjust pH to 70 with NaOH

Cell Lysis Buer

20 mM Na-HEPES (or Tris-HCl), pH 85

500 mM NaCl (or 501,000 mM NaCl)

1 mM EDTA (optional)

01% Triton X-100 (optional)

Nonionic detergents (0105% Triton X-100 or 0102% Tween 20) and protease inhibitors[eg, PMSF (20 M)] can also be included For a target protein sensitive to oxidation, 1 mM oTCEP [tris-(2-carboxyethyl)phosphine] or TCCP [tris-(2-cyanoethyl)phosphine] may be used

Column Buer20 mM Na-HEPES (or Tris-HCl), pH 85



Cleavage Buer20 mM Na-HEPES (or Tris-HCl), pH 85


50 mM DTT or -mercaptoethanol or cysteine*


*(use 2-mercaptoethanesulonic acid and HEPES buer or IPL see Appendix Il, page 27)

Stripping Solution03 M NaOH

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Fqu Akd Qui (FAQ):CigWhich vector should I use?

We routinely use pTXB1 with success The mini-intein o bacterial origin

usually allows or higher level o expression; however, we have not conducteda systematic scientic comparison between pTXB1 and the other IMPACTvectors

pTXB1 allows or the usion o the C-terminus o a target protein to the inteintag whereas with the pTYB21 vector, the N-terminus o the target protein isused to the intein tag It is conceivable that dierent target proteins, due tocertain structural constraints, may preer either a C-terminal or a N-terminalusion to allow proper olding o the usion precursor and or a high level oprotein expression

Check the "Guide to IMPACT Vectors and Applications" in our catalog and onour web site (Section 135 o the IMPACT FAQs on the web site) Since inteinsexhibit dierent preerences or the amino acid at the cleavage site, the usershould ollow guidelines or choosing a vector based on the inormation givenin Tables 1A and 1B These tables are only a guide, since the olding o theentire usion protein, not just a single amino acid, aects cleavage Since each

usion protein varies in expression level and cleavage we oten clone the targetprotein into more than one IMPACT vector

The choice o vector will also depend on the restriction map o the gene; thepresence o an internal NdeI or SapI site will aect the selection o the cloningvector

I the puried target protein is also intended or intein mediated protein liga-tion (IPL), C-terminal labeling or peptide ligation, pTXB1 should be used

I you wish to generate a target protein with a N-terminus other than methi-onine, you may use pTYB21, pTYB21 or the Intein 1 (Ssp DnaB intein) inthe pTWIN1 (NEB #N6951) or pTWIN2 (NEB #N6952) vector A N-terminalcysteine can be generated or IPL using Intein 1 (Ssp DnaB intein) in thepTWIN vectors

How do I design primers with the SapI site?

SapI (or its isoschizomer, BspQI) is a Type IIs enzyme; it cuts outside its rec-ognition sequence to give a staggered cut To design the primer please reer toTable 2, write out the cloning procedure and it will be apparent how compat-ible ends between the vector and insert are generated Further inormation isgiven in the IMPACT FAQ section on our web site

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epiWhat if I observe in vivo cleavage, where a band corresponding tointein-CBD but not the fusion protein, is detected in the crude cellextract?

First, to accurately assess the extent o in vivocleavage, the saples should beprepared in SDS Sample Buer without DTT or -mercaptoethanol, since boilingin DTT-containing Sample Buer may cause cleavage o the usion protein (seenote in Table 1A) I proteolysis is evident, try dierent hosts or include proteaseinhibitors In vivocleavage may be reduced by varying induction temperature Inecessary, perorm a Western blot with anti-target protein serum to dierentiatebetween proteolysis and intein-mediated cleavage Sometimes changing one orseveral residues between the target protein and the intein tag may reduce intein-mediated in vivocleavage However, the puried target protein will contain extraresidues ater cleavage

What if the fusion protein is insoluble?

I you have problems with solubility one o the rst things to try is varying theinduction temperature (15C or lower; some customers have used 812Cinduction temperatures) and/or the concentration o IPTG (001 mM04 mM)

A resh colony rom an overnight plate, maybe necessary or optimal expression

Resuspend the cell pellet in at least 100 ml Column Buer/L culture

During sonication, it is crucial to handle the sample cautiously I the proteinsolution gets warm or the solution oams there is a possibility that the proteinsare being denatured Use an ice-chilled water bath to keep the cell suspensioncool

I you wish to use a detergent in your Column and Cleavage Buers, werecommend 0105% Triton X-100 or 0102% Tween-20 Too muchdetergent will impair binding to the chitin column

I the usion is still insoluble you can use urea to resuspend the proteinI urea is used, several actors should be considered:

1 The binding eciency o the intein-tag to the chitin resin is lower at 4 M urea orhigher

2 The intein-mediated cleavage reaction should be carried out in 02 M urea

The ollowing reolding protocol has been successully applied to the recovery oinsoluble proteins

1 Resuspend the cell pellet rom 1 L o E. coliculture in 100 ml Cell Lysis Buer

2 Break cells by sonication

3 Spin down cell debris containing the inclusion bodies at 15,000 g at 4C or 30minutes

4 Pour out supernatant and resuspend pellet in 100 ml Breaking Buer

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5 Stir solution or 1 hour at 4C

6 Spin remaining cell debris down at 15,000 g and 4C or 30 minutes

7 Load supernatant into dialysis bag and dialyze against Renaturation Buer A, B,C, D and 2 times E Each step is against 1 L o a renaturation buer and shouldtake at least 3 hours at 4C During dialysis the buer should be continuously

stirred8 Centriuge the dialyzed solution containing the renatured protein at

15,000 g and 4C or 30 minutes to remove any remaining impurities orincorrectly olded protein which is again aggregated

9 Use a standard protocol or chitin chromatography and cleavage reaction Elutethe protein product and analyze both the eluate and chitin beads or cleavageeciency and protein solubility

suiCell Lysis Buer: 20 mM Tris-HCl, pH 85 and 05 M NaClBreaking Buer: 20 mM Tris-HCl, pH 85, 05 M NaCl, 7M Guanidine-HCl

Renaturation Buer A: 20 mM Tris-HCl, pH 85, 05 M NaCl, 8 M urea

Renaturation Buer B: 20 mM Tris-HCl, pH 85, 05 M NaCl, 6 M urea

Renaturation Buer C: 20 mM Tris-HCl, pH 85, 05 M NaCl, 4 M urea

Renaturation Buer D: 20 mM Tris-HCl, pH 85, 05 M NaCl, 2 M urea,01 mM oxidized glutathione, 1 mM reduced glutathione

Renaturation Buer E: 20 mM Tris-HCl, pH 85, 05 M NaCl, 01 mM oxidizedglutathione, 1 mM reduced glutathione

CavagWhat should I do if the fusion precursor is the major product on thechitin resin after target protein elution?

This means that the thiol-induced on-column cleavage is not ecient invariablyleading to a low yield o the target protein The ollowing options can be tried toincrease the cleavage eciency: (i) increase the duration o the on-column cleav-age (ii) increase the temperature rom 4C to room temperature (iii) increase thepH o the Cleavage Buer to 8590 (iv) change the residue(s) adjacent to theintein cleavage site

What does it mean if the target protein is not eluted after on-columncleavage but is present on the chitin beads after target proteinelution?

I both the target protein and the intein tag are present on the chitin beads aterelution, it suggests that the target protein becomes insoluble ater inducedon-column cleavage Increase the salt concentration (052 M NaCl) or add anonionic detergent to the Cleavage Buer to improve the solubility o the targetprotein A number o nonionic detergents examined (0105% Triton X-100 or0102% Tween 20) had little eect on binding or cleavage and may improvesolubility I urea is used to elute, some intein tag may co-elute with the targetprotein In this case, it may be necessary to repuriy and reold the target protein

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If my target protein is sensitive to DTT, are there alternative meansto induce the on-column cleavage?

I the activity o the target protein is aected by high concentrations o DTTor -mercaptoethanol, lower concentrations o DTT or -mercaptoethanol(510 mM) may be used or on-column cleavage However, longer incuba-

tion time or higher temperatures (up to room temperature) may be requiredor ecient cleavage Alternatively, 50 mM o reshly prepared hydroxylamine(or pTXB1 at pH 6) or cysteine solution (at pH 89) can be used to inducecleavage at 425C Be aware that when hydroxylamine or cysteine is usedwith C-terminal IMPACT vectors (pTXB1), they orm a stable covalent bondwith the C-terminus o the target protein One should determine whether aC-terminal hydroxamate or cysteine aects the activity o the target proteinWhen cysteine is used or cleavage with pTYB21, the cysteine is not attached

to the target protein

I you do not wish to use a thiol reagent or cleavage, the Intein1 (Ssp DnaBintein) in the pTWIN vectors may be used; cleavage is induced by temperature(room temperature) and/or pH (rom pH 85 to 6)

How do I remove DTT after cleavage?

Ater elution o the target protein, ree DTT can be removed rom the sampleby dialyzing at least twice against an appropriate buer (at pH 89)

For more FREQUENTLY ASKED QUESTIONS (FAQs) please reer to the web site www.neb.com Technical Reerence

Gene Expression and Purication Systems FAQs or IMPACT Kit.

rc:1 Chong, S, Mersha, FB, Comb, DG, Scott, ME, Landry, D, Vence, LM, Perler,

FB, Benner, J, Kucera, RB, Hirvonen, CA, Pelletier, JJ, Paulus, H, and Xu, M-Q(1997) Single-column purication o ree recombinant proteins using a sel-cleav-

able anity tag derived rom a protein splicing element Gene, 192, 2772812 Watanabe, T, Ito, Y, Yamada, T, Hashimoto, M, Sekine, S, and Tanaka, H (1994)

The role o the C-terminal domain and type III domains o chitinase A1 rom BacilluscirculansWL-12 in chitin degradation J. Bacteriol., 176, 44654472

3 Evans, TC, Benner, J, and Xu, M-Q (1998) Semisynthesis o cytotoxic proteinsusing a modied protein splicing element Protein Sci, 7, 22562264 (pTXB1; IPL)

4 Chong, S, Montello, GE, Zhang, A, Cantor, EJ, Liao, W, Xu, M -Q, and Benner,J (1998) Utilizing the C-terminal cleavage activity o a protein splicing element to

puriy recombinant proteins in a single chromatographic step Nucl. Acids Res, 26,51095115 (pTYB11)

5 Southworth, MW, Amaya, K, Evans, J, TC, Xu, M-Q and Perler, FB (1999)BioTechniques, 27, 110120

6 Chong, S, Williams, KS, Wotkowicz, C, and Xu, M-Q (1998) Modulation oprotein splicing o the Saccharomyces cerevisiaevacuolar membrane ATPase inteinJ. Biol. Chem., 273, 1056710577

7 Muir, TW, Sondhi, D and Cole, PA (1998) Proc. Natl. Acad. Sci. USA 95,67056710

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8 Dubendor, JW and Studier, FW (1991) Controlling basal expression in an induc-ible T7 expression system by blocking the target T7 promoter with lacrepressor J.Mol. Biol., 219, 4559

9 Peroza, EA and Freisinger, E (2008) Tris is a non-innocent buer during intein-mediated protein cleavage Protein Expr. Purif. 57, 217225

IMPACt U Pubicai:1 Grant, JE, Guo, LW, Vestling, MM, Martemyanov, KA, Arshavsky, VY, and Ruo-

ho, AE (2006) The N terminus o GTP gamma S-activated transducin alpha-subunitinteracts with the C terminus o the cGMP phosphodiesterase gamma-subunit J.Biol. Chem., 281, 61946202

2 Hemelaar, J, Borodovsky, A, Kessler, BM, Reverter, D, Cook, J, Kolli, N, Gan-Erdene, T, Wilkinson, KD, Gill, G, Lima, CD, Ploegh, HL and Ovaa, H (2004)

Specic and covalent targeting o conjugating and deconjugating enzymes oubiquitin-like proteins Mol. Cell Biol., 24, 8495

3 Morassutti, C, De Amicis, F, Bandiera, A and Marchetti, S (2005) Expressiono SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated systemProtein Expr. Purif., 39, 160168

4 Pezza, JA, Allen, KN, and Tolan, DR (2004) Intein-mediated purication o arecombinantly expressed peptide Chem. Commun., 24122413

5 Salazar, JC, Ahel, I, Orellana, O, Tumbula-Hansen, D, Krieger, R, Daniels, L,

and Soll, D (2003) Coevolution o an aminoacyl-tRNA synthetase with its tRNAsubstrates Proc. Natl. Acad. Sci. USA, 100, 1386313868

6 Vitali, F, Henning, A, Oberstrass, FC, Hargous, Y, Auweter, SD, Erat, M, and Al-lain, FH (2006) Structure o the two most C-terminal RNA recognition motis o PTBusing segmental isotope labeling EMBO J., 25, 150162

7 Wojtaszek, J, Kolaczkowska, A, Kowalska, J, Nowak, K and Wilusz, T (2006) LTCI,a novel chymotrypsin inhibitor o the potato I amily rom the earthworm Lumbricusterrestris Purication, cDNA cloning, and expression Comp. Biochem. Physiol B.

Biochem. Mol. Biol., 143, 465472For more reerences rom IMPACT Users please go to the Section 111 in the IMPACTFAQs

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Appdi I: Cig i pMxB10The control vector pMXB10 encodes the usion protein maltose binding pro-tein (MBP) Mxe GyrA intein Chitin Binding Domain (CBD) The polylinkerregions fanking the coding region or MBP can conveniently be used to clonea gene o interest (Figure 5) The 5 end o the target gene can be cloned

into the NdeI, SapI or NcoI site I the SapI or NcoI sites are used the primershould be designed so that the gene is in rame with the translational start inthe NdeI site For cloning the 3 end o the gene the SacI, HindIII, NotI, EcoRIor XhoI site can be used; make sure that the target protein is in rame with theintein However, ater intein cleavage the target protein will contain additionalamino acids at its C-terminus, including (LEY), which has been shown tohave a high rate o successul cleavage For the 3 site you may also use theSpeI site, present near the N-terminus o the Mxe GyrA intein, by designing

a primer that contains the intein sequence; this can result in a usion withoutany vector-derived residues ollowing cleavage

T7 Promoter

Intein3 MCS

CBDpMXB10 MBP5 MCS

Target Gene

CAT ATG GGA AGA GCC ATG GATGTA TAC CCT TCT CGG TAC CTA

Translation Start

5 MCS Region:

NdeI SapI NcoI

G AGC TCG AAG CTT GGC GGC CGC GAA TTC CTC GAG TAC TGCC TCG AGC TTC GAA CCG CCG GCG CTT AAG GAG CTC ATG ACG

Intein3 MCS Region:

SacI HindIII NotI EcoRI XhoI

Figu 5: pMxB10 vc ad muip cig i.

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Appdi II: Ii-mdiad Pi ligai(IPl) ad Pi labigThe IPL reaction, also reerred to as expressed protein ligation, allows theligation o a bacterially expressed protein or a synthetic peptide with anN-terminal cysteine residue to a protein with a C-terminal thioester througha native peptide bond (Figure 6; 3,7) In addition to protein purication,pTXB, and other C-terminal usion vectors (pTYB and pTWIN series), canbe used to generate a protein with a C-terminal thioester or IPL Typically2-mercaptoethanesulonic acid (MESNA) is used as the thiol reagent to induceintein-mediated cleavage; this produces a C-terminal thioester on the targetprotein The C-terminus o the target protein can then be covalently labeled orligated to a synthetic peptide with an N-terminal cysteine

The ollowing protocol illustrates a typical ligation or labeling experiment:

1 One o the components should have a nal concentration o at least051 mM For the ligation o a peptide to a protein we use 001 mMprotein with 051 mM peptide

2 Combine the components in the presence o 01 M Tris, pH 85 and10 mM MESNA and incubate overnight at 4C Alternatively, the reactioncan be incubated at 25C or 14 hours

3 The ligation may be visualized by a 10% or 12% SDS-PAGE as a shit inmobility o the ligated protein, or can easily be detected by Western blotusing an antibody specic or the peptide Though a puried peptide isnot required, it can usually yield a higher ligation eciency (75%90%)Add 20 l o 3X SDS Sample Buer (with DTT) to 40 l o the proteinsample Boil or 5 minutes and analyze by SDS-PAGE, with unligatedprotein as a control

To label a protein with biotin, a peptide with an N-terminal cysteine and abiotinylated residue can be used The biotinylated protein sample can beanalyzed by SDS-PAGE and detected by Western blot with anti-biotin antibodyCurrently NEB has 2 peptides with N-terminal cysteines or labeling: onecontains a fuorescein group (Flu-P1; NEB #P6606S) and the other a biotin(Bio-P1; NEB #P6607)

For cleavage, HEPES buer should be used (9) For long term storage oMESNA-tagged proteins, dialyze the protein into 5 mM Bis Tris, pH 65,

250 mM NaCl and store at 80C

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1: N-S Shift

2: Thiol MediatedCleavage

3: Peptide Attack

4: S-N Shift

Target ProteinCysO

NH

SH

Chitin

CBD

Intein2

Cys

Intein2

3 2 2SO -CH -CH -SH-

S

O

NH2

Target Protein

S

Cys

NH2

O

Target Protein Peptide

N

SH

Cys

H

O

Target Protein Peptide

O

Target ProteinS-CH -CH -SO32

-2

Cys

SH

NH2

Peptide

Figu 6: Mchaim ii-mdiad

pi igai (IPl).

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Appdi III: squcig PimThere are a variety o sequencing primers available or checking the target geneollowing its insertion into an IMPACT vector The annealing position and direc-tion o sequencing or each primer are shown in Figure 7 For urther detailsplease reer to the web site wwwnebcom

#S1285S

#S1271S

#S1271S

CBDInteinMBP

#S1248S

5 MCS

pMXB103 MCS

Intein

#S1248S

MCSpTYB21

#S1263S

CBD

#S1248S

InteinMCS CBD

#S1285S

pTXB1

T7 Promoter

Figu 7: IMPACt Ki vc ad qucig pim.

Appdi IV: rach U Auac samThe buyer and user have a non-exclusive sublicense to use this system or anycomponent thereo or RESEARCH PURPOSES ONLY, based upon agreement tothe ollowing assurances

Transer o the host cells that contain the cloned copy o the T7 gene 1 to thirdparties is explicitly prohibited This limitation applies to E. colistrain ER2566which is provided in combination with the IMPACT system or in combinationwith appropriate vectors or said system

A license to use this system or any components thereo or commercial pur-poses may be obtained rom New England Biolabs, Inc

Commercial Laboratory Buyer and User: Use o the host cells that contain thecloned copy o T7 gene 1, the gene or T7 RNA polymerase or any purposeother than in combination with the IMPACT system is explicitly prohibited

Use o the host cells that contain the copy o the T7 gene 1, the gene or T7RNA polymerase with any other vector(s) containing a T7 promoter to direct the

production o RNA or protein requires a license rom Brookhaven National Labo-ratory Inormation about research-use or commercial-use license agreementsmay be obtained rom the Oce o Technology Transer, Brookhaven NationalLaboratory, Building 475D, PO Box 5000, Upton, New York, 11973-5000;telephone: 631-344-7134, ax: 631-344-3729

You may reuse this non-exclusive research license agreement by returningthe enclosed materials unused. By keeping or using the enclosed materials,you agree to be bound by the terms o this sub-license.

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product catalog # siZe

IMPACt Ki e6901s

Kit components sold separately

ptxB1 Vc n6707s 10 g

ptyB21 Vc n6709s 10 g

pMxB10 C Pamid n6903s 10 g

Chii Bad s6651s/l 20/100 m

Ai-Chii Bidig Dmai sum s6654s 0.05 m

Bu ladig Bu Pack B7703s 8 m

companion products

t7 ep Cmp E. coli(High eicic)

C2566H 20 ub

neB 10-ba Cmp E. coli(High eicic)

C3019H 20 ub

neB tub Cmp E. coli(High eicic)

C2984H 20 ub





ptxB3 Vc n6708s 10 g



pKyB1 Vc n6706s 10 g

ptWIn1 Vc n6951s 10 g

ptWIn2 Vc n6952s 10 g

ptWIn-MBP1 Vc n6953s 10 g

t7 Uiva Pim (20-m) s1248s 0.5A260

ui

Ii rv Pim (24-m) s1261s 0.5A260 ui

Ii Fwad Pim (24-m) s1263s 0.5A260

ui

t7 tmia rv Pim (19-m) s1271s 0.5A260

ui

MxeIi rv II Pim s1285s 0.5A260

ui

SspDaB Ii Fwad Pim s1269s 0.5A260

ui

odig Imai

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Notice to Buyer/User:The Buyer/User has a non-exclusive license or the IMPACT Kit or any components thereo orRESEARCH PURPOSES ONLY Inormation presented herein is accurate and reliable to the best o our knowledge andbelie, but is not guaranteed to be so Nothing herein is to be construed as recommending any practice or any productin violation o any patent or violation o any law or regulation It is the users responsibility to determine or himselor hersel the suitability o any material and/or procedure or a specic purpose and to adopt such saety precautionsas may be necessary The Buyer and User have a non-exclusive sub-license tot use this system or any componentthereo or RESEARCH PURPOSES ONLY, based upon agreement to the ollowing assurances Transer o the hostcells that contain the cloned copy o the T7 gene 1 to third parties is explicitly prohibited This limitation applies toE. colistrain ER2566 which is provided in combination with IMPACT Kit or in combination with appropriate vectorsor said system A license to use this system or any components thereo or commercial purposes may be obtainedrom New England Biolabs, Inc: Commercial Laboratory Buyer/User: Use o the host cells that contain the cloned

copy o T7 gene 1, the gene or T7 RNA polymerase or any purpose other than in combination with the IMPACT

Kit is explicitly prohibited Use o the host cells that contain the copy o T7 gene 1, the gene or T7 RNA polymerasewith any other vector(s) containing a T7 promoter to direct the production o RNA or protein requires a license romBrookhaven National Laboratory Inormation about research-use or commercial-use license agreements may be ob-tained rom the Oce o Technology Transer, Brookhaven National Laboratory, Building 475D, PO Box 5000, Upton,New York 11973-5000; Telephone: 631-344-7134, Fax: 631-344-3729

product catalog # siZe

MthrIr1 Ii rv Pim s1270s 0.5A260

ui

Fu-P1 Ppid P6606s 0.29 mg

Bi-P1 Ppid P6607s 0.29 mg

CGC 549 Ppid P6013s 10 m

CGC 649 Ppid P6014s 10 m

Quick ligai Ki M2200s/l30/150aci

Quick Buig Ki e1201s/l20/100aci

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USA

nw egad Biab, Ic.

240 Cu rad

Ipwich, MA 01938-2723

tph: (978) 927-5054

t F: (UsA od) 1-800-632-5227

t F: (UsA tch) 1-800-632-7799

Fa: (978) 921-1350

-mai: [email protected]

www.b.cm

Canadanw egad Biab, ld.t F: 1-800-387-1095Fa: (905) 837-2994Fa t F: 1-800-563-3789-mai: [email protected]

China, Peoples Republicnw egad Biab (Bijig), ld.tph: 010-82378265/82378266Fa: 010-82378262-mai: [email protected]

Germanynw egad Biab GmbHtph: +49/(0)69/305 23140F Ca: 0800/246 5227 (Gma)Fa +49/(0)69/305 23149F Fa: 0800/246 5229 (Gma)-mai: [email protected]

Japan

nw egad Biab Japa, ld.tph: +81 (0)3 5669 6192-mai: [email protected]

United Kingdom

nw egad Biab (UK), ld.tph: (01462) 420616Ca F: 0800 318486Fa: (01462) 421057Fa F: 08-- 435682

-mai: [email protected]

cloning & mapping

dna amplification & pcr

rna analysis

protein expression & analysis

gene expression & cellular analysis

manual e 6901

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