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Protein Expression and Purification 15, 389–400 (1999)Article ID prep.1999.1038, available online at http://www.idealibrary.com on

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emoval of N-Terminal Polyhistidine Tags from Recombinantroteins Using Engineered Aminopeptidases

ohn Pedersen,1 Conni Lauritzen, Mads Thorup Madsen,2 and Søren Weis DahlNIZYME Laboratories, Dr. Neergaards Vej 17, DK-2970 Hørsholm, Denmark

eceived December 7, 1998, and in revised form January 19, 1999

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We have developed a specific and efficient method foromplete removal of polyhistidine purification tagsHisTags) from the N-termini of target proteins. Theethod is based on the use of the aminopeptidase dipep-

idyl peptidase I (DPPI), either alone or in combinationith glutamine cyclotransferase (GCT) and pyroglu-

amyl aminopeptidase (PGAP). In both cases, the HisTags cleaved off by DPPI, which catalyzes a stepwise exci-ion of a wide range of dipeptides from the N-terminus ofpeptide chain. Some sequences, however, are resistant

o DPPI cleavage and a number of mature proteins haveonsubstrate N-termini which protects them against di-estion. For such proteins, HisTags composed of an evenumber of residues can be cleaved off by treatment withPPI alone. When the target protein is unprotectedgainst DPPI, a blocking group is generated enzymati-ally from a glutamine residue inserted between theisTag and the target protein. A protein with a HisTag-ln extension is incubated with both DPPI and GCT. Asbove, the polyhistidine sequence is cleaved off by DPPI,ut when the glutamine residue appears in the N-erminus, it is immediately converted into a pyroglu-amyl residue by an excess of GCT and further DPPIigestion is prevented. The desired sequence is finallybtained by excision of the pyroglutamyl residue withGAP. All the enzymes employed can bind to immobi-

ized metal affinity chromatography (IMAC) matrices,nd in this paper we demonstrate a simple and highlyffective process combining IMAC purification of His-agged proteins, our aminopeptidase-based method forpecific excision of HisTags and use of subtractive IMACor removing processing enzymes. Typical recoveriesere 75–90% for the enzymatic processing and subtrac-

ive IMAC. The integrated process holds promises forse in large-scale production of pharmaceutical pro-eins because of a simple overall design, use of robust

1 To whom correspondence should be addressed. Fax: 1455761407. E-mail: JP@unizyme.dk.

2 Present address: Danisco Biotechnology, Langebrogade 1, DK-

411 Copenhagen K, Denmark. a

046-5928/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

nd inexpensive matrices, and use of enzymes of eitherecombinant or plant origin. © 1999 Academic Press

Key Words: polyhistidine tags; cleavage; immobilizedetal affinity chromatography; dipeptidyl peptidase I;

rotein purification.

During the past few years, a number of purification tagystems have been developed to facilitate and standard-ze purification of recombinant proteins. In these sys-ems, a terminal polypeptide or protein tag with bindingpecificity suitable for affinity purification is fused to therotein of interest, most frequently to the N-terminus1–10). The addition of a histidine-rich peptide tag (poly-istidine tag; HisTag) to the target protein is a simplend well-established approach for generating a novel af-nity for metal ions, making one-step purifications pos-ible by using immobilized metal affinity chromatogra-hy (IMAC).3 IMAC matrices have a number ofdvantages including high protein-binding capacity andigand stability, low cost, and use of mild elution condi-ions. Furthermore, because of their chemical nature,MAC matrices can easily be sanitized and regenerated,aking them suitable for large-scale applications.The HisTag needs not be removed for some applica-

ions of the purified proteins. However, when the re-ombinant protein is intended for structural/physiolog-cal studies or pharmaceutical use, the HisTag must beemoved to obtain the protein with the correct aminocid sequence, thus avoiding unpredictable properties.ne way of removing HisTags is by cleavage with

hemical reagents. This method has several drawbacksncluding the use of protein-destroying conditions and

3 Abbreviations used: IMAC, immobilized metal affinity chroma-ography; DPPI, dipeptidyl peptidase I; GCT, glutamine cyclotrans-erase; PGAP, pyroglutamyl aminopeptidase; HT-, HisTag-; HP-,istidine patch; hIL-1b, human interleukin 1b; MBP, maltose bind-

ng protein; hGH, human growth hormone; hTNFa, human tumorecrosis factor a; pyroGlu, pyroglutamyl; IPTG, isopropyl-b-D-thio-alactopyranoside; SDS–PAGE, sodium dodecyl sulfate–polyacryl-

mide gel electrophoresis.

389

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390 PEDERSEN ET AL.

oxic chemicals (1, 4). Another approach is to applyndoproteases that mainly recognize specific aminocid sequences, e.g., coagulation factor Xa, thrombin,nd enterokinase (1, 3, 4, 9–12). These enzymes, how-ver, are often ineffective because their cleavage ratesre affected by the accessibility of the cleavage site andhe amino acid residues adjacent to it (9, 11, 12). Fur-hermore, the use of endoproteases may result in un-anted internal cleavage of the protein because of theresence of internal noncanonical cleavage sites.To overcome these problems, we have developed a

pecific method for removal of N-terminal HisTags byhe use of exopeptidases. The method is based on these of dipeptidyl peptidase I (DPPI; dipeptidyl amino-eptidase I; cathepsin C; EC 3.4.14.1) (13, 14) whichatalyzes a stepwise cleavage of dipeptides from the-terminus unless (a) the amino group of the N-termi-us is blocked, (b) the site of cleavage is on either sidef a proline, or (c) the N-terminal residue is eitherysine or arginine. Studies have previously demon-trated the efficiency with which DPPI removes certain-terminal extensions from proteins that have natural

top points for DPPI in the N-termini of their nativeorms (9, 15–17). Here we show how DPPI alone or inombination with glutamine cyclotransferase (GCT;C 2.3.2.5)(18) and pyroglutamyl aminopeptidase

PGAP; EC 3.4.19.3)(19) allows efficient and completeemoval of N-terminal HisTags from proteins irrespec-ive of their N-terminal amino acid sequence. All thenzymes employed bind to IMAC matrices which haveeen utilized in the design of a simple process for thexcision of HisTags.

ATERIALS AND METHODS

aterials

Cysteamine–HCl, Gly-Phe-p-nitroanilide, chickenystatin, trans-epoxysuccinyl-L-leucylamido-(4-gua-idino)butane (E-64), benzonase, and dried papaya

atex were obtained from Sigma. Pyroglutamine-p-itroanilide and Gln-Trp-Glu were from Bachem, Swit-erland. Sephadex G-25 F, CM-Sepharose FF, Chelate-epharose FF, Sephacryl S-300 HR, phenyl–SepharoseF, HisTrap, NAP 5, NAP 10, Mono S HR 5/5, andono Q HR 5/5 were from Amersham Pharmacia Bio-

ech. Glucagon was obtained from Novo Nordiskarmaka (Bagsvaerd, Denmark).

onstruction of Escherichia coli Expression Vectors

Expression vectors for all the his-tagged proteins wereonstructed essentially as follows. The protein encodingequences were amplified by PCR (polymerase chain re-ction) using plasmid DNA as template. Plasmids carry-ng nucleotide sequences encoding hGH and hIL-1b wereindly provided by Novo Nordisk. The plasmid (BBG18)

ncoding hTNFa was from R&D Systems, the pMAL-c u

lasmid carrying the MBP sequence was from New En-land Biolabs, and the PGAP sequence was obtained onlasmid pBPG1 (19). Oligonucleotide primers were de-igned to introduce the N-terminal histidine tag encodingequences in frame with the target sequences, and bothhe upstream and the downstream primers included ap-ropriate restriction sites to facilitate cloning of the prod-cts into the pTrcHisA vector (Invitrogen). A 63 His-tag-ncoding sequence already present on the commercialector was deleted.

xpression of His-Tagged Proteins (HT Protein)in E. coli

E. coli strain TOP10 (Invitrogen) transformed withach of the expression vectors was cultivated in a SOBedium (HT15-hGH, HT15-hIL-1b, HT15-MBP, andT(n)-hTNFa) or a 23 YT medium (HT-PGAP) supple-ented with 50 mg/ml ampicillin. Volumes of 500–600l in 2-liter shake flasks were grown at 30°C (HT15-hGH

nd HT15-hIL-1b) or 37°C (HT15-MBP, HT(n)-hTNFand HT-PGAP), and gene expression was induced byddition of IPTG to 0.1 mM (HT15-hGH and HT15-hIL-b) or 0.5 mM (HT15-MBP, HT(n)-hTNFa, and HT-GAP) at OD600 5 0.2 (HT15-hGH and HT15-hIL-1b) orD600 5 0.4–0.6 (HT15-MBP, HT(n)-hTNFa, and HT-GAP). Cells were harvested after 4–5 h (HT15-MBPnd HT(n)-hTNFa), 6–7 h (HT15-hGH and HT15-hIL-b), or 22 h (HT-PGAP) of induction.

urification of HT Proteins

The following purification steps were performed at°C. Cells from approximately 1.2 liters of culture werearvested by centrifugation and resuspended in 80 ml

ysis buffer (25 mM Tris–HCl, 300 mM NaCl, pH 8.0).ysis was performed by a freeze/thaw cycle and subse-uent treatment with lysozyme (25 mg/ml) and benzo-ase (48 units/ml) for 1 h. After centrifugation theupernatant was loaded onto a Ni21-chelate–SepharoseF column (2 cm2 3 6 cm) equilibrated with lysisuffer. The column was then washed with lysis bufferollowed by buffer A (25 mM Bis–Tris–HCl, 300 mMaCl, 10% glycerol, pH 6.0). HT protein was eluted bylinear gradient (85 ml) from buffer A to buffer B

buffer A including 1 M imidazole) at a flow rate of 1l/min. Fractions containing the desired protein were

ooled. HT15-MBP, HT15-hIL-1b, HT15-hGH, andT(n)-hTNFa were stored at 218°C in 20 mM sodiumhosphate, 150 mM NaCl, pH 7.0. Purified HT-PGAPas stored at 218°C in 6 mM Tris–HCl, 40 mM NaCl,mM EDTA, 2 mM cysteamine, 50% glycerol, pH 8.0.

ssays for DPPI, GCT, and PGAP

For all enzymes, one unit is defined as the amount ofnzyme which converts 1 mmol of substrate per minute

nder the conditions used. DPPI (0.01–0.025 units)

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391AMINOPEPTIDASE REMOVAL OF N-TERMINAL POLYHISTIDINE TAGS

as assayed at 37°C in 20 mM citric acid, 150 mMaCl, 1 mM EDTA, 5 mM cysteamine, pH 4.5, contain-

ng 4 mM Gly-Phe-p-nitroanilide as substrate (20).GAP (0.01–0.025 units) was assayed at 37°C in 20M Tris–HCl, 100 mM NaCl, 5 mM EDTA, 5 mM

ysteamine, pH 8.0, containing 2 mM pyroglutamyl-p-itroanilide as substrate (21). GCT (0.5–2 units) wasssayed at 37°C in 50 mM Tris–HCl, pH 8.0, contain-ng 2.5 mM Gln-Trp-Glu as substrate. After 2 min, theeaction mixture was diluted fivefold with cold 10 mMris–HCl, pH 8.0, and immediately injected onto aono Q column (HR 5/5) equilibrated with 10 mMris–HCl, pH 8.0. The product (pyroGlu-Trp-Glu) waseparated from the substrate by eluting the columnith a linear NaCl gradient in 10 mM Tris–HCl, pH.0. The portion of Gln-Trp-Glu converted to pyroGlu-rp-Glu detected at 280 nm was calculated from theespective peak areas.

urification of Natural DPPI and Recombinant His-Tagged DPPI (HT-DPPI)

Natural DPPI was prepared from turkey liver essen-ially according to Metrione et al. (14). The purificationrocedure included the following steps: extraction, au-olysis, ammonium sulfate precipitation (40–70% sat-ration), gel filtration on Sephacryl S-300 HR, desalt-

ng on Sephadex G-25 F, and anion-exchangehromatography on DEAE–Sepharose FF. HT-DPPIas prepared as described by Lauritzen et al. (22).oth natural DPPI (4–5 units/mg protein) and HT-PPI (8–9 units/mg protein) were stored at 220°C in.5 mM sodium phosphate buffer, 150 mM NaCl, 2 mMysteamine, 50% glycerol, pH 6.8.

urification of GCT

GCT was prepared from crude papaya latex, essen-ially according to Messer and Ottesen (18), except thatM–Sepharose FF replaced the CM–Sephadex em-loyed by Messer and Ottesen. Purified GCT in 10 mModium phosphate, 50 mM NaCl, pH 7.0, was treatedith 1 mM cystamine and 20 mM E-64 for 5 h at room

emperature to inhibit traces of cysteine protease ac-ivity. The final GCT preparation (80–100 units/mgrotein) was stored at 220°C in 4 mM sodium phos-hate, 20 mM NaCl, 50% glycerol, pH 7.0.

urification of Recombinant PGAP and His-TaggedPGAP (HT-PGAP)

PGAP was purified by hydrophobic interaction chro-atography on a phenyl–Sepharose FF column essen-

ially as described earlier (19). HT-PGAP was purifiedy IMAC as described above for His-tagged proteins.he PGAP and HT-PGAP preparations (7–8 units/mg

rotein) were stored at 220°C. 7

mmobilization of HT-PGAP

One hundred fifteen units of HT-PGAP in 10 ml 20M sodium phosphate, 50 mM NaCl, pH 7.0, wasixed with 7.5 ml chelating Sepharose FF, prechargedith Zn21 and equilibrated with 20 mM sodium phos-hate, 50 mM NaCl, pH 7.0. After incubation for 15in at room temperature with occasional mixing, theatrice was packed into a 20-ml column, washed withtotal of 30 ml buffer, and stored at 4–8°C until use.

nzymatic Treatment of Glucagon

Treatment of glucagon with DPPI and HP-GCT waserformed by mixing 25 ml glucagon in 0.01 M HCl (125g; 0.036 mmol), 452 ml 100 mM sodium phosphateuffer, pH 7.0, 10 ml HP-GCT (375 mU), and 12.5 mlPPI (12.5 mU) (reaction A). In a control reaction

reaction B), HP-GCT was omitted. The mixtures werencubated at 37°C, and at different times portions of 50l were mixed with 950 ml 20 mM citric acid/NaOH, 7

urea, pH 4.6. Five hundred-microliter aliquotshereof were analyzed on a Mono S HR 5/5 columnsing a 20-ml gradient of 0–500 mM NaCl in 20 mMitric acid/NaOH, 7 M urea, pH 4.6. In a preparativexperiment, specific removal of His1-Ser2-Gln3 fromlucagon was carried out by mixing 400 ml glucagon in.01 M HCl (2 mg; 0.57 mmol) with 1420 ml 100 mModium phosphate, pH 7.0, 80 ml HP-GCT (3 units), and00 ml DPPI (0.2 units), and then incubation at 37°C.fter 20 min, an aliquot of 1500 ml was mixed with 750l 100 mM sodium phosphate buffer, pH 7.0. One thou-and microliters of the diluted sample was desalted onSephadex G-25 column (NAP 10) equilibrated with 10M sodium phosphate, pH 7.0 (sample A). Then 300 ml

luate was mixed with 6 ml chicken cystatin (1 mg/ml)nd after incubation for 30 min at room temperature tonactivate DPPI, 10 ml PGAP (0.1 units) was added.he mixture was incubated for 16 min at 37°C, afterhich the sample was mixed with 200 ml 10 mM so-ium phosphate, pH 7.0, and desalted on a Sephadex-25 column (NAP 5) equilibrated with 10 mM sodiumhosphate, pH 7.0 (sample B).

nzymatic Treatment of His-Tagged Proteins

HT-DPPI cleavage of HT15-hIL-1b, HT15-MBP, andT15-hGH (5 to 6 mg) was carried out in 20 mM sodiumhosphate, 150 mM NaCl, pH 7.0, at a protein concen-ration of 1–1.5 mg/ml. HT-DPPI activated with 10 mMysteamine for 5 min at 37°C was added to the protein atratio of either 1:200 or 1:40 (w/w) and the mixtures were

ncubated at 37°C for up to 60 min. The progress of theleavage reaction was followed by SDS–PAGE. After thenzymatic reaction, the mixture was passed through a-ml HisTrap column charged with Ni21 and equilibratedith 20 mM sodium phosphate buffer, 150 mM NaCl, pH

.0. HT-DPPI cleavage of HT15-MBP was further ana-

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392 PEDERSEN ET AL.

yzed by ion-exchange HPLC. At the times indicated, 100l of sample was diluted into 1900 ml 20 mM citric acid/aOH, pH 4.0, and 1 ml portions hereof were analyzed onMono S HR 5/5 column at a flow rate of 1 ml/min using20-ml gradient of 0–1 M NaCl in 20 mM citric acid/aOH, pH 4.0.HT-DPPI/HP-GCT cleavage of HT15-Gln-hTNFaas carried out by adding a mixture of activated HT-PPI and HP-GCT to HT15-Gln-hTNFa in 20 mM

odium phosphate, 150 mM NaCl, pH 7.0, at a ratio of:400 (w/w) for HT-DPPI and 1:40 (w/w) for HP-GCTnd incubating at 37°C for 30 min. Following the en-ymatic reaction, the mixture was subjected to subtrac-ive IMAC as above. Cleavage of pyroGlu-hTNFa withT-PGAP in solution was then carried out by addingctivated HT-PGAP at a ratio of 1:3 (w/w) and incubat-ng at 37°C for 60 min. Finally HT-PGAP was removedy IMAC as above. In a simplified process the HT-PPI/HP-GCT digest was pumped at a flow rate of 0.3l/min through a 1-ml Ni21 charged HisTrap column

erially connected to a 5-ml column containing immo-ilized HT-PGAP (75 units). The flowthrough (mea-

IG. 1. Schematic summary of the the overall cleavage stategy. Seeext for further details.

ured by absorbance at 280 nm) containing hTNFa was p

ollected. The total process was followed by SDS–AGE analysis.

ESULTS

oncept of the Method

Generation of untagged target protein involves these of either one or three exoenzymes depending on the-terminal sequence of the target protein. All three

nzymes have affinity for IMAC matrices by virtue ofhe designed HisTags of the recombinant enzymes HT-PPI and HT-PGAP or by virtue of an assumed histi-

IG. 2. Scheme of the combined cleavage and purification stategy.A) Procedure for proteins having a N-terminal DPPI stop point. (Bnd C) Procedures for proteins without N-terminal DPPI stop points.A) After cleavage with HT-DPPI, simultaneous removal of HT-PPI, residual HisTag-protein, and copurified contaminants ischieved by passage of the cleavage mixture over an IMAC column.B and C) After cleavage with HT-DPPI and HP-GCT, the cleavage

ixture containing pyroGlu-protein is subjected to subtractiveMAC as above. In B, the flowthrough (pyroGlu-protein) is thenreated with HT-PGAP in solution followed by a second subtractiveMAC step to remove HT-PGAP. In C, the pyroglutamyl residue isleaved off by direct transfer of the Ni21-column flowthrough to an21 column with immobilized HT-PGAP. Complete processing of the

yroglutamyl is controlled by regulation of the flow rate.

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393AMINOPEPTIDASE REMOVAL OF N-TERMINAL POLYHISTIDINE TAGS

ine patch on the surface of natural GCT (HP-GCT). Inny case, the metal-binding polyhistidine sequence ofhe target protein is first cleaved off by HT-DPPI whichatalyzes a stepwise removal of N-terminal dipeptidesxcept if (a) the amino group of the N-terminus islocked, (b) the site of cleavage is on either side of aroline, or (c) the N-terminal residue is either lysine orrginine. This ability of certain residues to function asPPI stop points is exploited to ensure the integrity of

he desired protein by preventing excessive digestion.A range of mature proteins have DPPI stop points of

ype b or c. Accordingly, N-terminal HisTags contain-ng an even number of amino acid residues can beompletely and specifically removed by treatment withT-DPPI alone (Fig. 1A). The enzymatic treatment is

hen followed by removal of both HT-DPPI, residualnconverted HisTag protein and copurified contami-ants by subtractive IMAC (Fig. 2A).If the N-terminus of the target protein does not con-

TABLE 1

Natural N-Terminal Sequences of the Expressed Proteins

Protein N-terminal sequence

IL-1b (human interleukin 1b) APVRSLNCTLBP (maltose binding protein) KIEEGKLVIW

GH (human growth hormone) FPTIPLSRLFTNFa (human tumor necrosis factor a) VRSSSRTPSD

Note. The amino acids acting as DPPI stop-points are shown inold. Amino acid sequences verified by amino acid sequence analysisre underlined.

TAB

Amino Acid and Encoding Nucleot

HisTag Am

M R G S HHT 1 ATG.CGT.GGA.TCC.CAT

M R H H HHT 3 ATG.CGT.CAT.CAT.CAT.

M H G H GHT 8 ATG.CAT.GGT.CAT.GGT

M K G H GHT 9 CAT.GGC.CAT.GCA.CAT

M H A R GHT 10 ATG.CAT.GCT.CGA.GGG

M H A K AHT 11 ATG.CAC.GCA.AAA.GCT

M H A K AHT 12 ATG.CAC.GCA.AAA.GCT

M H A K AHT 14 ATG.CAC.GCA.AAA.GCT

M K H Q HHT 15 ATG.AAA.CAC.CAA.CAC

M K H H AHT 17 ATG.AAA.CAT.CAC.GCT.

M K H H H

HT 18 ATG.AAA.CAT.CAT.CAT.CAT

ain blocking residues, a type a stop point consisting ofn N-terminal pyroglutamyl (pyroGlu) is generated en-ymatically by coincubation with HP-GCT, which cat-lyzes the cyclization of N-terminal glutamine residueso pyroglutamyl. The glutamine residue to be con-erted should be inserted between the sequences of theisTag and the target protein. An uneven competitionetween HP-GCT in excess and HT-DPPI ensures im-ediate cyclization of the inserted glutamine when theisTag is digested and the glutamine appears in the-terminus (Fig. 1B, step 1). This pyroGlu-extended

orm of the target protein is protected against furtherT-DPPI digestion. Following the coincubation, HP-CT and HT-DPPI are removed from the product and

he target sequence is obtained by cleaving off theyroglutamyl residue with HT-PGAP (Fig. 1B, step 2).Detagging of proteins lacking natural stop points is

erformed in either two steps (Fig. 2B) or in a single stepnvolving the use of two serially connected columns (Fig.C). In both cases the product is first incubated withT-DPPI and HP-GCT. The resulting pyroGlu-extended

arget protein is then passed through a Ni21-chelate col-mn (as in Fig. 2A) to remove the processing enzymes,nconverted product, and residual contaminants. Afterhis IMAC column, the two setups shown in Figs. 2B andC differ. In Fig. 2B the pyroGlu product is collected,ncubated with HT-PGAP in solution, and passedhrough a second Ni21-chelate column to remove HT-GAP. This setup makes it easy to collect samples fornalyses of intermediate products and is ideal for smallcale preparations. In Fig. 2C, the pyroglutamyl residue

2

Sequences for Different HisTags

acid sequence and coding region

H H H H H G M A ST.CAT.CAT.CAT.CAT.GGT.ATG.GCT.AGC.-H H H

T.CAT.CAC. 2H G H G H G HT.GGT.CAT.GGC.CAT.GGT.CAC.-H A H G H G H G HT.CAT.GGC.CAT.GGT.CAC.CAG.GGT.CAC.-H G H A H G H G H G HT.GGC.CAT.GCA.CAT.GGT.CAT.GGC.CAT.GGT.CAC.-

H A H A H A H G H G HC.GCT.CAC.GCA.CAC.GCC.CAC.GGC.CAT.GGT.CAT.-H A H A H A H A H G HC.GCT.CAC.GCC.CAC.GCT.CAC.GCC.CAT.GGT.CAC.-H A H A H A H G H A HC.GCT.CAC.GCA.CAC.GCC.CAT.GGA.CAC.GCA.CAC.-Q H Q H Q H Q H QA.CAT.CAA.CAT.CAA.CAT.CAA.CAT.CAA.-H H Q H HT.CAT.CAA.CAT.CAT-H H H

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394 PEDERSEN ET AL.

s cleaved off the pyroGlu-extended product by directransfer of the Ni21-column eluate to a Zn21 column withmmobilized HT-PGAP. Complete processing is achievedy regulation of the flow rate.

emoval of a Polyhistidine Tag from Proteins Havinga Natural DPPI Stop Point

Human interleukin 1b (hIL-1b), maltose binding pro-ein (MBP), and human growth hormone (hGH) have-terminal sequences which constitute natural DPPI

top points (see Table 1). These three proteins extendedith a 16-residue N-terminal polyhistidine tag (HT15,

ee Table 2) were expressed in E. coli and purified byMAC on Ni21-chelate–Sepharose FF. The HT-DPPIleavage of purified HT15-hIL-1b, HT15-MBP, andT15-hGH, followed by subtractive IMAC according toig. 2A, is illustrated by SDS–PAGE analysis in Figs.A–3C. A nearly quantitative conversion of the HT15usion proteins (Figs. 3A–3C, lane 2) to proteins with

igrations in accordance with those expected for thentagged proteins was observed after 30–60 min (Figs.A and 3B, lanes 3–6, and Fig. 3C, lanes 3–7). The smallmount of unconverted HT15 fusion proteins still present

IG. 3. SDS–PAGE analysis of HT-DPPI cleavage of (A) HT15-hIL1b,MAC. A, lanes 2–6, samples taken at 0, 2, 5, 10, and 30 min, respectiv, 2, 5, 30, and 60 min, respectively; lane 7, MBP after subtractive IMA0 min, respectively; lane 8, hGH after subtractive IMAC. Molecular w

fter 30 to 60 min of incubation was not affected by either f

rolonged treatment with HT-DPPI or by increasedmounts of enzyme. After the cleavage the reaction mix-ures (Figs. 3A and 3B, lane 6, and Fig. 3C, lane 7) wereassed through a Ni21-chelate–Sepharose column and-terminal amino acid sequencing of the three resultingroducts (Figs. 3A and 3B, lane 7, and Fig. 3C, lane 8)evealed the sequences of the authentic proteins (seeable 1). The yield of purified protein obtained after theT-DPPI cleavage and subtractive IMAC ranged be-

ween 75 and 90%.The use of SDS–PAGE to monitor the digestion of

mall HisTag extensions of large proteins (e.g., MBP)ay result in insufficient resolution and lack of verifi-

ation of the sequential removal of dipeptides witholecular masses of only 0.2 kDa (Fig. 3B). As an

lternative, we successfully applied cation-exchangehromatography at pH 4.0 to confirm the stepwise re-ease of dipeptides containing positively charged histi-ine residues during the conversion of HT15-MBP toBP. It was assumed that HT-DPPI cleavage of HT15-BP would result in the sequential loss of seven pos-

tive charges due to removal of the dipeptide Met-Lysnd the following six His-Gln dipeptides. The result

HT15-MBP, and (C) HT15-hGH fusion proteins followed by subtractivelane 7, hIL1b after subtractive IMAC. B, lanes 2–6, samples taken atlane 8, HT15-MBP. C, lanes 2–7, samples taken at 0, 2, 5, 10, 20, andht markers (sizes in kDa) are shown in lane 1.

(B)ely;C;

rom the time course of cleavage of HT15-MBP with

FIG. 4. Ion exchange (IE)-HPLC analysis of HT-DPPI cleavage of HT15-MBP. See text for further details.

Fa

IG. 5. Time course of treatment of glucagon with DPPI and GCT (A) or with DPPI alone (B). At intervals, samples were taken and

nalyzed by cation-exchange chromatography on a Mono S column (see Materials and Methods).

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397AMINOPEPTIDASE REMOVAL OF N-TERMINAL POLYHISTIDINE TAGS

T-DPPI (Fig. 4) was in accordance with this assump-ion because seven products eluting earlier than un-reated HT15-MBP were generated sequentially dur-ng the reaction. This illustrates that cation-exchangePLC can be useful for measuring the progress ofisTag cleavage.

IG. 6. Mono S analysis of glucagon (A), pyroGlu3-glucagon(4-29) ob-ained by treatment of glucagon with DPPI and GCT (B) and glucagon(4-9) obtained by treatment of pyroGlu3-glucagon(4-29) with PGAP (C).

IG. 7. SDS–PAGE analysis at various stages during the enzymatind HP-GCT was used at a ratio of 1:400 (w/w) and 1:40 (w/w), respectn solution at a ratio of 1:3 (w/w) (A) or with HT-PGAP immobilized o–5, HT15-Gln-hTNFa treated with HT-DPPI 1 HP-GCT, samples taubtractive IMAC; lane 7, the reaction mixture after treatment of pyy subtractive IMAC; lane 9, hTNFa subjected to a second HT-DPPIreated with HT-DPPI and HP-GCT, samples taken after 10, 20, anMAC and treatment with HT-PGAP immobilized onto Zn21-chelareatment. Molecular weight markers (sizes in kDa) are shown in la

f 20–24 and 6.5 kDa (22) resulting in multible bands on SDS–PAGE.

emonstrating GCT-Catalyzed Generation of a DPPIStop Point

When removing HisTags from proteins without aatural DPPI stop point, it is essential to obtain in-tantaneous cyclization of the glutamine residue in-erted between the HisTag and the protein to avoidPPI cleavage beyond the stop point. Glucagon, whichas the N-terminal sequence His1-Ser2-Gln3-Gly4-hr5-Phe6-Thr7, was chosen as a model substrate for

llustrating the effectiveness of the cyclization reac-ion. Glucagon was treated at 37°C with either a mix-ure of DPPI and HP-GCT or with DPPI alone. Thenzymatic modification of glucagon was followed byation exchange on a Mono S column as illustrated inig. 5. Treatment of glucagon with DPPI and HP-GCTas assumed to result in a loss of two positive chargest pH 4.6 due to the removal of the N-terminal His1-er2 dipeptide and the cyclization of Gln3. The result ofhe cation-exchange HPLC analysis was in accordanceith this assumption because a single product elutingarlier than the untreated glucagon was observed (Fig.A). The action of HP-GCT was apparent when chro-atograms of DPPI/HP-GCT-treated glucagon were

ompared to the analogous chromatograms of glucagonhich had only been treated with DPPI. Initially, a

ingle product eluting between unmodified glucagonnd the DPPI/HP-GCT-treated glucagon (presumablyyroGlu3-glucagon (4–29)) was observed. This products suggested to arise from the loss of the N-terminalis1-Ser2 dipeptide which has a net charge of 11.ater, a mixture of components appeared as a result of

urther degradation beyond Gln3 (Fig. 5B).Using optimized conditions (glucagon at 1.0 mg/ml

nversion of HT15-Gln-hTNFa to hTNFa. A combination of HT-DPPIly, followed by subtractive IMAC and cleavage with either HT-PGAPZn21-chelate–Sepharose FF (B). A, lane 2, HT15-Gln-hTNFa; lanesafter 10, 20, and 30 min, respectively; lane 6, pyroGln-hTNFa after

lu-TNFa with HT-PGAP; lane 8, hTNFa after removal of HT-PGAPatment. B, lane 2, HT15-Gln-hTNFa; lanes 3–5, HT15-Gln-hTNFa0 min, respectively; lane 6, hTNFa after simultaneous subtractiveSepharose FF. Lane 7, hTNFa subjected to a HT-DPPI control. HT-DPPI is composed of several subunits with molecular weigths

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398 PEDERSEN ET AL.

nd a reaction time of 20 min), a single product of thePPI/HP-GCT treatment was obtained as judged from

ation-exchange chromatography (Fig. 6B). This prod-ct was subjected to N-terminal sequencing but noequence could be detected, indicating the presence of alocking N-terminal pyroglutamyl residue. After inhi-ition of DPPI with chicken cystatin (a cysteine pro-ease inhibitor), the glucagon product was furtherreated with PGAP and the reaction product was againnalyzed by cation-exchange chromatography (Fig.C). More than 98% of the peak corresponding to theresumed pyroGlu3-glucagon(4–29) was converted to aeak with a retention time similar to that of the prod-ct assumed to be des(His1-Ser2-Gln3)-glucagon (gluca-on(4–29)). This indicates regeneration of one positiveharge as a result of the pyroGlu3 removal and regen-ration of an unblocked N-terminus. Amino acid se-uencing of this product revealed the sequence Gly-hr-Phe-Thr-Ser corresponding to the N-terminal oflucagon(4–29). Accordingly, the sequence His1-Ser2-ln3 had been selectively removed from glucagon by

he combined action of DPPI, HP-GCT and PGAP.

pecific Removal of a Polyhistidine Tag from aProtein without a Natural DPPI Stop Point

Human tumor necrosis factor a (hTNFa) has an N-erminal sequence which does not constitute a naturalPPI stop point (see Table 1). The fusion proteinT15-Gln-hTNFa was expressed in E. coli and purifiedy IMAC. Purified HT15-Gln-hTNFa (Fig. 7A, lane 2)as treated with HT-DPPI and HP-GCT at 37°C. Aearly quantitative conversion of HT15-GlnhTNFa

18.6 kDa) to a protein with a migration similar to thatxpected for pyroGlu-hTNFa (17.5 kDa) was observedfter 30 min (Fig. 7A, lanes 3–5). The reaction mixtureFig. 7A, lane 5) was then passed through a Ni21-helate–Sepharose column to remove HT-DPPI, HP-CT, unconverted HT15-Gln-hTNFa, and residual

ontaminants. The eluate (Fig. 7A, lane 6) was sub-ected to N-terminal sequencing but no sequence coulde detected indicating the presence of a blocking N-erminal pyroglutamyl residue. The presumed pyro-lu-hTNFa was then treated with HT-PGAP for 1.5 ht 37°C (Fig. 7A, lane 7) after which HT-PGAP wasemoved by passing the product through a Ni21-helate–Sepharose column. The similarity of the prod-ct (Fig. 7A, lane 8) to native hTNFa was confirmed by-terminal sequencing, resulting in the known se-uence of mature hTNFa (Val1-Arg2-Ser3-Ser4-Ser5-rg6-Thr7-Pro8). The yield of hTNFa from 6.1 mgT15-Gln-hTNFa was 4.3 mg corresponding to 70%.To further investigate the efficiency of the HT-

GAP-catalyzed removal of pyroglutamyl residues, anliquot of the fully processed hTNFa was subjected to aecond HT-DPPI treatment (125 mg/mg hTNFa, 2 h)

nd SDS–PAGE. A quantitative HT-DPPI-catalyzed t

onversion of deprotected hTNFa into truncated formsith increased electrophoretic mobility was observed,

onfirming an efficient HT-PGAP-catalyzed removal ofhe pyroglutamyl residue. The most degraded form waselieved to be des(Val1-. . .-Arg6)-hTNFa due to theresence of a blocking proline (Pro8) (Table 1).

implified Removal of a Polyhistidine Tag EmployingImmobilized HT-PGAP

The procedure for removal of a polyhistidine tag fromprotein without a natural DPPI stop point was sim-

lified to cut down the number of hands on steps asutlined in Fig. 2C. Purified HT15-Gln-hTNFa (Fig.7B,ane 2) was treated with HT-DPPI and HP-GCT (Fig.B, lanes 3–5) as above to obtain pyroGlu-hTNFaFig.7B, lane 5). After completion of the reaction, the

ixture was pumped at a flow rate of 0.3 ml/minhrough two serial connected columns, the first con-aining 1 ml Ni21-chelate–Sepharose FF and the sec-nd containing 5 ml Zn21-chelate–Sepharose FF chargedith 75 units HT-PGAP. The protein flowthrough from

he passage of the two columns (Fig. 7B, lane 6) had thexpected N-terminal sequence of hTNFa (Table 1). Thefficiency of the HT-PGAP-catalyzed removal of theyroglutamyl residue was tested as above and found toe quantitative (Fig. 7B, lane 7). The yield of purifiedTNFa from 20 mg HT15-Gln-hTNFa was 18 mg, cor-esponding to 90%.

ptimization of the Polyhistidine Tag Design

The precise amino acid sequence of a polyhistidineag and the nucleotide sequence selected to encode itre of great importance for the overall performance of

IG. 8. SDS–PAGE analysis of HT-DPPI/HP-GCT treatment (ratiof 1:400 w/w and 1:40 w/w, respectively) of HT15-Gln-hTNFa (lanes–5), HT17-Gln-hTNFa (lanes 6–9), and HT18-Gln-hTNFa (lanes0–13) after 0, 10, 20, and 30 min of incubation, respectively. Mo-ecular weight markers (sizes in kDa) are shown in lane 1.

he resulting construct during expression, posttransla-

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399AMINOPEPTIDASE REMOVAL OF N-TERMINAL POLYHISTIDINE TAGS

ional processing, purification, and tag removal. Toddress these aspects in greater detail, gene fusionsncoding hTNFa and a number of HisTags of distinctesign were constructed (Table 2).HisTag 18, 17, and 15 are examples of the most

ommon -(His)3-6-, -(His-His-X)2-3-, and -(His-X)3-6- poly-istidine motifs and each of these peptide tags werefficiently processed by HT-DPPI and HP-GCT duringncubation for 30 min (Fig. 8). HT1-Gln-hTNFa whichlso represents the -(His)3-6- motif was, however, onlylowly cleaved (results not shown) due to the inclusionf a poor DPPI dipeptide substrate (Gly3-Ser4). Thisllustrates the importance of avoiding slowly cleavedipeptide sequences (13) in the HisTag. In general weound that the dipeptides Xaa-His (where Xaa is Gly,la, Met, or His) and His-Xbb (where Xbb is Lys, Arg,la, Gln, or Gly) are excellent DPPI substrates. Still,

ncorporation of Gly-His as the penultimate C-terminalipeptide (Gly13-His14 in HT11 and HT14) in tags fusedo hTNFa resulted in a markedly decreased cleavageate and accumulation of an intermediate cleavageroduct. Replacing this dipeptide with Ala-His (as inT12) eliminated the problem.Because a HisTag must be composed of an even num-

er of amino acid residues to be processed correctly byPPI, the penultimate tag residue should be bulky (e.g.,ys, Arg, His, Glu) to minimize in vivo processing of Met1

y the host methionine aminopeptidase (23,24). It is ourxperience that some processing may still be observednd to preclude erroneous cleavage, the penultimate res-due should also be a potential DPPI stop point as Lys2 inT15, HT17, and HT18 (see Table 1). With Lys in the

econd position, the retention of Met1 will be nearly quan-itative and any des(Met1)-Lys-HisTag protein formedill escape HT-DPPI processing and be removed by the

ubtractive IMAC.The nucleotide sequences encoding the polyhistidine

ags finally appeared to have a strong effect on the ex-ression levels (25–28). Little or no product could beetected when hTNFa was fused to HisTag 8, 9, or 10,hereas others (in particular HisTag 3 and 15) resulted

n the expression of about 100 mg/liter of culture. Thexpression levels correlated with the absence of mRNAecondary structures in the vicinity of the AUG startodon as predicted by the MFOLD program (29).

ISCUSSION

In this paper we have described a novel enzymaticethod for specific removal of N-terminal HisTags

rom recombinant proteins. This aminopeptidase ap-roach makes the method independent of any sequencen the protein that can give rise to undesired internalleavage and also ensures the formation of the desired-terminus of the target protein. Problems with un-

pecific cleavage and the creation of an incorrect N-

erminus are often unsolved when endoproteases or p

hemical methods are used for removal of purificationags. To fully exploit the potential of the combinedisTag/aminopeptidase approach, we applied enzymes

hat all have the ability to bind to IMAC matrices.The polyhistidine peptide fusions were cleaved off byT-DPPI, which was shown to be active against each of

he three most common polyhistidine motifs, but carehould be taken when selecting the amino acid andncoding nucleotide sequences. We found that expres-ion levels and the extent of posttranslational process-ng and erroneous degradation are parameters greatlynfluenced by the particular tag design. HisTag 15, 17,nd 18 all resulted in good expression and performedery well as purification tags and in combination withhe presented system for polyhistidine tag removal.

hen HT15/17/18-Gln-hTNFa was incubated withT-DPPI at a ratio of 400:1 (w/w), this resulted inearly complete processing in less than 30 min. How-ver, subsequent SDS–PAGE analyses of these andther reaction mixtures revealed that a small fractionf the preparations was always insensitive to the HT-PPI treatment. This could not be observed by cation-xchange chromatography presumably because of ei-her insufficient detection sensitivity or a true absencef unmodified, his-tagged protein. If the latter is thease, it points to target protein aggregation as theause of HT-DPPI resistance, but Met1 oxidation orormylation may also explain these findings. In anyase, the subtractive IMAC will remove not only therocessing enzymes and co-purified contaminants butlso the unwanted forms of the target protein whichight be very difficult to eliminate by other means.A rapid HP-GCT-catalyzed conversion of a glu-

amine residue to pyroglutamyl is essential when re-oving HisTags from proteins without a DPPI stop

oint. The successful formation and subsequent re-oval of these engineered DPPI stop points by HP-CT and HT-PGAP, respectively, was demonstratedy amino acid sequencing, cation-exchange chromatog-aphy, and SDS–PAGE analyses of samples taken dur-ng the processing of both the model substrate gluca-on and of His-tagged hTNFa.The reactions leading to the removal of HisTags from

roteins without a DPPI stop point are both more exten-ive and enzyme consuming than the reactions for pro-eins with a natural DPPI stop point. This is due to theelatively low activity of HT-PGAP which necessitateshe use of more enzyme. To minimize the actual con-umption of HT-PGAP and to simplify HisTag removalrom proteins lacking natural stop points, we successfullyoupled the HT-DPPI/HP-GCT remover column to a sec-nd IMAC column with immobilized HT-PGAP. The ben-fit of this setup is a simple two-step process, resulting ineduced handling and expenses.

The simple and effective two-step process consists ofminopeptidase cleavage and subtractive IMAC and the

rocess facilitates the specific removal of HisTags from

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400 PEDERSEN ET AL.

roteins of interest with minimal manipulation, resultingn typical recoveries of 75 to 90%. Another advantage ofsing the present process design is the fact that an IMACtep is performed twice, both prior to and followingisTag removal. In this way contaminants that bind and

lute with the HisTag protein on the initial IMAC puri-cation column bind again along with the added en-yme(s) and residual unconverted HisTag protein on theecond IMAC column applied after the enzymatic reac-ion. The target protein is then recovered in theowthrough fractions of either the IMAC or the coupledMAC/HT-PGAP column(s). We successfully combinedhis aminopeptidase approach for removal of HisTagsith the IMAC purification strategy to purify essentiallyomogeneous hIL-1b, hGH, MBP, and hTNFa with theesired and correct N-terminal sequences. These com-ined processes hold promises for use in large-scale pro-uctions of pharmaceutical protein and peptide productsecause of the simple overall design, the use of robust andnexpensive matrices, and the use of enzymes of eitherecombinant or plant origin.

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6. Kim, J.-S., and Raines, R. T. (1993) Ribonuclease S-peptide as acarrier in fusion proteins. Protein Sci. 2, 348–356.

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