Engineered Tobacco Etch Virus (TEV) Protease Active in the Secretory

Journal of Biotechnology 212 (2015) 159–166

Contents lists available at ScienceDirect

Journal of Biotechnology

j ournal homepage: www.elsevier.com/locate/jbiotec

Engineered tobacco etch virus (TEV) protease active in the secretory

pathway of mammalian cells

a a,b,1 a,∗

Francesca Cesaratto , Alejandro López-Requena , Oscar R. Burrone , a,∗∗

Gianluca Petris

International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy

Immunobiology Division, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba

a r t i c l e i n f o a b s t r a c t

Article history: Tobacco etch virus protease (TEVp) is a unique endopeptidase with stringent substrate speciﬁcity. TEVp

Received 14 June 2015

has been widely used as a puriﬁed protein for in vitro applications, but also as a biological tool directly

Received in revised form 25 August 2015

expressing it in living cells. To adapt the protease to diverse applications, several TEVp mutants with

Accepted 27 August 2015

different stability and enzymatic properties have been reported. Herein we describe the development of

Available online 30 August 2015

a novel engineered TEVp mutant designed to be active in the secretory pathway. While wild type TEVp

targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalyti-

Keywords:

cally inactive enzyme, a TEVp mutant with selected mutations at two veriﬁed N-glycosylation sites and

TEV protease

Sec-TEV at an exposed cysteine was highly efﬁcient. This mutant was very active in the endoplasmic reticulum

Glycosylation (ER) of living cells and can be used as a biotechnological tool to cleave proteins within the secretory

ER pathway. As an immediate practical application we report the expression of a complete functional mon-

ERAD oclonal antibody expressed from a single polypeptide, which was cleaved by our TEVp mutant into the

Secretory pathway two antibody chains and secreted as an assembled and functional molecule. In addition, we show active

TEVp mutants lacking auto-cleavage activity.

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction occurs between Q and the short aliphatic G or S residue (Parks

et al., 1994). A TS with the sequence GHKVFM/S is also present

In tobacco etch virus part of the Nuclear Inclusion a (NIa) gene in the protease’s C-terminal portion between residues 213–219,

encodes a 27 kDa protease named tobacco etch virus protease which is intramolecularly recognised and cleaved at M218 (Kapust

(TEVp) (Dougherty and Parks, 1991). TEVp is homologous to serine- et al., 2001; Parks et al., 1995). Conﬂicting results exist on the

proteases but, unlike them, it has a cysteine residue (C151) in its proteolytic activity of the auto-cleaved protein fragment (Kapust

catalytic site. Indeed, the single point mutation C151A completely et al., 2001; Nunn et al., 2005; Parks et al., 1995). To overcome the

abolishes protease activity (Parks et al., 1995). problem of auto-cleavage, several TEVp mutants of amino acid 219

The minimal TEVp substrate speciﬁcity is encoded in the seven have been characterized (Kapust et al., 2001). Reduced activity of

amino acid long sequence EXXYXQ-S/G (TEVp cleavage Site, TS) the auto-cleaved protein has been associated to inhibitory prop-

where X can be any amino acid (Dougherty et al., 1988). Up erties of the cleaved C-terminal end (amino acids 219–242) that

to now, despite this apparent low speciﬁcity and the variety of depended on residues 235–242, which were supposed to tightly

peptides that could be included as TS, the best recognized and bind to the active site (Nunn et al., 2005). A TEVp truncated at amino

cleaved TS is identical to the protease’s natural substrate pep- acid 218 was found functional and did not undergo auto-inhibition

tide (ENLYFQ-G/S) (Kostallas et al., 2011). The proteolytic cleavage (Nunn et al., 2005) while TEVp truncated at residue 219 was also

found effective in a controllable split-TEV system (N-TEV residues

1–118 and C-TEV residues 119–219) (Gray et al., 2010). Interest-

ingly, mutation of C-terminal residues SELVYSQ (236–242) with ∗

Corresponding author.

∗∗ NEGGGLE highly increased TEVp puriﬁcation through a C-terminal

Corresponding author. Present address: CIBIO, University of Trento, Via delle

6His-tag (Wu et al., 2009), suggesting these modiﬁcations blocked

Regole 101, Mattarello 38123, Italy.

TEVp auto-cleavage.

E-mail addresses: [email protected], [email protected] (G. Petris).

Present address: Laboratory of Clinical Immunology, Department of Microbiol-

ogy and Immunology, KU Leuven, 3000 Leuven, Belgium.

http://dx.doi.org/10.1016/j.jbiotec.2015.08.026

160 F. Cesaratto et al. / Journal of Biotechnology 212 (2015) 159–166

Because of its remarkable speciﬁcity, stability and activity in plates (about 5 × 10 cells/well) by standard calcium phosphate

several buffer conditions, TEVp is widely used as a biotechnological technique. Approximately 18 h after transfection, medium was

tool, for instance as a reagent for endoproteolytic removal of afﬁnity changed and cells were further incubated for at least 7–8 h, before

tags (Melcher, 2000; Sun et al., 2012). medium and cell harvesting. When indicated the proteasome

Expression of TEVp was found to be safe not only in bacterial, inhibitor MG132 (Sigma) was added at a concentration of 10 ␮M

yeast, plant and mammalian cells, but also in living organisms like for 4 h. P3 × 63 cells were cultured in RPMI medium (RPMI, Life

Drosophila (Ceriani et al., 1998; Henrichs et al., 2005; Pauli et al., Technologies) supplemented with 10% fetal calf serum (FCS, Life

2008; Satoh and Warren, 2008; Taxis et al., 2009) Technologies).

During viral infection, TEVp accumulates mainly in the nucleus

of plant cells before being released from the full-length Nla precur-

2.3. Cell extract preparation and western blotting

sor protein, while when expressed alone it largely localises in the

cytoplasm (Ceriani et al., 1998).

Transfected HEK-293T cells were washed with PBS and lysed

With the aim of targeting TEVp to the ER for biotechnological

with SDS-lysis buffer (100 mM Tris–HCl, pH 6.8, 6% SDS, 30 mM

applications, we engineered a mutated version that showed strong

NEM, 1% protease inhibitors cocktail (Sigma)) and sonicated.

cleavage activity on substrates localised to the ER lumen. Here we

For SDS-PAGE samples were boiled 10 min in SDS-gel-loading

show its ability to speciﬁcally clip a tag from membrane proteins

buffer (25 mM Tris–HCl, pH 6.8, 1% SDS, 10% glycerol, 175 mM ␤-

in living mammalian cells and to assist in the post-translational

mercaptoethanol) before gel loading. Gels were blotted onto PVDF

maturation of a single ORF encoding the secretory L and H chains of

membranes (Millipore), reacted with anti-SV5 (Life Technologies)

an IgG mAb, allowing proper assembly and secretion of the mature

or anti-roTag (Petris et al., 2014a) mAb followed by incubation

and functional H2L2 protein.

with a HRP-labeled anti-mouse whole IgG antibody (KPL) for ECL

detection. Where indicated, as loading or transfection control,

mouse anti-␣-tubulin (Calbiochem) antibody was used followed

2. Materials and methods

by appropriate HRP-labeled secondary antibody. Where indicated,

cellular lysates or eluted material were treated with Peptide-N-

2.1. Constructs

Glycosidase-F (PNGase-F) or Endoglycosidase Hf (Endo-Hf) (New

England Biolabs) according to manufacturer instructions.

A codon-optimized version of TEVp was cloned into pcDNA3

upstream the SV5-tag (GKPIPNPLLGLD) and used as the basis to

perform all TEVp mutants, obtained by site-directed mutagenesis 2.4. Cytoﬂuorimetric analysis

(QuikChange Site Directed Mutagenesis Kit, Stratagene), using

primers: N23Q fw/rv (CTATCTGCCACCTGACTcAgGAAAGCGACG- HEK-239T cells, co-transfected with sec-TEVp wt or QSG and

GACATACC/GGTATGTCCGTCGCTTTCcTgAGTCAGGTGGCAGATAG); Tetherin-SV5-TS-roTag, were incubated with anti-SV5 tag mAb

C130S fw/rv (GTCTCCGACACCTCTTcTACATTCCCTTC- (Invitrogen), or anti-roTag mAb (Petris et al., 2014a) and then with

TAGTG/CACTAGAAGGGAATGTAgAAGAGGTGTCGGAGAC); T173G an Alexa Fluor 488-conjugated goat anti-mouse IgG (Jackson).

fw/rv (CCATTCAGCCAGCAACTTCggAAATACTAACAATTACTTCA- P3 63 cells were incubated with puriﬁed 14F7 mAb (Carr et al.,

CCTC/(GAGGTGAAGTAATTGTTAGTATTTccGAAGTTGCTGGCTG- 2000) or with supernatants of cells expressing 14F7 mAb from a sin-

AATGG); C110S fw/rv (GAGGGAGGAACGCATCaGCCTGGTGACTAC- gle polypeptide co-transfected or not with sec-TEVp QSG, followed

CAACTTCC/GGAAGTTGGTAGTCACCAGGCtGATGCGTTCCTCCCTC). by ﬂuorescein-conjugated anti-mouse IgG (KPL) and analyzed in a

TEVp was modiﬁed by inserting at the N-terminus the cod- FACSCalibur (Becton Dickinson). mAb 1E10 (Vázquez et al., 1998)

ing sequences of an immunoglobulin secretion signal (Li et al., was used as idiotype control.

1997) and cloned into pcDNA3 vector, yielding sec-TEVp. Sec-TEVp

235 was obtained by substitution of the wild type (wt) C-terminal

2.5. Analysis of structures

sequence with AACGAAGGGGGCCTGGAA.

The SV5-TS-MHC-I␣-roTag, and the SV5-MHC-I␣-TS-roTag

TEVp mutants were evaluated on published TEVp structures

were derived from a construct encoding the cDNA of the A2 allele of

(PBD code: 1LVB and 1LVM (Phan et al., 2002)) using Swiss-

the human MHC-I␣ chain previously described (Petris et al., 2011),

PdbViewer 4.0.4 or PyMOL.

containing at the N-terminus the coding sequences for a secre-

tion signal and the SV5 tag (GKPIPNPLLGLD) and at the C-terminus

3. Results

the roTag tag (SISSSIFKNEG) (Petris et al., 2014a). The TEV cleav-

age site (ENLYFQ/G) was included either in the ER luminal position

3.1. ER-targeted wild type TEVp is inactive in the secretory

downstream of SV5 or in the cytosolic tail just upstream roTag. The

pathway

construct for Tetherin (N65,92A) (Petris et al., 2014b), with a dele-

tion of the 20C-terminal residues that serve as a signal for the GPI

In order to localise TEVp to the secretory pathway of mam-

anchor was C-terminal tagged with SV5 followed by TS and roTag.

malian cells (sec-TEVp), the enzyme was engineered by fusing at

The single ORF pcDNA3 vector, encoding the full length L and H

the N-terminus a strong immunoglobulin signal peptide (Li et al.,

chains from mAb 14F7 (Carr et al., 2000), was obtained by cloning

1997). The SV5-tag was added to the C-terminus for detection. sec-

the coding sequences of the two chains (Rodríguez et al., 2007),

TEVp activity within the lumen of the ER was assayed in HEK-293T

engineered by the addition of an immunoglobulin secretion signal

cells by co-expression with a double-tagged MHC-I␣ chain reporter

peptide at the N-terminus and including the linker SSGSENLYFQGT ≈

( 48 kDa), previously used in other studies (Petris et al., 2011),

with a TS (underlined) between the two chains.

which contains the SV5 tag at the N-terminus (luminal side) and

roTag at the C-terminus cytosolic side (Petris et al., 2014a). A TS was

2.2. Cell culture and transfection included downstream of SV5 in ER luminal position (Fig 1A). Upon

cleavage, SV5 is removed and the digested polypeptide (≈43 kDa)

HEK-293T cells were cultured in Dulbecco’s modiﬁed Eagle’s can still be detected by anti-roTag mAb. Despite sec-TEVp was

medium (DMEM, Life Technologies), supplemented with 10% fetal well expressed migrating in SDS-PAGE as a 37 kDa band, no pro-

calf serum (FCS, Life Technologies). Cells were transfected in 6-well teolytic cleavage of the MHC-I␣ reporter was observed (Fig. 1B).

F. Cesaratto et al. / Journal of Biotechnology 212 (2015) 159–166 161

Fig. 1. Wild type TEVp is not active in the ER. (A) scheme of the MHC-I␣ reporter construct with a cleavage site (TS) in luminal position. SV5 and roTag were used as tags at the

N- and C-terminus. (B) WB of cell extracts from 293T cells co-expressing MHC-I␣ reporter and the indicated TEVp versions. Blots were developed for SV5 or roTag as indicated.

WB of the SV5-tagged TEVp is also shown. (C) schematic representation of the TEVp structure (adapted from pdb ﬁle 1LVB, (Phan et al., 2002)) with the N-glycosylation sites

N23 and N171 shown in red and N52 and N68 in green. Peptide substrate is indicated in light blue. (D) WB of cell extracts from HEK 293T cell transfected with wt sec-TEVp

treated with endo-glycosydases Endo-Hf and PNGase F. With exception of panel D blots are representative of at least three independent experiments.

As expected, the control cytosolic TEVp (cyt-TEVp) and the inac- (C19, C110, C130, and C151) are present in TEVp primary sequence,

tive mutant C151A did not cleave the luminal TS (Fig. 1B). While of which C151 is in the catalytic site. Considering solvent acces-

sec-TEVp was readily detected in cell extracts of transfected cells, sibility in the TEVp structure (1LVB, (Phan et al., 2002)), C130 is

the non-ER-targeted cyt-TEVp was not because of its well-known the most exposed one, while side chains of C19 and C110 are

auto-cleavage activity, which leads to the removal of the C-terminal buried within the molecule. In addition, residue C130 is highly

tag. This result is consistent with lack of activity of sec-TEVp in reactive: in diverse crystal structures it either forms a disulphide

the ER lumen. The expected apparent molecular mass of cyt-TEVp bridge between TEVp molecules or reacts with ␤-mercaptoethanol

is 26 kDa, as observed for the cyt-C151A mutant, while the one present in solution (Nunn et al., 2005). We therefore tested a panel

observed for sec-TEVp was of ≈37 kDa, as a consequence of N- of sec-TEVp mutants (schematically shown in Fig. 2A) in HEK-293T

glycosylations. In fact, four potential N-glycosylation sites (NXS/T, cells using the TS-tagged MHC-I␣ reporter. As shown in Fig 2B,

where X is any amino acid except P) are present in TEVp primary similarly to wild type (wt) sec-TEVp and the catalytically inac-

sequence, localised in various regions of the enzyme structure (N23, tive C151A, the single mutants N23Q and C130S with apparent

N52, N68, and N171) (Fig. 1C), which can account for the altered molecular masses of 35 and 37 kDa, respectively, did not cleave the

PAGE migration. Indeed, post-lysis treatment with glycosidases reporter, while the double mutants N23Q-C130S (QS) and C130S-

Endo-Hf and PNGase-F resulted in the expected 26 kDa protein T173G (SG) (both of ≈35 kDa) showed only partial activity (Fig. 2B,

(Fig 1D). N-glycosylation is also likely one of the main reasons that lanes 5, 6). To prevent N-glycosylation at N171, the mutation of

impair sec-TEVp activity in the ER lumen as two N-glycosylation T173 into glycine was preferred, because the alternative N171Q

sites, N23 and N171, could compromise correct folding: N23 is mutation was detrimental for sec-TEVp activity (Fig 2B, lane 10). In

buried inside the TEVp molecule, while N171 is in close proximity fact, a variant containing the three mutations N23Q-C130S-T173G

to the catalytic pocket (Fig 1C, labeled in red). In contrast, glycosyla- (QSG) (≈35 kDa) completely cleaved the MHC-I␣ reporter (Fig 2B,

tion of N52 and N68 that are exposed on the folded TEVp structure lane 7 and 9). Addition of the C110S mutation into sec-TEVp QSG

unlikely affect enzymatic activity (Fig. 1C, labeled in green). variant had a deleterious effect on its activity (Fig 2B, lane 8). Sim-

ilarly, mutation C19S blocked TEV activity (not shown). Further

demonstration of the QSG cleavage activity in the ER lumen was

3.2. Engineering an active sec-TEVp

obtained with BST-2/Tetherin, a different membrane reporter pro-

tein. Tetherin is a dimeric type II trans-membrane protein stabilised

To design an active sec-TEVp variant, we took into consider-

by three disulﬁde bonds between the two parallel monomers with a

ation the two major post-translational modiﬁcations of proteins

GPI anchor at the C-terminus that is well expressed and exposed on

in the ER lumen: N-glycosylation and cysteine oxidation, which

the cell surface. This is also true for the non-glycosylated (N65,92A)

may hamper correct folding of sec-TEVp. Four different cysteines

162 F. Cesaratto et al. / Journal of Biotechnology 212 (2015) 159–166

Fig. 2. A sec-TEVp mutant active in the ER lumen. (A) schematic representation of the relevant residues in the wild type (wt) TEVp sequence, mutated into the indicated

amino acids in the different sec-TEVp versions. C151, indicated in red is essential for catalytic activity. (B) WB developed with SV5 or roTag of the MHC-I␣ reporter with

TS in luminal position, co-expressed with the indicated sec-TEVp mutants. Arrowhead indicates un-cleaved reporter; arrow cleaved reporter. (C) scheme of the Tetherin

reporter construct with a cleavage site (TS) in luminal position, between SV5 and the C-terminal roTag. (D) WB of extracts from cells co-expressing Tetherin-SV5-TS-roTag

with sec-TEVp wt or QSG variant. (E) Cytoﬂuorimetry of HEK 293T cells co-expressing Tetherin-SV5-TS-roTag with sec-TEVp wt or QSG variant, analysed with anti-SV5 (left

panel) or anti-roTag (right panel) followed by Alexa488-conjugated goat anti-mouse antibody. Blots and cytoﬂuorimetric analysis are representative of at least 3 independent

experiments.

mutant (≈24 kDa) with the GPI acceptor sequence replaced by the (Kapust et al., 2001), but QSG mutant was clearly more efﬁcient, as

SV5-TS-roTag double-tag sequence (Fig. 2C). sec-TEVp QSG efﬁ- S219P cleaved only around 50% of the MHC-I␣ reporter.

ciently cleaved Tetherin molecules bearing the TS (cleaved Tetherin Interestingly, while wt cyt-TEVp did not cleave the lumi-

≈

22 kDa), as shown by WB (Fig. 2D) and cytoﬂuorimetry (Fig. 2E). nal TS, sec-TEVp variants including the wt were able to cleave

(albeit partially) the reporter MHC-I␣ with TS in the cytosolic tail

3.3. Activity of TEVp mutants in the cytosol. (Fig. 3C). They also showed, as expected, different levels of N-

glycosylation. The cytosolic activities of sec-TEVp enzymes targeted

The sec-TEVp mutants QS and QSG were the two most active to the ER lumen are most likely due to the fraction of molecules

ones in the ER lumen. To compare their activity with wt cyt-TEVp spontaneously targeted to the ER-associated degradation path-

and the largely used mutants S219 V and S219P (reported to have way (ERAD). Molecules entering ERAD are retro-translocated to

reduced auto-cleavage activity), we directly co-expressed them in the cytosol, where they become de-glycosylated by the endoge-

the cytosol of HEK-293T cells (by removing the sec signal peptide) nous cytosolic PNGase before proteasomal degradation (Misaghi

with an MHC-I␣ reporter containing the TS in the cytosolic tail just et al., 2004). As N-glycosylation prevented wt sec-TEVp activity

upstream of roTag (≈48 kDa) (Fig. 3A). As shown in Fig. 3B, wt cyt- in the ER lumen, it was expected that the de-glycosylated frac-

TEVp and mutants S219 V and QS digested almost completely the tions resulted in an active enzyme. In fact, de-glycosylated wt

reporter MHC-I␣ to a 45 kDa fragment and they were themselves sec-TEVp accumulated in the cytosol upon treatments of cells with

auto-cleaved. In comparison, mutant QSG was slightly less efﬁcient MG132, which blocks degradation by the proteasome (Fig. 3D).

in the cytosol and not completely auto-cleaved. It was expressed Consistently, mutant QSG with two N-glycosylations sites mutated

at levels comparable to mutant S219P, reported to be very stable showed higher relative activity in the cytosol (Fig. 3C, lane 6).

F. Cesaratto et al. / Journal of Biotechnology 212 (2015) 159–166 163

Fig. 3. Activity of sec-TEVp mutants in the cytosol. (A) Scheme of the MHC-I␣ reporter with TS in cytosolic position. (B) WB of the MHC-I␣ reporter co-expressed with

TEVp mutants expressed in the cytosol (without leader peptide). Arrowhead indicates un-cleaved reporter; arrow cleaved reporter. (C) WB of the MHC-I␣ reporter with

TS in cytosolic position co-expressed with the indicated cytosolic (cyt) or sec-TEVp mutants. Arrow indicates un-cleaved reporter; arrowhead cleaved reporter. (D) WB of

wt sec-TEVp from cells untreated or treated with the proteasome inhibitor MG132. Arrowhead indicates de-glycosylated sec-TEVp. Blots are representative of at least 3

independent experiments.

3.4. Functional un-cleavable versions 3.5. Functional maturation of a single chain mAb by mutant QSG

The ER-active QSG mutant showed partial auto-cleavage activ- The activity of sec-TEVp QSG was further tested on the

ity. We thus tested two alternative QSG versions differing in the expression of the model full-length IgG monoclonal antibody

C-terminal end: one corresponding to the truncated form (end- (mAb) 14F7, which recognises the N-glycosylated GM3 ganglioside

ing with M218, sec-TEVp QSG-218) and one in which the TEVp (GM3(Neu5Gc)) (Carr et al., 2000). We prepared a construct with a

C-terminus (from M235) was changed (residues 236–242) from single ORF encoding an N-terminal signal peptide (sec) followed by

SELVYSQ to MNEGGLE (sec-TEVp QSG-235), similarly to mutations the full-length L and H chains of the IgG antibody that were sepa-

introduced in the TEVp C-terminus to improve its puriﬁcation (Wu rated by the 7-amino acid-long TS (schematically shown in Fig. 5A).

et al., 2009). As shown in Fig. 4A, both variants showed activity in We expected that upon co-expression with the ER-functional QSG

the ER lumen on the MHC reporter (Fig. 4A, lanes 5, 6) and reduced mutant, the single polypeptide would be cleaved into the two func-

auto-cleavage (shown only for mutant 235, as the 218 was not tional chains (L and H), thus allowing proper IgG assembly and

tagged). Interestingly, in agreement with a previous report with secretion. As shown in Fig. 5B, in the presence of the active QSG

shortened TEVp (Nunn et al., 2005), the truncated QSG-218 form protease the single polypeptide of around 75 kDa was found fully

was active both in the ER (Fig. 4A, lane 5) and in the cytosol (Fig. 4B, cleaved into the two chains (L: 25 kDa; H: 50 kDa) both in cellu-

lane 3). In addition, the cytosolic wt-235 and QSG-235 versions lar extracts and supernatants, while in the absence of the protease

lacked auto-cleavage activity while remaining fully active on the the two chains remained fused. The secreted material was mostly

cytosolic reporter substrate (Fig. 4B, lanes 4, 6). In contrast to sec- formed by the expected 150 kDa of the fully assembled H2L2, as

TEVp wt and sec-TEVp wt-235, which were not secreted (and not shown in a non-reducing WB (Fig. 5B, right panel). Surprisingly,

active), sec-TEVp QSG-235 was found actively secreted in the cell the un-cleaved L-H was also found secreted. However, the mature

culture supernatant (Fig. 4C, lanes 1, 2 and 5, 6). We expect sec- (cleaved) 14F7 IgG produced by cells co-expressing the QSG mutant

TEVp QSG-218 to be also actively secreted (since it is not tagged it was able to bind the antigen on the surface of P3 × 63 cells, while

could not be conﬁrmed), as it has the same glycosylation muta- the signal with the un-cleaved one was very low (Fig. 5C). Thus, the

tions of sec-TEVp QSG-235. As reported above, the QSG variant active sec-TEVp QSG is a valuable tool to speciﬁcally and efﬁciently

retained auto-cleavage activity and was therefore undetectable in cleave functional polypeptides into the ER lumen without affecting

the supernatant. assembly and secretion.

164 F. Cesaratto et al. / Journal of Biotechnology 212 (2015) 159–166

Fig. 5. Processing and maturation of a full antibody molecule translated from a

single ORF upon co-expression with sec-TEVp QSG variant. (A) schematic represen-

tation of the construct encoding in a single ORF the full-length mAb 14F7. L and

H chains are separated by TS. A single signal peptide (sec) was present at the N

terminus of the polyprotein. Upon co-expression with the ER active sec-TEVp QSG

mutant the two processed L and H chains assembled into a mature H2L2 species. (B)

WB of cellular extracts or supernatants of the sec-L-(TS)-H construct after expres-

Fig. 4. TEVp un-cleavable versions. (A) WB showing activity of sec-TEVp wt and

sion without or with the sec-TEVp QSG. Supernatants were run under reducing and

QSG mutants with modiﬁed C-termini on the MHC-I␣ reporter with TS in luminal

non-reducing conditions, as indicated. (C) Cytoﬂuorimetric analysis of P3 × 63 cells

position (upper panels) and resistance to auto-cleavage. (B) WB showing activity

that reacted with puriﬁed mAb 14F7 (positive control), or with supernatants from

of cyt-TEVp wt and QSG mutants with modiﬁed C-termini on the MHC-I␣ reporter

cells transfected with the 14F7 sec-L-(TS)-H construct alone (un-cleaved) or with

with TS in cytosolic position (upper panels) and resistance to auto-cleavage. (C)

sec-TEVp QSG (mature) followed by an Alexa488-conjugated goat anti-mouse anti-

WB of cell extracts (E) and supernatants (S) from cells expressing sec-TEVp wt-235

body. Blots and cytoﬂuorimetric analysis are representative of at least 3 independent

and variants QSG and QSG-235. Blots are representative of at least 3 independent experiments.

experiments.

oxidation. In fact, two N-glycosylations at N23 and N171 were

4. Discussion able to strongly impair sec-TEVp activity in the ER lumen. Of note,

while the conservative mutation of N23 into Q was well tolerated,

TEVp has been widely used both for in vitro applications and the same type of replacement in position 171 was much more

in vivo studies by expressing it in the cytoplasm to process pro- critical due to involvement of N171 in interactions with the sub-

teins with a cleavage site (Ceriani et al., 1998; Henrichs et al., 2005; strate (Phan et al., 2002). In fact, the sec-TEVp N171Q was almost

Melcher, 2000; Pauli et al., 2008; Satoh and Warren, 2008; Sun inactive within the ER lumen. In silico simulation of N171Q substi-

et al., 2012; Taxis et al., 2009). We observed, however, that the tution showed that the bulkier glutamine side chain clashed with

wt enzyme could not be similarly exploited within the secretory either the substrate or other TEVp residues in almost all theoretical

pathway by targeting it to the ER via a secretion signal peptide isomeric conformations, consistent with the experimental results.

at the N-terminus, as it was totally inactive on luminal substrate Conversely, mutation of the surface-exposed residue T173 into

proteins harboring a TS. We speculated this was due to inactiva- glycine was predicted to have no adverse effect on protein struc-

tion by characteristic post-translational modiﬁcations occurring ture, while preventing N171 glycosylation, as we experimentally

in the ER environment, such as N-glycosylation and cysteine observed. Interestingly, in contrast to our ﬁndings in mammalian

F. Cesaratto et al. / Journal of Biotechnology 212 (2015) 159–166 165

cells, it was recently reported a screening of sec-TEVp mutants in internal cleavage site and delimitation of VPg and proteinase domains.

Virology 183, 449–456, http://dx.doi.org/10.1016/0042-6822(91)90974-G.

yeast, where even wild type sec-TEVp was found to be active in

Gray, D.C., Mahrus, S., Wells, J.A., 2010. Activation of speciﬁc apoptotic caspases

the ER (Yi et al., 2013). This could be the consequence of lower N-

with an engineered small-molecule-activated protease. Cell 142, 637–646,

glycosylation efﬁciency in yeast cells upon over-expression of the http://dx.doi.org/10.1016/j.cell.2010.07.014.

sec-TEVp. Henrichs, T., Mikhaleva, N., Conz, C., Deuerling, E., Boyd, D., Zelazny, A., Bibi, E., Ban,

N., Ehrmann, M., 2005. Target-directed proteolysis at the ribosome. Proc. Natl.

Mutation of C130 into S also improved TEVp activity in the

Acad. Sci. U. S. A. 102, 4246–4251, http://dx.doi.org/10.1073/pnas.0408520102.

ER lumen, most likely due to interference of this highly exposed Kapust, R.B., Tözsér, J., Fox, J.D., Anderson, D.E., Cherry, S., Copeland, T.D., Waugh,

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bly into mature H L molecules in conditions of strict 1:1 molar

2 2 AAV-mediated in vivo long-term expression and antitumour activity of an

ratio of each polypeptide, as in the case here described, as opposed anti-ganglioside GM3(Neu5Gc) antibody. Gene Ther., 1–8, http://dx.doi.org/10.

1038/gt.2015.71.

to conditions in which L chain is expressed in excess in relation to

Principe, M., Ceruti, P., Shih, N., Chattaragada, M.S., Migliorini, P., Cappello, P.,

the H chain.

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two ganglioside-associated idiotypic networks. Immunobiology 212, 57–70,

http://dx.doi.org/10.1016/j.imbio.2006.08.005.

G.P. and F.C. were partially supported by ICGEB pre-doctoral

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fellowships. tether inhibits exocytic transport. Trafﬁc 9, 1522–1529, http://dx.doi.org/10.

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