Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site

David P. Humphreys1, Lloyd M. King, Shauna M. West, Andrew P. Chapman, Mukesh Sehdev, Matthew W. Redden, David J. Glover, Bryan J. Smith and Paul E. Stephens

Celltech Therapeutics, 216 Bath Road, Slough, Berkshire SL1 4EN, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The peptide sequence NDKTHC was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized {gamma}1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu2+, such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni2+. Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.

Keywords: copper(II)/cupric/Fab'/FLAG peptide/protein cleavage


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many proteins of scientific or commercial interest are made as fusions with protein domains that confer beneficial purification, production or temporary functional properties. The removal of these fusion domains by proteolysis can be variably ineffective or expensive, particularly if this is to be done on a large scale. In an effort to identify a process for large-scale and inexpensive proteolysis, we had previously studied the tetrapeptide sequence NDKTHC as a target for Cu2+-catalysed protein cleavage (Humphreys et al., 1999Go).

The upper hinge tetrapeptide sequence NDKTHC had been identified as the site of cleavage of a highly purified recombinant humanized {gamma}1 antibody by traces of Cu2+. The sequence was cleaved between the Lys and Thr residues. The degree of cleavage increased with increasing temperature, length of time and pH of incubation and also with increasing amounts of available Cu2+ ions. The NDKTHC sequence was also cleaved by Cu2+ when present in a peptide (Smith et al., 1996Go).

We had previously used a humanized {gamma}1 Fab' with a C-terminal FLAG peptide as a model protein. An NDKTHC site was introduced between the hinge and the FLAG peptide, after mutagenesis of the native NDKTHC sequence in the upper hinge to create a `null' site. Dimerization of the Fab' to F(ab')2 via the hinge cysteines, followed by incubation at 62°C, pH 9.0 for 8–28 h demonstrated site-specific cleavage and loss of the FLAG peptide. The F(ab')2 remained in the dimeric state, proving that the `null' site was not cleaved by Cu2+. Treatment with Cu2+did not affect the protein since it was intact and retained antigen binding ability of the F(ab')2. Although cleavage of the NDKTHC sequence had approached 86% efficiency, the cleavage reactions were considered too long and harsh for many applications.

The cleavage site is thought simply to act as a Cu2+-specific chelator, with amide bond cleavage caused by a non-specified Cu2+-catalysed free radical reaction. In an attempt to improve the efficiency of the cleavage reaction, we made here single and double conservative amino acid substitutions in the cleavage site. Fine tuning of the proposed chelation site might result in a higher affinity for Cu2+, better access to ancillary reaction molecules (H2O/OH/OH·) and possibly altered metal ion specificity. Changes made to the NDKTHC sequence in short peptides was known to result in altered Cu2+-specific cleavage (Allen and Campbell, 1996Go). Hence we made D -> E, K -> R, T -> S single substitutions and D + T -> E + S and K + T -> R + S double substitutions in our model protein. Two of these were found to be cleaved much more efficiently by Cu2+ than the wild-type sequence and four of them were found to be measurably cleaved (20–30%) by Ni2+.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nomenclature of new cleavage variants

The NDKTHC sequence (CUT 1) was the basis of all of the new variants, which were numbered sequentially and annotated in order to highlight the residue that had changed in each case. For example, CUT 2E has the sequence NEKTHC and CUT 3R has the sequence NDRTHC. The abbreviations LC, HC and di-HC are used to denote light chain, heavy chain and dimeric heavy chain, respectively.

Construction of new cleavage variants of `Null 2 FLAG' expression plasmid

During construction of pDPH76 `Fab 40.4 hinge 1 {Delta} inter Null 2 FLAG' unique BspEI and ScaI restriction sites were introduced into the middle hinge and `elbow' spacer regions, respectively. New cleavage site coding regions were introduced as annealed BspEI–NotI oligonucleotide cassettes into BspEI–NotI restricted plasmid pDPH76 (Humphreys et al., 1999Go). Codons preferred by Escherichia coli were used (Wada et al., 1991Go) The sequence of the oligonucleotide encoded region was confirmed by sequencing of both strands using a PRISM cycle sequencing kit and an ABI 373A sequencing machine.

pDPH76 `Fab 40.4 hinge 1 {Delta} inter Null 2 FLAG' was derived from plasmid pDPH40 which encodes `Fab 40.4 hinge 1/2 {Delta} inter'. pDPH40 has a SpeI restriction site introduced at the 3' end of the heavy chain (HC) coding region, where the interchain disulphide Cys had been mutated to a Ser, along with an adjacent Ser to Thr change (Humphreys et al., 1997Go).

Production and purification of `Null 2 FLAG' F(ab')2. High cell density bacterial fermentations of strain W3110 bearing the plasmids shown in Table IGo were performed as described previously (Humphreys et al., 1997Go). Extraction of periplasmic material,protein G purification of Fab', production and phenyl-Sepharose purification of F(ab')2 were performed as described previously (Humphreys et al., 1998Go). F(ab')2 protein was concentrated and buffer exchanged with several volumes of PBS using an Amicon pressurized stirred cell with a 10 kDa cut-off membrane to concentrations between 1.0 and 5.0 mg/ml and sterilized with a 0.22 µM filter for storage at –70°C.


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Table I. Plasmid and oligonucleotide details
 
Cu2+ cleavage reactions. F(ab')2 was at 0.33 mg/ml in all reactions, Tris and other buffers were used at a final concentration of 50 mM (from 50 mM stock solutions). Tris was used at pH 9.0 unless stated otherwise. All metal ions tested were chloride salts from 10x stocks for each concentration tested dissolved in H2O. Reaction volumes were most commonly 15 µl in 500 µl Eppendorf tubes, but were also scaled up to 500 µl. To eliminate evaporation during lengthy high-temperature incubations (55–62°C), reactions were layered with paraffin oil. All incubations were carried out in duplicate in a Biometra TRIO-Thermoblock with heated lids for 15 h unless indicated otherwise. Samples of 1.33 µg were withdrawn through the paraffin oil for immediate SDS–PAGE or into 10 mM EDTA (final concentration) and stored at –20°C during time-course experiments.

SDS–PAGE analysis of `Null 2 FLAG' cleavage by Cu2+, surface plasmon resonance and mass spectrometry. These were all performed as described previously (Humphreys et al., 1999Go), except that 1.33 µg of protein per lane was used for SDS–PAGE.

N-terminal sequencing of cleaved FLAG tail from F(ab')2

Large-volume cleavage reactions had paraffin oil removed by pipetting and the low molecular weight cleaved tails separated from the F(ab')2 by centrifugation through a 10 kDa cut-off Minicon spin column (Amicon). 100 µl of each peptide containing solution (50 mM Tris, 2.5 mM CuCl2, pH 9.0), was passed through poly(vinylidene difluoride) (PVDF) membrane in a Prosorb device (PE Biosystems). Peptides were trapped on the membrane, whereas salts passed through it. Polybrene (Biobrene, PE Biosystems) was added to each PVDF membrane in order to minimize peptide washout: 500 µg Biobrene in 7 µl of 30% (v/v) methanol in 0.1% (v/v) TFA were applied to each PVDF sample for 15s, then excess solution was removed prior to thorough drying. The PVDF membranes were each sequenced in a PE Biosystems 494 Procise protein sequencer according to the manufacturer's guidelines.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Characterization of new cleavage variants

Initially three single substitution variants (CUT 2E, CUT 3R and CUT 4S) were made and analysed for any cleavage by Cu2+. Surprisingly, all three were found to be cleaved by Cu2+, although CUT 2E was clearly the least efficient. Therefore, we decided to make two double substitution variants (CUT 5ES and CUT 7RS). Other proposed variants containing the D -> E substitution were not made, i.e. CUT 6ER and CUT 8ERS. All five Fab's were analysed by SDS–PAGE and mass spectrometry. Under non-reducing denaturing conditions all five were found to contain a consistent amount (~25%) of a band that migrated just in front of the di-HC band. However, the HC was single banded upon reduction (Figure 1Go, lanes 6–10) and gave single mass determinations (see Table IIGo), showing this fast running HC band to be a gel artifact. The fast running artifactual band may be a result of strong non-covalent interactions involving the new cleavage sites or may be due to the much higher protein concentration and different storage conditions (–70°C). These proteins were found to be degraded slowly if stored at 4°C over a period of months, presumably by traces of metal ions in PBS.



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Fig. 1. SDS–PAGE of purified proteins and typical cleavage reactions. Lanes 1– 5 show non-reduced CUT 4S uncleaved (lane 1), cleaved with 2.5 mM Cu2+, pH 9.0, 62°C for 8 h (lane 2), 15 h (lane 3) and 24 h (lane 4) and 2.5 mM Ni2+, pH 9.0, 62°C for 24 h (lane 5). Reducing SDS–PAGE of proteins are shown in lanes 6–10: CUT 2E (lane 6), CUT 3R (lane 7), CUT 4S (lane 8), CUT 5ES (lane 9) and CUT7 RS (lane 10). Arrows H and L denote positions of HC and LC, respectively, and arrows U, S and D show the positions of uncleaved, singly cleaved and doubly cleaved di-HCs, respectively. Molecular weight markers are on the left and from the top are 250, 148, 60, 42, 30, 22, 17, 6 and 4 kDa.

 

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Table II. Mass spectrometric analysis of new CUT site Cu2+-catalysed cleavage
 
Effect of pH on efficiency of cleavage by Cu2+

We had seen with the CUT 1 sequence that elevation of pH resulted in increased cleavage efficiencies, such that pH 9.0 was used for all subsequent cleavage tests as a balance between maximal cleavage efficiency and minimal harshness of the cleavage conditions (CUT 1 was ~50% cleaved after 15 h at pH 9.0, 62°C). Figure 2Go shows that after 15 h at 62°C three of the new variants (CUT 4S, CUT 5ES and CUT 3R) are cleaved well at pH 8.0. CUT 2E and CUT 7RS are cleaved less well in general and require a pH of 8.0 before showing any significant cleavage. The pH requirement of four of the proteins (CUT 3R, CUT 4S, CUT 5ES and CUT 7RS) reaches a plateau at pH 8.0, whilst CUT2E preferentially requires pH 9.0. The buffer at pH 8.0/62°C actually has a real value of pH {approx}7.0 owing to the temperature-dependent nature of Tris buffers (Dawson et al., 1986Go). This reduced pH requirement for CUT 4S and CUT 5ES represents a significant improvement, as it will allow the use of milder cleavage reactions that may be more generally applicable to other proteins.



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Fig. 2. Effect of pH on efficiency of cleavage of new CUT variants.

 
Cleavage of new variants by Ni2+

We tested the new proteins for cleavage by transition metal and other metal ions. As seen previously with CUT 1 (Humphreys et al., 1999Go), Ca2+, Fe3+, Mg2+, Mn2+ and Zn2+ at 2.5mM did not cleave the new target sites after incubation at 62°C, pH 9.0 for 15 h (data not shown). However, Ni2+ was variably found to cleave the new sites (Figure 3Go). After incubation for 24 h cleavage was seen to exceed 30% for CUT 4S and CUT 5ES, whilst even the less efficient CUT 3R and CUT 7RS were found to be cleaved ~20%. Hence, by making minor alterations to the chelation site, we have been able to effect changes in the metal ion specificities of the sites. This is unlikely to have a practical impact where Ni2+ affinity purification of His-tag proteins is performed, since these procedures would be in the order of <1 h and <=25°C. Since Ni2+ had been able to cleave some of these sites, we looked at what molecular properties (such as electron structure, ionic radius and hydration energy) Cu2+ has that are partially mimicked by Ni2+ and whether other ions could also cleave these sites. Co2+ appeared to catalyse trace cleavage (<=10%) of CUT 4S and CUT 5ES, but the extent of cleavage was difficult to estimate owing to poor separation of the singly cleaved and uncleaved di-HC species. Cr2+ and Cr3+ did not cleave any of these proteins at pH 9.0 (data not shown).



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Fig. 3. Cleavage of new CUT variants by Ni2+.

 
Maximizing efficiency of cleavage of CUT 4S and CUT 5ES by Cu2+

Damage of proteins by metal ions has been found to be accelerated by H2O2 and or ascorbate (Samuni et al., 1981Go; Tang et al., 1996Go) and cleavage of peptides by metal ions accelerated by chaotropic agents (Allen and Campbell, 1996Go). However, with CUT 4S, we find no increase in cleavage efficiency: by the inclusion of H2O2 between 1% (v/v) and 0.00001% (v/v) with and without 50 µM ascorbic acid, ascorbic acid between 5 mM and 5 µM, guanidine chloride between 100 and 16.6 mM, sodium chloride between 500 and 100 mM and buffers based on sodium phosphate, sodium acetate or sodium hydrogencarbonate (data not shown). In addition, pre-heating the Tris and Cu2+ components in solution at 62°C for 24 h in an attempt to pre-generate the unknown cleavage active species did not increase the efficiency of cleavage (data not shown).

We then analysed the time, temperature and pH dependence of Cu2+ cleavage of our two best cleavage sites: CUT 4S and CUT 5ES. Figure 4Go shows that CUT 4S is cleaved more efficiently than CUT 5ES (Figure 5Go), that there is very little difference between cleavage at pH 8.0 and 9.0 and that significant levels of cleavage can be achieved by incubation at 55°C. With CUT 1 at pH 9.0 the maximum cleavage achieved was ~86% after 28.5 h. CUT 5ES reaches ~92% cleavage after 30 h under similar conditions, but is improved over CUT 1 in that this is also achieved at pH 8.0. CUT 4S is much improved in that it reaches 100% cleavage after 24 h at either pH 8.0 or 9.0. More importantly, the time dependence for CUT 4S is vastly improved over that of CUT 1; 50% cleavage is reached in ~15 h for CUT 1 but in only ~6 h for CUT 4S and practical levels of cleavage (~80%) are achieved in ~14 h with CUT 4S.



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Fig. 4. Effect of pH and temperature on the Cu2+ cleavage of CUT 4S.

 


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Fig. 5. Effect of pH and temperature on the Cu2+ cleavage of CUT 5ES.

 
The improvement in the cleavage efficiency of CUT 4S did not permit reduction of the temperature of incubation to allow the use of very mild conditions. Reduction to 55°C resulted in a significant impact on the cleavage efficiency of CUT 4S (~50% cleavage after 30 h).

Specificity of Cu2+-catalysed protein cleavage

Mass spectrometry was used to estimate the accuracy of cleavage of these new sites by Cu2+. Table IIGo shows that all of the sites were cleaved in the expected place, i.e. in the middle of the four residues, between the basic and polar/hydrophilic residues. Both the LC and HC species were consistently found to contain extra mass units in multiples of ~25. These are commonly seen, are non-reducible and are thought to be Na+ adduct series of the proteins. The HC is the more susceptible of the two chains and only the LC can be detected in an unmodified form. However, the relative mass differences between the cleaved and uncleaved proteins are an accurate measurement of the cleavage of the FLAG tail. Use of larger amounts of these proteins allowed the estimation of the extent of cleavage to be made. These mass spectrometric data show that CUT 4S is ~90% cleaved after 15 h at 62°C, pH 9.0, supporting the SDS–PAGE analysis. The rank order of cleavage efficiency estimated by this method is CUT 4S > CUT 5ES > CUT 3R > CUT 2E {approx} CUT 7RS, which also supports that found by the SDS–PAGE analysis. N-terminal sequencing of the C-terminal peptides liberated by the cleavage reactions showed that all of the singly modified cleavage sites were cleaved in the predicted site. The following sequences were deduced: CUT 2E NTHTIEGSC, CUT 3R NTHTIEGSTXDC, CUT 4S NSHTIEGSTC. In addition, the efficiently cleaved double mutant CUT 5ES was also cleaved accurately: NSHTIEGSTXDYKKC (X = no residue identified). Use of spin colums to isolate the cleaved tails also demonstrates that there are no other prevalent fragments of the cleaved F(ab')2 < 10 kDa.

Effect of cleavage conditions on protein function

BIACORE measurements on Cu2+ cleaved and uncleaved CUT 4S and CUT 5ES show no significant difference between the affinities of the proteins after being subjected to a 15 h high-temperature `alkaline' incubation with Cu2+ (Table IIIGo). This technique had been used previously to probe for metal ion damage to CDR residues or to the structural integrity of the Fab' arm (Humphreys et al., 1999Go). These new variants show as before that the incubation conditions do not cause substantial non-specific cleavage of the Fab' protein.


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Table III. Effect of incubation conditions on affinity of new CUT variants of `Null 2 FLAG F(ab')2
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have used conservative amino acid substitutions of the previously reported Cu2+ cleavage site (CUT 1) in an attempt to improve the cleavage efficiency. We have shown here that these changes can result in faster cleavage, use of lower pH buffers, lower temperatures and even altered metal ion specificity. The mechanism of action of these changes is not known, although it seems most likely that it is effected through change of the Cu2+ chelation properties of the sites. In particular, the ability of Ni2+ to cleave two of the variants (CUT 4S and CUT 5ES) with modest efficiency was a surprise. We did not alter the histidine residue of the cleavage site, since there is no obvious substitute residue.

The individual and combined effects of the substitutions are difficult to rationalize. The T -> S change was the single most beneficial change. This may potentially generate a slightly more volumous/flexible chelation site than the original. The single worst change was D -> E, which may have resulted in a less volumous/flexible chelation site. However, when these two changes were combined in CUT 5ES, the effectiveness of the T -> S change was hardly diminished. The K -> R change produced a site that was essentially unchanged from CUT 1 in its cleavage efficiency at pH 9.0. This too may have resulted in a less volumous/flexible chelation site. However, when this was combined with the T -> S change it produced the least efficient of all six sites tested so far. These complications validate the empirical approach used here.

What are the unique properties of Cu2+ that make it proficient as a catalyst of protein cleavage? Ionic valency, radius, hydration and chelation/coordination properties are likely to be important factors. Cu2+ properties are partly explained by its electron structure (1s2, 2s2, 2p6, 3s2, 3p6, 3d10, 4s1) and small size. Cu2+ has to break its (stabilizing) full 3d shell to reach the cupric (II) state. Its hydration energy is the highest of the divalent ions of the first transition series and it preferably forms tetravalent coordination complexes (Cotton and Wilkinson, 1972Go). Ni2+ has very similar hydration and coordination behaviour, but its ionic radius is smaller than that of Cu2+. Co2+, on the other hand, has a coordination behaviour and ionic radius almost identical with those of Cu2+, but a lower hydration energy. Cr seemed the most likely to match the electron behaviour of Cu, owing to its half-full 3d shell and single 4s electron (electron structure 1s2, 2s2, 2p6, 3s2, 3p6, 3d5, 4s1). However, the ionic radius of Cr2+ is larger than that of Cu2+ and the hydration energy is lower, which may explain why it does not catalyse cleavage of these peptide sites. In short, the order of activity seen here corresponds with the extended Irving–Williams series that describes the coordination of metal ions with proteins: Cu2+ > Ni2+ >= Zn2+ > Co2+ > Fe2+ > Mn2+ > Mg2+ > Ca2+ (Irving and Williams, 1953Go).

Here and in our related previous paper we tried many different biochemical approaches to improve the efficiency of cleavage of the target sequences by metal ions including different buffers, pHs, metal ions, use of H2O2 ± ascorbate, use of chaotropic agents and even a pre-incubation of Tris and Cu2+ at 62°C to try to generate a `reactive species'. Apart from pH, these biochemical approaches failed to provide significant improvements of the cleavage reaction. In fact, the only significant improvements have come from the mutagenic, protein engineering approach taken here. A detailed understanding of the biochemistry of the cleavage reaction may certainly yield further improvements. However, the small-scale and complex biochemistry of the model protein-based experiments performed here preclude such an analysis. Peptide-based experiments may help in this respect. Since protein engineering yielded the most improvements so far, we wonder whether there may be other short peptide sequences that can chelate and be cleaved efficiently by other transition metal ions, perhaps improving on that seen with NDKSHC and Cu2+? In particular, we wonder whether inclusion of flexible spacing residues such as Gly between some of the chelating residues of NDKSHC might create a more efficient cleavage site due to higher affinity coordination of Cu2+.

In summary, the tetrapeptide sequence NDKSHC has considerably improved efficiency as a site for cleavage by Cu2+ ions. This target sequence was also found to be cleaved more efficiently when part of a small peptide (Allen and Campbell, 1996Go). It conserves the specificity of cleavage demonstrated for CUT 1 and so leaves just two amino acids on the `mature' part of a protein fusion. This feature need not be a problem, where it could be incorporated in a part of a protein that already contains DK or SH as an inherent part of their sequence (such as where the FLAG peptide NDYKDDDDKC or the enterokinase target sequence NDDDDKC is already used). In fact, there are now four possibilities for inclusion of a Cu2+ cleavage site of different sequences in order that two of the amino acids may be hidden in `native' sequence: two high-activity sites, NDKSHC and NEKSHC, and two medium-activity sites, NDKTHC and NDRSHC.


    Acknowledgments
 
We thank Alan Lyons and Daniel Farley for expert DNA sequencing and Dominic Reeks for help with fermentations.


    Notes
 
1 To whom correspondence should be addressed. E-mail: dhumphre{at}celltech.co.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Allen,G. and Campbell,R.O. (1996) Int. J. Pept. Protein Res., 48, 265–273.[ISI][Medline]

Cotton,F.A. and Wilkinson,G. (eds) (1972) Advanced Inorganic Chemistry. Interscience, New York.

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Received October 12, 1999; revised January 12, 2000; accepted January 19, 2000.





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