IPR2022-00045, No. 2035 Exhibit - Graslund 2000 (P.T.A.B. Aug. 19, 2022) (2024)

`

`T.Graslund et al.
`
`more than 90% of the proteins have an isoelectric point
`between 4 and 7 (Link et al., 1997). Therefore, ion-exchange
`handles with positive charge recruited from lysines or arginines
`would theoretically be ideal, as it should allow for cation­
`exchange chromatography at high pH values where there
`is potentially less adsorption of contaminating E.coli host
`cell proteins.
`An alternative to the use of a charged peptide handle
`described by Sassenfeld and Brewer would be to utilize a
`protein domain capable of independent folding for accommoda­
`tion of charge. It should have the potential to be more
`proteolytically stable while still being able to confer a strong
`charge polarity in a fusion protein. Such charged domains can
`either be designed de novo based on e.g. helical motifs
`(Hoshino et al., 1997; SzilaketaZ., 1997) or by adding charges
`to an existing protein, recruited as a scaffold. In this work, we
`investigated the three-helix bundle domain Z as a scaffold for
`the accommodation of positive charge to allow for cation­
`exchange chromatography at high pH values.
`
`Materials and methods
`Escherichia coll strain RRIAM15 (Ruther, 1982) was used as
`a host for the cloning work and protein expression. Synthetic
`oligonucleotides were purchased from Interactiva (Ulm,
`Germany). DNA restriction and modifying enzymes were
`purchased from Amersham Pharmacia Biotech (Uppsala,
`Sweden) and In Vitro (Stockholm, Sweden) and used according
`to the manufacturers’ recommendations. Base composition of
`the constructed vectors was verified by dye-primer solid-phase
`DNA sequencing (Hultman et al., 1991).
`Reversed-phase high-performance liquid chromatography
`(RP-HPLC) was performed on an HP 1090 instrument from
`Hewlett-Packard (Waldbronn, Germany) using a C-18 column.
`All ion-exchange experiments were performed on an Akta-
`Explorer 100 system (Amersham Pharmacia Biotech).
`Molecular modelling
`Molecular modelling was performed on a Silicon Graphics
`Octane workstation (Access Graphics, Amstelveen, The
`Netherlands) using Sybyl (Tripos, St. Louis, MO). Energy
`minimization was carried out using a Kollman-all force field
`(Weiner et al., 1984, 1986) with standard parameters. The
`model of Zwt was constructed by adding hydrogens to the N-
`terminus of the NMR structure (Protein Databank entry 2SPZ,
`http://www.rcsb.org/pdb). Charges were loaded on to the mole­
`cule and five layers of water were added to the system. Energy
`minimization was carried out while keeping Zwt static, followed
`by energy minimization of the whole system. The mutants
`were constructed by exchanging the appropriate amino acids,
`followed by their manual alignment. Charges were loaded on
`to the system and it was energy minimized while keeping the
`backbone and non-mutated residues static. Five layers of water
`were added and energy minimization was performed while
`keeping the protein static and finally the whole system was
`energy minimized. Electrostatic surface potentials were calcu­
`lated using Delphi (Gilson and Honig, 1987; Nicholls and
`Honig, 1991). Molecular surfaces were built in Grasp (Nicholls
`et al., 1991).
`Assembly of Z variants
`Methods for recombinant DNA work were performed essen­
`tially as described by Sambrook et al. (1989). Oligonucleotides
`coding for helices 1 and 2 of the three Z variants were
`704
`
`assembled in a stepwise fashion analogous to the Z-library
`constructed by Nord et al. (1995). The base compositions of
`the constructs were verified and the resulting vectors were
`labelled pKNl-Zwt, pKNl-Zbasic) and pKNl-Zbasle2, respect­
`ively. Each one codes for one Z variant followed by a
`streptococcal Protein G-derived albumin-binding domain
`(ABD) (Eliasson et al., 1991).
`Construction of expression vectors
`The vectors pKNl-Zwt, pKNl-Zbasicl and pKNl-Zbasjc2 was
`used as templates in PCR {[96°C for 15 s; 59°C for 20 s;
`72°C for 90 s (30 times)], 72°C for 7 min] using the primers
`Zsub ।: 5' -CCCCG A ATTCCGTAG AC AAC A A ATTC A AC A A-
`3' and GRTO-I0: 5'-CCGGCCGGCTGCAGTTAATGGTGA-
`TGGTG ATGGTGTTTCGGCGCCTG AGC ATC ATTTAG-3'.
`The resulting PCR product was ligated to pGEM-5zf(+)
`(Promega, Madison, WI) and base composition was verified.
`The pGEM-5zf(+) construct was then restricted with EcoRi
`and PstI and the genes coding for the Z variants were isolated
`and ligated with pTrpABD (Kraulis et al., 1996) that had been
`cut with the same enzymes. The resulting vectors each code
`for a Z variant including a trp-leader followed by a His6-tag
`under control of the trp-promoter. The different vectors were
`named pTrp-Zwt, pTrp-Zbasic] and pTrp-Zbasic2.
`Production and purification of Zwl, Zbasic/ and ZbasiC2
`The pTrp constructs were grown at 30°C for 20 h in 1 1 of
`TSB + YE medium (30 g/1 tryptic soy broth (Lab M, Topley
`House), 5 g/1 yeast extract (Difco), 50 mg/1 kanamycin). Zwt
`was purified using IgG affinity chromatography as described
`(Nilsson et al., 1987), followed by RP-HPLC. Cell cultures
`containing Zbasic| or Zbasic2 were centrifuged and the resulting
`pellet was resuspended in -30 ml of buffer A (10 mM
`phosphate, pH 7) supplemented with 450 mM NaCl. The cells
`were then disrupted by sonication followed by centrifugation
`to remove cell debris. The supernatant containing soluble
`intracellular material was filtered through a 1.2 |im hydrophilic
`filter (Sartorius, Gottingen, Germany) and loaded on a 20 ml Q-
`Sepharose FF column, equilibrated with buffer A supplemented
`with 450 mM NaCl. The flow-through (50 ml) was collected
`and diluted to 300 ml with buffer A and loaded on a 20 ml
`S-Sepharose FF column that had been equilibrated with buffer
`A. The column was washed extensively and bound proteins
`were eluted using a linear NaCl gradient from 0 to 1 M.
`Fractions containing Zbasicl or Zbasic2 were pooled and further
`purified by RP-HPLC. The purity of Zwt, Zbasicl and Zbasic2
`was determined by loading ~4 pg of each protein on a 16%
`Tris-Tricine gel (Novex Electrophoresis, Frankfurt, Germany)
`and running it according to the manufacturer’s recommenda­
`tions (see Figure 3).
`Circular dichroism measurements of Z variants
`Data were collected using a lasco J-720 spectropolarimeter
`(Jasco, Tokyo, lapan) equipped with a thermostable water­
`bath for temperature control. The buffer used in all experiments
`was 10 mM phosphate, pH 7.0. Scan data were collected
`between 250 and 190 nm at 25 °C with a scanning speed of
`50 nm/min and each spectrum is an average of three consecutive
`scans. Data for the thermal melts were collected as the change
`in 0222 nm. The temperature gradient was 60°C/h.
`Cation-exchange analysis of Zwt, Zbasic/ and Zbasic2
`Cation-exchange analysis was performed on the purified pro­
`teins, by loading ~1 mg of each protein on to a 1 ml Resource
`S column (Amersham Pharmacia Biotech) that had been
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`Helix 3
`
`pH 7.5). The column was washed with running buffer and
`elution was performed using a linear gradient of NaCl from 0
`to 1 M.
`
`Charge engineering of Z
`
`Fig. 1. Amino acid alignments of the Z variants. Horizontal lines indicate
`amino acid identity and the full amino acid sequence of Zwl is listed at the
`top. The three boxes indicate the a-helices of Zwl as determined by Tashiro
`et al. (1997).
`
`equilibrated with 5 column volumes (CV) of running buffer
`(50 mM phosphate at pH 3 and 20 mM ethanolamine at pH
`9). The column was washed with 10-15 CV of running buffer,
`with subsequent elution with a linear gradient of 0-2 M NaCl
`for experiments performed at pH 3 or 0-1 M NaCI for all
`other experiments. The length of the NaCl gradient was 20 CV
`in all experiments and the chromatograms were recorded as
`the absorbance at 214 nm.
`Production and purification of Zbasic2-ABD
`Cells containing pKNl-Zbasic2 were grown in 1 1 batches in
`TSB + YE medium supplemented with ampicillin (100 mg/1)
`at 30°C for 24 h with induction by adding 240 mg of isopropyl-
`p-D-thiogalactoside (IPTG) at OD = 1. Cells were harvested,
`lysed by sonication, followed by affinity chromatography on
`human serum albumin (HSA) Sepharose as described by
`Nygren et al. (1988). Eluted fractions were pooled, freeze-
`dried and redissolved in 20% acetonitrile in water supplemented
`with 0.25% (v/v) of pentafluoropropionic acid. Zbasic2-ABD
`was further purified using RP-HPLC.
`Cation-exchange chromatographic analysis of Zbasic2~ABD
`Purified Zbasic2-ABD fusion protein was loaded on a 1 ml
`Resource S column previously equilibrated to the different pH
`values investigated. The column was washed with 10 CV of
`equilibration buffer and elution was performed using a linear
`gradient of 0-1 M NaCl. The buffers used were 20 mM 1,3-
`diaminopropane, pH 8.5 and 10.5, 20 mM ethanolamine, pH
`9.5, 50 mM phosphate, pH 7.5, and 20 mM bis-Tris propane,
`pH 6.5.
`Purification of Zbasic2-ABD from a whole cell lysate
`Purified Zbasic2-ABD fusion protein was spiked into a whole
`cell lysate from E.coli (50 |J.g fusion protein/ml cell lysate).
`The mixture was loaded on a 1 ml Resource S cation-exchange
`column equilibrated with running buffer (50 mM phosphate,
`
`Results
`Rationale for the design of Zbasic variants
`In this work we used the Z domain as a scaffold for creating
`highly charged protein domains. The Z domain is a compact
`58 amino acid (7 kDa) three-helix bundle derived from the B
`domain of staphylococcal protein A (Nilsson et al., 1987). It
`contains no cysteines and is highly soluble. The Z domain has
`been shown to help in solubilizing fused target proteins in vitro
`(Samuelsson et al., 1994) and is also stable against proteolysis
`in a number of different hosts (Stahl and Nygren, 1997). In
`addition, this domain has earlier been shown to be highly
`permissive to mutations in helices one and two in studies
`where it was used as a scaffold for combinatorial mutagenesis
`to isolate variants with novel binding specificities using phage
`display selection technology (Nord et al., 1995, 1997). An
`indication that the Z domain could also accommodate numerous
`basic amino acids on its surface came from panning experi­
`ments against the highly acidic target protein EB200, derived
`from Pfalciparum (Ahlborg et al., 1997), in which a highly
`basic variant (ZEB4) containing multiple positively charged
`amino acids was selected (Figure 1). That variant was shown
`to have a secondary structure content similar to that of the Zwt
`protein as determined by circular dichroism spectroscopy
`(E.Gunneriusson, personal communication). However, ZEB4
`contained two cysteines at positions 25 and 32 that were
`undesirable in a purification handle. Therefore, the first gene
`construct encoding a highly charged ion-exchange handle was
`ZEB4, with the two point-mutations C25S and C32S, designated
`ZbasicI. Its amino acid sequence is shown in Figure 1.
`A second variant where a total of 10 amino acids in helices
`one and two were changed into arginines was also constructed.
`It was labelled Zbasic2 and its amino acid sequence is also
`shown in Figure 1. All three proteins, Zwt and the two mutated
`proteins, were fitted with codons coding for six histidines in
`their C-terminal end in order to make it possible to purify them
`through immobilized metal affinity chromatography (IMAC)
`(Porath et al., 1975)
`
`Fig. 2. Model of the electrostatic potentials of the three Z variants. Colouring of Zwl left (A), Zbasicl middle (B) and Zbasic2 right (C) shows positive potential
`as blue and negative potential as red. The authors note the gradual increase in positive electrostatic potential from left to right which is in agreement with
`their behaviour in cation-exchange chromatography (see text).
`
`705
`
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`T.Graslund et al.
`
`1 2 3 M
`
`- 17,200
`- 14:600
`- 8,240
`-6,380
`- 2,560
`
`Fig. 3. SDS-PAGE analysis of purified proteins from E.coli cultures. Lane
`1: -4 jig of Zbasic2 from pooled material after the RP-HPLC step. Lane 2:
`-4 pg of Zbasic| from pooled material after the RP-HPLC step. Lane 3: -4
`pg of Zwl from pooled material after IgG affinity chromatography followed
`by RP-HPLC. Lane M: low molecular weight marker.
`
`Molecular modelling of Z variants
`Based on the solution structure of Z (Tashiro et al., 1997),
`energy-minimized models of Zw[, Zbasic] and Zbasic2 were
`calculated (data not shown). The model of Zwt shows a compact
`anti-parallel three-helix bundle very similar to the starting
`NMR structure. The models of ZbasicI and Zbasic2 have similar
`backbone traces but Zbasic2 has a larger molecular surface
`owing to its many arginines. ZbasicI, on the other hand, has a
`smaller molecular surface than Zwt. On close examination of
`the structures, we note that the arginines can participate in
`multiple hydrogen bonds, both with the backbone and with
`other amino acids in their vicinity, that was not possible
`for their substituents in Zwt. Electrostatic potentials for the
`molecular surfaces of the three Z variants were also determined
`and are displayed in Figure 2. This shows ZW1 to have a slightly
`hydrophobic molecular surface with about equal elements of
`positive and negative potential. Zbasjcl has a less hydrophobic
`molecular surface and a large positively charged area with
`islands of negative charge interspersed. Zbasic2 has an area (of
`-500 A2) with extremely high positive charge.
`Expression and purification of Z variants
`Zwt, Zbasici and Zbasic2 were successfully expressed as intracellu­
`lar proteins in E.coli. Zwt was purified on a resin containing
`immobilized IgG for capture and purification (Nilsson et al.,
`1987), after which it was -95% pure. Zbasicl and Zbasic2, on
`the other hand, do not bind IgG since several of the amino
`acids involved in IgG binding (Deisenhofer, 1981; Jendeberg
`et al., 1995) has been changed. They were instead purified
`using cation-exchange chromatography at pH 7 and purified
`to 90% purity for ZbasicI and >95% purity for Zbasic2 (data not
`shown). The material from the initial purification of the three
`variants was further purified by RP-HPLC to more than 95%
`purity, as can be seen in Figure 3.
`Structural characterization of Zw„ Zbasicl and Zbasic2
`In order to characterize the secondary structure of the Zbasic
`variants, their absorption of far-ultraviolet circular polarized
`light was measured between 190 and 250 nm. The CD spectra
`collected for Zwt, Zbasicl and Zbasic2 are shown in Figure 4. The
`double minima for all three proteins at 222 and 209 nm and
`a high peak around 195 nm are characteristic for proteins
`with a high a-helical content. Tashiro and co-workers have
`determined the structure of Zwt and they have assigned 65%
`of the amino acids in a-helical conformation (Tashiro et al.,
`706
`
`Fig. 4. Circular dichroism spectra of the Z variants between 190 and 250
`nm. Spectra were collected on a Jasco J-720 spectropolarimeter at 25°C in
`10 inM phosphate buffer. pH 7. Protein concentrations were between 10 and
`15 pM and the cuvette used had a pathlength of 1 mm. The scanning speed
`was 50 nm/min and each spectrum is the average of three consecutive
`scans.
`
`Fig. 5. Temperature denaturation curves of Z variants. Spectra were
`recorded at 222 nm in 10 mM potassium phosphate buffer at pH 7. Protein
`concentrations were between 1 and 1.5 pM and the cuvette had a pathlength
`of 10 mm. The temperature gradient used was 60°C/h.
`
`Table I. Characteristics of the Z variants
`
`Z variant
`
`zwl
`2-basic 1
`Zbasic2
`
`Tm
`(°C)
`
`75
`62
`40
`
`©222 nm (25°C)
`deg*/cm2*/dmol 11 (g/mol)
`
`Measured
`pl
`
`Calculated
`pl
`
`-14700
`-9969
`-11971
`
`9518
`9326
`9787
`
`6.4
`8.2
`>9.3
`
`6.01
`9.10
`10.53
`
`1997). The 0222 nm response for the mutants are -70 and -80%
`for Zbasic| and Zbasic2, respectively, of the response for Zwt.
`Based on the similarity of the three spectra, we concluded that
`both Zbasici and Zbasic2 also have a high a-helical content
`although lower than that of Zwt, which is to be expected owing
`to the numerous mutations introduced.
`Temperature stability measurements of Zw„ Zbasici and Zbasic2
`The thermal denaturation of the three Z variants is shown in
`Figure 5. The melting curves show only one transition region
`each. Therefore, a two-state folding mechanism was assumed.
`The melting points were calculated using standard techniques
`(Creighton, 1997) and are displayed in Table I. Zwt melts at
`the highest temperature (75°C) while Zbasici melts at a lower
`
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`Charge engineering of Z
`
`10
`
`20
`
`30
`
`40
`
`Fig. 7. Cation-exchange chromatogram of Z(,asjc2-ABD at pH 7.5. The
`running buffer consisted of 50 mM phosphate. About I mg of purified
`protein was loaded on a 1 ml Resource S column and elution was
`performed with a linear NaCl gradient from 0 to 1 M. The absorbance was
`recorded at 280 nm.
`
`isoelectric point for Zwt was determined to 6.4 and for Zbasicl
`to 8.2. Zbasic2 focuses at the negative electrode since its pl is
`higher than that which can be resolved on the gel used (pH
`3.5 to 10). Its pl was determined as >9.3 as that corresponds
`to the marker of highest pl that can be resolved on the gel.
`Cation-exchange chromatographic characterization of
`Zbasic2~ABD
`In order to investigate if the chromatographic properties of the
`Zbasic2 domain were preserved also after fusion to a target
`protein, Zbasic2 and an albumin-binding domain (ABDpI 5)
`derived from streptococcal protein G were fused on the genetic
`level. The gene fusion, Zbasic2-ABD, was expressed as a
`periplasmically secreted protein and the resulting gene product
`was purified to hom*ogeneity (see Materials and methods).
`Purified Zbasic2-ABD fusion protein was loaded on a cation­
`exchange column at different pH values and eluted with a
`linear gradient of NaCl from 0 to 1 M. As previously seen for
`the free Zbasic2 domain, this fusion protein could be recovered
`by cation exchange at pH values ranging from 6.5 to 10.5 (see
`the example in Figure 7). However, at pH > 7.5, apparently
`lower yields were obtained (see Discussion).
`Purification of Zhasic2~ABD from a whole cell lysate by
`cation-exchange chromatography
`In order to investigate the usefulness of the positively charged
`Zbasic2 domain as a purification tag, Zbasic2-ABD fusion protein
`was spiked into a total cell extract from E.coli and loaded on a
`cation-exchange column at pH 7.5. The resulting chromatogram
`(Figure 8) demonstrates that it was possible to capture the
`protein by the cation-exchange column and also that the
`selectivity was high, leading to a product of >90% purity.
`
`Discussion
`An attractive strategy to facilitate recombinant protein recovery
`is to fuse a charged moiety to the recombinant protein, thus
`allowing efficient adsorption by an ion-exchange resin (for a
`review, see Ford et al., 1991). Previously, large efforts have
`been made to construct handles consisting of 5-15 arginines.
`However, arginine handles are often susceptible to trypsin-like
`proteases in the recombinant host and the yield of intact
`707
`
`Fig. 6. Cation-exchange chromatograms of the three Z variants. About 1 mg
`of each protein was loaded on a 1 ml Resource S column. (A) Overlay of
`the chromatograms recorded at pH 3. (B) Overlay plot of the
`chromatograms obtained at pH 9.
`
`temperature (62°C) and Zbasic2 melts at the lowest temperature
`(40°C). All three proteins show a slow loss of a-helicity at
`the pre- and post-transition regions.
`Cation-exchange chromatographic characterization of the Z
`variants
`Purified proteins (1 mg) were loaded on a cation-exchange
`column. At pH 3 all three proteins were binding quantitatively
`to the resin as shown in Figure 6A using linear NaCl gradient
`elution. Zwt was eluted at an NaCl concentration corresponding
`to a conductivity of 60 mS/cm, whereas Zbasicl was more
`tightly bound to the ion-exchanging groups and therefore
`eluted at a higher NaCl concentration corresponding to
`80 mS/cm. Zbasic2, the most basic variant, was eluted at
`110 mS/cm corresponding to the highest NaCl concentration.
`At pH 9 the Zwt protein was only detected in the flow-
`through (Figure 6B), whereas both Zbasicl and Zbasic2 were still
`quantitatively adsorbed at this pH. Both mutants were eluted
`at lower NaCl concentrations than at pH 3, corresponding to
`20 and 40 mS/cm, respectively (Figure 6B). These ion­
`exchange results showed that Zwt was positively charged at
`pH 3 but negatively charged at pH 9 (Figure 6A and B). Zbasicl
`and Zbasic2, on the other hand, were positively charged at both
`pH 3 and 9 (Figure 6A and B). Zbasicl lost its binding to the
`cation-exchange column when the pH was increased above 9.
`Zbasic2 was completely adsorbed on the column at both pH 10
`and 11. However, at pH 11 the amount of Zbasic2 eluted from
`the column was significantly reduced. The pl values were also
`measured using isoelectric focusing and the values are shown
`along with theoretically calculated values in Table I. The
`
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`T.Graslund et al.
`
`Fig. 8. Cation-exchange chromatogram showing the purification of the
`Zbasic2-ABD fusion protein spiked into a whole cell lysate from E.coli. The
`purification was performed using 50 mM phosphate (pH 7.5) as running
`buffer and elution was performed using a linear gradient of NaCl from 0 to
`I M. Inset: SDS-PAGE analysis of samples collected at different stages of
`the procedure. Lane 1, starting material; lane 2, flow-through; lane 3, pooled
`main peak fractions. The absorbance was recorded at 215 nm.
`
`product is small. Also, severe growth defects and a high
`proportion of revertants not expressing the charged handle
`have been reported (Skerra et al., 1991). The use of charged
`handles has therefore not been commonly used recently. In
`this paper, we suggest an alternative strategy that might be
`more attractive for such applications. The charge is introduced
`by protein design into a highly ordered protein domain, here
`exemplified by the stable three-helix bundle domain, from the
`bacterial receptor protein A. This domain Z, normally acidic,
`could be made at least three pl units more basic. The use of
`the Z domain as compared with charged peptides has several
`advantages. First, the protein A domain is highly soluble and
`can increase the solubility of fusion proteins that otherwise
`are insoluble (Samuelsson et al., 1994). Second, basic amino
`acids in ordered structures are poorly recognized by trypsin­
`like proteases (Wang et al., 1989; Jonasson et al., 1996). The
`likelihood of obtaining large amounts of full-length fusion
`proteins with intact charge is thereby significantly increased.
`Third, the introduction of charges into a highly ordered
`structure allows the charge to be distributed in a predicted
`way over a large surface, i.e. 500 A2 (Figure 2). Two strategies
`were used to design the new Z variants. First, a variant selected
`from a phage combinatorial library by binding to an acidic
`protein was used as a basis for limited directed mutagenesis.
`Two cysteines obtained in the protein after selection were
`replaced by serines. Altogether 13 residues out of 58 differ
`compared with the parental Z molecule. In the second variant,
`Zbasic2, even more charge was introduced into helices one and
`two. In total, 10 of the surface-exposed amino acids were
`changed into positively charged residues. For the design of
`this variant, comparison of the two basic amino acids lysine
`and arginine shows that each 5-guanido group in arginine has
`two potential ways of stabilizing the molecule through hydro­
`gen bonding with its 0-N and y-N compared with the single
`NH4 group of lysine. In addition, arginine has been shown to
`participate more readily in strong hydrogen bonding networks
`such as in sperm whale myoglobins, where the variant with
`Arg45 is more stable than the variant that has a lysine at
`708
`
`Table II. Cation-exchange chromatographic characterization of Z variants
`
`PH
`
`3
`9
`10
`II
`
`^wi
`
`60
`
`ND1’
`ND
`
`^basie 1
`
`^basic2
`
`80
`20
`-a
`ND
`
`110
`40
`35
`+c
`
`The values represent the conductivity (mS/cm) of the buffer when the
`protein is released from the cation exchange resin.
`aNo protein is adsorbed by the ion-exchange resin.
`bND, not determined.
`cSome protein is adsorbed.
`
`position 45 (Matthew et al., 1985). Arginine was also expected
`to give less charge repulsion than lysine since its charge is
`delocalized over a larger van der Waals area. Arginine was
`therefore chosen as a better amino acid to incorporate in the
`ion-exchange handle than lysine. Two of the amino acids
`changed in Zbasicl were left unmodified as they help stabilize
`the protein; Hl 8 forms the C-cap of helix one and E25 is
`involved in N-capping of helix two (Olszewski et al., 1996;
`Tashiro et al., 1997).
`Both new variants could be produced in E.coli and purified
`by different means, indicating that they were stable molecules
`and correctly folded despite their high content of positively
`charged amino acids. Far-ultraviolet CD measurements on the
`mutants showed that their a-helical content is of the same
`order of magnitude as that of their parental molecule Zwt.
`Thermal denaturation of the mutants showed that they have
`significantly lower melting points than the wild-type protein.
`This indicates that it is favourable to start with a scaffold with
`high thermal stability, as was done in this work to ensure that
`the stability of the protein after charge engineering does not
`decrease to very low values.
`An interesting observation was the highly varied migration
`in the SDS electrophoresis analysis of the various variants
`(Figure 3). The theoretical molecular weights differ by only
`0.5 kDa between the variants, while the apparent sizes, judged
`by the electrophoresis, differ by -2 kDa. Analysis using
`electrospray mass spectrometry confirmed that the proteins
`have molecular weights identical with the theoretical values.
`A comparison between the wild-type Z domain and the
`engineered variants in cation-exchange experiments demon­
`strated a dramatic difference in chromatographic performance.
`At low pH values the basic variants were eluted at much
`higher salt concentrations than the wild-type variant and at
`elevated pH values only the engineered variants could be
`adsorbed on the chromatographic resin (Table II). These pH-
`dependent binding properties followed the expected