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ARTICLES

Process Engineering Strategy for Recombinant Protein Recovery from Canola by Cation Exchange Chromatography

Chenming Zhang and Charles E. Glatz*

Department of Chemical Engineering, Iowa State University, Ames, Iowa 50011

The suitability of canola as a recombinant protein production host was evaluated in terms of the potential for simple chromatographic recovery by ion exchange. To investigate the influence of the charge of a recombinant protein on recovery from canola, a series of mutants of T4 lysozyme of varying charge were used to model the situation of transgenic expression by being spiked into nontransgenic canola protein extracts. This mixture was then fractionated by cation exchange chromatography. Two different means of charge modification were compared, namely, point mutations and fusions. Point mutations proved the better means for adding positive charges. A linear relationship between the protein charge and the eluent conductivity, which could be used to guide the genetic engineering for protein recovery from canola, was found. It showed that an increase of +1 charge on T4 lysozyme increased the required conductivity (molarity) of the eluent by 0.068 mS/cm (27.8 mM NaCl). For this specific case, T4 lysozyme with a nominal charge of +7 moves the point of elution into a valley between two major native canola protein peaks, where substantial one-step enrichment can be obtained. Equivalent charge changes provided by polyarginine fusions gave very wide elution patterns that were ascribed to either proteolytic degradation within the polyarginine fusion or interaction of the polyarginine with polyanions present in canola. While the above results came after a dialysis step to adjust the canola extract, elimination of the dialysis step did not significantly influence the purification behavior of the cation-exchange column. However, a more severe resin regeneration scheme was needed to maintain the column’s performance.

Introduction

Plant agriculture is attracting attention for its poten- tial to replace microorganisms for production of large quantities of recombinant proteins. Through genetic engineering of plants or “bio-farming”, numerous recom- binant proteins, have been expressed successfully (White- lam, 1995; Goddijn and Pen, 1995; Ponstein et al., 1996). Bio-farming of oil seeds, such as canolasour focus here, offers economies in scale-up, harvesting, storage, and transport. However, the limited knowledge of expression control and lack of effective and economic downstream protein purification processes limits the use of plants as recombinant protein production hosts.

Canola, long used as an oil source and animal feed, has been explored as a possible host (van Rooijen and Moloney, 1995). The native canola proteins are mainly from two groupssa neutral, high molecular weight, 12S group and a basic, low molecular weight, 2S (or 1.7S) group (Bhatty et al., 1968; Schwenke, 1994). The 2S proteins have an isoelectric point of around 11 and have molecular weights between 12 000 and 17 000 (Lönnerdal

and Janson, 1972; Schwenke et al., 1973; Swanljung, 1970). The amino acid compositions of proteins in this group are similar, and the proteins consist of two polypeptide chains linked by disulfide bridges (Lönnerdal and Janson, 1972). The main charge character comes from two exposed regions with high accumulation of the basic amino acids lysine and arginine (Schwenke, 1994).

Native canola proteins have been partly characterized by various chromatographic methods (Bhatty et al., 1968; Lönnerdal and Janson, 1972; Rabb et al., 1992; Ama- rowicz et al., 1993). With focus on the characterization of each individual native protein, a combination of different chromatographic methods, which most often included size exclusion and ion exchange chromatogra- phy, were employed to obtain the best possible resolution between chromatographic elution protein peaks. 2S proteins were shown to be chromatographically and electrophoretically homogeneous, and they were strongly adsorbed by carboxymethylcellulose (CM-cellulose) (Bhat- ty et al., 1968). Along with 2S proteins, there is another fraction of proteins, the intermediate fraction (IF), that can be weakly adsorbed by cation exchange at neutral pH (Rabb et al., 1992). These works showed that canola native proteins give relatively simple cation exchange

* To whom correspondence should be addressed at 2114 Sweeney Hall, Department of Chemical Engineering, Iowa State University, Ames, IA 50011. Fax: (515) 294-2689. E-mail: [email protected].

12 Biotechnol. Prog. 1999, 15, 12−18

10.1021/bp980110y CCC: $18.00 © 1999 American Chemical Society and American Institute of Chemical Engineers Published on Web 12/31/1998

chromatographic profiles. However, none of them evalu- ated canola seeds as a recombinant protein production host.

Chromatographic methods have been used in plant protein purification (Jervis and Pierpoint, 1989) because of their high resolving power, especially when very large production of a protein is not crucial. Ion exchange chromatography (IEC) is widely used in purifying pro- teins of distinctive charges, and genetic engineering methods could be used to confer distinctive charges (Flachel and Friehs, 1993). This can be done by site- directed mutagenesis or attachment of a charged fusion tail. While the former method can best keep the integrity of a protein by avoiding unexpected proteolytic decom- position, the latter is more flexible in “designing” the charged character without interfering in its stability or functioning (Ford et al., 1991; Wilkinson et al., 1995; Uhlen and Moks, 1990). In addition, the fusion method could be the only option when detailed structural infor- mation of a protein required for carrying out site-directed mutagenesis is not available.

Our goals here are to characterize the native canola proteins from the recombinant protein purification stand- point and investigate the effect of protein charge on a single-step chromatographic purification. To investigate the charge effect, T4 lysozyme was chosen to be the target protein, because it and its mutants, both point mutation and fusions, are readily available in our lab. Cation exchange chromatography is adopted throughout this work because the target proteins are positively charged at neutral pH. The stability of different mutants in canola protein extract is also addressed in this paper. Producing a supply of new transgenic seed is a major and expensive task, and for this reason, screening of possible genetic strategies by means of spiking studies is very useful to narrow the number of mutations/fusions to pursue.

Materials and Methods T4 Lysozyme. T4 lysozyme is a basic protein with an

isoelectric point above 9.0 and carries nine positive charges at neutral pH. It has 164 amino acids, and its molecular weight is 16 800. The T4 lysozyme mutants used in this study were developed using both site-directed mutagenesis and fusions (Table 1). The point mutants were developed by replacing surface lysine residues with glutamic acids (Sun et al., 1991). The three mutants are K16E (single mutant), K16/135E (double mutant), and K16/135/147E (triple mutant), respectively, with the number designating the position of the replaced lysines to give variants with charges of +7, +5, and +3 at neutral pH. The fusions were polyarginine tails of two (U1) or four (U2) arginines attached to the C-terminal of the triple mutant (Fan and Glatz, 1998), providing net charges of +5 and +7, respectively.

Enzyme Production and Protein Assays. T4 lysozyme production was induced in Escherichia coli by the addition of isopropyl-â-D-thiogalactopyranoside (IPTG). Extracted protein was purified by cation exchange chro- matography (Luther and Glatz, 1994).

Lysozyme activity was measured by the clearing of a Micrococcus lysodeiktikus cell suspension (Perry et al., 1965). The concentration of protein was obtained by using Bio-Rad protein assay (Bio-Rad, Hercules, CA); the pure T4 lysozyme solution content was also determined by A280 based on e280

1% ) 1.28 (Poteete et al., 1991). Preparation of Protein Extract. Nontransgenic

canola seed was provided by Pioneer Hi-bred Interna- tional (Johnston, IA). Canola seed was ground, dehulled, and flaked before oil extraction. The defatted canola meal was obtained by two stages, 30 min each, of cold n-hexane extraction with a ratio of 1 g of seed/20 mL of solvent. The final oil content of the canola meal was <2% (w/w).

Native canola proteins were extracted into 50 mM sodium phosphate (NaPi) buffer (pH 7.0) at 1:10 (w/v) for 30 min with mild stirring. The protein concentration of the extract after centrifugation and filtration by a 0.22 µm syringe filter was 30 ( 5 mg/mL. Then the protein extract was dialyzed (1:400; Spectra/Pro1 membrane tube; MWCO 6-8000; Spectrum, Laguna Hills, CA) at 4 °C for at least 36 h against 20 mM NaPi (pH 7.0), which was the equilibrating buffer for ion exchange chroma- tography, with one fresh buffer change at around 18 h into dialysis. After dialysis, the extract was filtered through a 0.22 µm syringe filter again to remove pre- cipitates developed during dialysis. The protein concen- tration of the final filtrate was 12 ( 1 mg/mL.

Chromatography. The flowchart of the experimental procedures is shown in Figure 1. Native canola proteins were characterized by both cation exchange and anion exchange chromatography. Cation exchange chromatog- raphy (CEC) was then used throughout since the target protein, T4 lysozyme, is positively charged at the experi- mental condition of pH 7.0. During each chromatographic run, 6 mL of sample was loaded onto the column. After 60 mL of washing by 20 mM NaPi, pH 7.0 (buffer A), linear gradient elution from buffer A to 20 mM NaPi with 500 mM NaCl, pH 7.0 (buffer B), was applied to elute bound proteins. Meanwhile 5 mL fractions were collected for later activity assay to identify where the spiked T4 lysozyme was eluted. A 1 mL/min flow rate was used for all the chromatographic experiments.

As a simpler alternative, dialysis was eliminated and the canola protein extract was loaded onto a column directly after centrifugation and filtration (Figure 1). All other parameters were unchanged.

Column regeneration was accomplished by serial wash- ing with 20 mL of buffer B, 20 mL of 0.1 M HCl, and 20 mL of buffer B (20/20/20), and then washing to baseline by buffer A. After using nondialyzed sample, regeneration volumes were increased to 40/40/40, respectively.

All columns were packed to a bed height of 6.5 ( 0.2 cm in 1 × 10 Econo columns (Bio-Rad, Hercules, CA). Chromatography was performed by using a fast perfor- mance liquid chromatography (FPLC) system controlled by BioLogic software (Bio-Rad, Hercules, CA).

Chemicals and Materials. All chemicals were pur- chased from Fisher (Itasca, IL). M. lysodeiktikus and all the resins were purchased from Sigma (St. Louis, MO).

Results and Discussion

Characterization of Native Canola Proteins. Fig- ure 2 shows the chromatographic profiles of native canola protein for cation and anion exchange chromatography. Both chromatograms show rather simple elution patterns of native canola proteins. With cation exchange chroma- tography, all bound canola proteins can be eluted into two major peaks with a wide, low background valley in

Table 1. Charge of T4 Lysozyme Mutants and Fusions at pH 7.0

T4 lysozyme net charge

wild type 9.0 K16E, single mutant 7.0 K16/135E, double mutant 5.0 K16/135/147E, triple mutant 3.0 U1, two arginines 5.0 U2, four arginines 7.0

Biotechnol. Prog., 1999, Vol. 15, No. 1 13

between. This provides a potential elution site for a genetically engineered protein. With anion exchange chromatography, there are three visible native canola protein peaks. Gaps between the peaks also provide potential elution sites for negatively charged proteins at neutral pH. Moreover, both of the chromatograms show patterns similar to those obtained by Finlayson (Finlay- son, 1966), who used a pH gradient in both weak anion and cation exchange chromatography.

From our experimental results (Figure 3), the only difference between using strong or weak cation exchange chromatography is the ionic strength at which the peaks are eluted, even though CM type resin does provide a broader valley to target a protein. The simple chromato- graphic elution behavior of 2S proteins of canola (Bhatty et al., 1968) is evident with a single elution peak eluted at high salt concentration.

T4 Lysozyme Purification. Figure 4 shows the elution profiles of canola samples spiked with different point-mutated T4 lysozymes, and Figure 5 shows the corresponding activity assay results. Figure 6 shows the activity assay results for canola samples spiked with fusion T4 lysozymes.

From Figures 4 and 5, the charges of T4 lysozymes have a significant influence on their retention behavior. The single-mutant T4 lysozyme spiked canola sample gives a noticeable peak in the space between the two native canola protein peaks (Figure 4), while other point mutants overlap with one or the other of the two native peaks. Sixty percent of the single-mutant T4 lysozyme

loaded onto the column was recovered from this peak, which also had 90% of the lysozyme activity detected in fractions collected after gradient elution started. Figure 6 shows the gel electrophoresis result of the IEC fractions of the single-mutant T4 lysozyme spiked canola sample. Besides the lysozyme band, there are four detectable

Figure 1. Flowchart for the extraction and fractionation. One alternative eliminates the dialysis step.

Figure 2. Comparison of native canola protein elution profiles between cation and anion exchange chromatography: cation exchanger, sulfopropyl Sephadex (SP); anion exchanger, diethyl- [2-hydroxypropyl]aminoethyl Sephadex (QAE). All the chro- matographic experiments were carried out using the same linear gradient program.

Figure 3. Comparison of weak (CM) and strong (SP) cation exchange columns in the separation of native canola proteins. CM ) carboxymethyl Sephadex.

Figure 4. Comparison of chromatograms of canola samples spiked with different T4 lysozymes by cation exchange chro- matography. A 2 mL sample of T4 lysozyme stock solution (∼1 mg/mL) was added to 8 mL of canola protein extract. Cation exchanger: SP. CC ) canola control; WTSC ) wild-type T4 lysozyme spiked canola sample; SSC ) single-mutant T4 lysozyme (K16E) spiked canola sample; DSC ) double-mutant T4 lysozyme (K16/135E) spiked canola sample. The linear gradient started at 1.7 h. The peak before the gradient started was due to incomplete dialysis. The experiment was repeated with 72 h of dialysis and showed no such peak.

14 Biotechnol. Prog., 1999, Vol. 15, No. 1

bands indicating four contaminating proteins, two of them with a molecular weight around 35 000 (too weak to be seen) and the other two with a molecular weight of less than 14 000 (one of them can be seen in Figure 6, and the other one has a slightly higher molecular weight). However, the estimated lysozyme purity from the band intensity on the gel was higher than 90%.

On the other hand, the fusion-spiked canola extracts show lysozyme activity spread over many fractions with two main activity peaks (Figure 7). U2 T4 lysozyme, which carries the same charge as single-mutant T4 lysozyme, does show an activity peak at the same location where single-mutant T4 lysozyme would elute. However, only 66% of the total lysozyme activity was recovered in this peak compared to 90% for single-mutant T4 lysozyme activity. This indicates a low recovery for the fusion strategy. Of the two main activity peaks seen for each fusion protein (Figure 7), the later one corresponds to where the point mutant with the same charge would be eluted, while the earlier one corresponds to that of triple- mutant T4 lysozyme without the fusion tail. Evidently, the tails were degraded by proteases in the canola extract. A less likely explanation is that polyanions found in canola, mainly pectic acid and phytic acid (Schwenke et al., 1991; Evans et al., 1982; Siy and Talbot, 1982), are present in the extract and interfere with binding of

the positively charged fusion proteins and the ion ex- changer. Pectic acid (cellulose sulfates) and phytic acid (polyphosphate) binding to the high charge density area of the tail would block the intended stronger binding of fusions to the column.

Nevertheless, T4 lysozyme and its mutants demon- strate the ability to improve the ease of purification from canola by manipulation of charge. Single-mutant T4 lysozyme with a positive charge of 7 at neutral pH can be enriched 35 times from a canola protein extract by this single chromatographic step. Other point mutant T4 lysozymes were eluted with one of the two major native canola protein peaks. Subjected to the influence of polyanions and protease activity, polyarginine fusions do not show advantages over point mutations in the canola system. An alternative would be to use polyanionic fusions, which in microbial systems have not had the same susceptibility to proteolysis as polyarginine fusions (Ford et al., 1991; Zhao et al., 1990).

Figure 8 shows the relationship between the conduc- tivity at elution (y, mS/cm) and the charge at pH 7 (x) of each mutant. The results are fitted by a linear relation- ship on point mutant and WT T4 lysozymes:

On the basis of the above equation, one additional positive charge on a protein would increase the conduc- tivity of an eluent by 0.068 mS/cm, which corresponds to an increase of 27.8 mM NaCl in 20 mM NaPi (27.8 mM NaCl/charge). Previous studies on human urogas- trone (Sassenfeld and Brewer, 1984) and â-galactosidase (Zhao et al., 1990; Heng and Glatz, 1993) showed differ- ent increments in elution buffer ionic strength per added charge. In the case of human urogastrone, five additional arginines at the carboxyl terminus increased the con- centration of NaCl of the eluent from 50 to 400 mM in Tris/urea buffer (5 M urea, 40 mM Tris-acetate, pH 5.5), which corresponds to 70 mM/charge. In the case of â-galactosidase, polyaspartic acid tails with 1, 5, and 11 aspartic acid residues at the carboxyl terminus provided additional charges to change the elution profile of â-ga- lactosidase in anion exchange chromatography (AEC) (Zhao et al., 1990) and hollow fiber ion exchange mem- brane adsorption (HFIEM) (Heng and Glatz, 1993). In AEC, an average of 18.2 mM NaCl/charge (0.1 M NaPi, pH 6.0) increase was reported, and for HFIEM, the increase per additional charge was 13.6 mM NaCl (0.089 M NaPi, pH 5.7) during elution. These are summarized

Figure 5. Superposition on the native canola profile of lysozyme activity assays from separate injections of each of the point mutant series spiked canola extracts. The absorbance at 280 nm of canola extract (UV-CC) is shown at the left ordinate, and the activity (Act.) of lysozyme is shown at the right ordinate. TSC ) triple-mutant T4 lysozyme (K16/135/147E) spiked canola sample. Other acronyms are as in Figure 4.

Figure 6. SDS-PAGE of those fractions from IEC of canola extract spiked with single-mutant T4 lysozyme (K16E) showing lysozyme activity: lane 1, single-mutant T4 lysozyme stock solution; lanes 2-7, IEC fractions comprising the peak in Figure 4 (UV-SSC). Lane 4 corresponds to the activity peak in Figure 5 (SSC-Act.). All fractions were concentrated about 26-fold before being loaded onto the gel.

Figure 7. Relative retention times of fusion T4 lysozymes (U1 and U2) compared to that of native canola proteins. Cation exchanger: SP.

y ) -0.078 + 0.068x (R2 ) 0.99)

Biotechnol. Prog., 1999, Vol. 15, No. 1 15

in Table 2. The difference in increment of NaCl per additional charge during elution is probably due to the pH of the buffers used, the systems from which proteins are to be purified, the protein size, and the retention medium used in separation.

Nonetheless, with the above equation, a reasonable prediction of when a protein would be eluted from the column could be made given the charge it carries. This can guide how the genetic engineering work should be done for a recombinant protein which would be produced with canola as the production host. There are two targeting elution sites for positively charged recombinant proteins; one is between the two native canola protein peaks, and the other is after the second native canola peak. If, without altering the identity (secondary struc- ture and activity) of a protein, the protein can be genetically engineered to carry a +13 charge, from the relationship we obtained, it would be eluted at a buffer conductivity of 0.81 ( 0.01 mS/cm, which corresponds to a salt concentration of 300 ( 4 mM NaCl in 20 mM NaPi. When this charge level is not possible, the protein can be targeted to the gap between the native canola protein peaks with a charge of +7. However, since this equation was obtained by studying a relatively small protein (∼17 kDa), the application to large proteins should be done relative to the observed elution point of the unal- tered protein.

Process Modification. Eliminating the dialysis step for buffer exchange would simplify the process and shorten the processing time. Two alternatives were examined to eliminate the dialysis step: one was to change the protein extraction buffer to the column equilibrating buffer, and the other was to eliminate the dialysis without changing the extraction buffer.

Figure 9 shows the comparison between the chromato- grams of canola protein samples extracted by 20 mM NaPi and 50 mM NaPi (pH 7.0), respectively, but without dialysis before loading (Figure 1). The total protein extracted at 20 mM NaPi is 25% less than that at 50 mM NaPi, but from the peak areas in the chromatograms in Figure 9 (peaks 1 and 2), the total basic proteins extracted are similar. Nevertheless, the space between the two native canola peaks remains the same for both extraction buffers used.

Figure 10 shows the chromatogram of the single- mutant T4 lysozyme spiked canola protein extract (50 mM NaPi) without the dialysis step. The spiked single- mutant T4 lysozyme still elutes between the two native canola protein peaks. The single-mutant peak shifts to the right in Figure 10 compared to that in Figure 3, because the washing time after loading the sample was increased by 120 min (1 mL/min) to obtain a good baseline before the linear gradient was started.

Compared to dialyzed samples (Figure 2), the nondia- lyzed canola samples (Figure 9) show a larger peak after loading the samples and before the gradient elution was started. This group of proteins was partially eliminated during sample dialysis largely due to precipitation, which

was significant at the end of dialysis. Gel electrophoresis (not shown here) on fractions with T4 lysozyme activity (Figure 10) shows higher intensities than that of the corresponding fractions of dialyzed sample (UV-SSC in Figure 4, SSC-Act. in Figure 5) on corresponding bands in Figure 6. In addition, there were four more very weak bands for the fractions of nondialyzed sample, two at 30 kDa and the other two around 66 kDa. So, the dialysis step not only exchanges the buffer to a lower ionic strength (50 mM NaPi to 20 mM NaPi) but also acts as a preseparation step to eliminate some native canola proteins, predominantly some weakly binding proteins. However, the eliminated proteins do not influence the purification performance of a highly charged (such as +7.0 or more) recombinant protein beyond introducing slightly higher concentrations of contaminating proteins in corresponding fractions. In addition, the recovery of single-mutant T4 lysozyme from a nondialyzed sample (>80%) is significantly higher than the recovery from a dialyzed sample (∼60%). This is probably because some T4 lysozyme was precipitated during dialysis.

Table 2. Comparison of the Increment in Salt Concentration per Added Charge for Protein Elution

protein human urogastrone â-galactosidase T4 lysozyme molecular weight 6 000 116 400 16 800 separation method CEC AEC HFIEC CEC system E. coli fermentation E. coli fermentation E. coli fermentation canola extract buffer and pH 5 M urea, 40 mM Tris-acetate,

pH 5.5 0.1 M NaPi, pH 6.0 0.089 M NaPi, pH 5.7 0.02 M NaPi, pH 7.0

elution method gradient step step gradient ∆mM NaCl/charge 70 18.2 13.6 27.8 reference Sassenfeld and Brewer, 1984;

Gregiry and Preston, 1977 Zhao et al., 1990 Heng and Glatz, 1993 this work

Figure 8. Eluent conductivity at which each T4 lysozyme is eluted at pH 7.0. The line is that regressed to the values for point mutants.

Figure 9. Comparison of different extraction buffers when dialysis was not performed before IEC. Cation exchanger: SP. The linear gradient elution started at ∼1.7 h.

16 Biotechnol. Prog., 1999, Vol. 15, No. 1

The drawback of no dialysis is that the fouling of the column was severe, which was mainly due to plugging by various precipitates, e.g., lipids, proteins, and other fine particles. This altered the flow rate/pressure drop through the column. Furthermore, when 50 mM NaPi buffer was used in extraction, the column fouling was more serious, since the equilibrating buffer was 20 mM NaPi. The decrease of the salt concentration might cause some proteins to precipitate once the sample was loaded onto the column. Regeneration with 0.5 M NaOH was needed to recover column performance.

Conclusions Canola as a recombinant protein production host offers

opportunity for selective recovery of the recombinant protein by IEC. A charge change of -2 in WT T4 lysozyme permits its selective recovery in a single cation exchange chromatographic step. A linear relationship between the protein charge and the eluent ionic strength was found. This could be used to guide a genetic engi- neering work on a recombinant protein with canola as a host. Charge distribution on T4 lysozymes is not very important from our results. Fusions and point mutations have the same retentions when their charges are the same. However, the performance of the polyarginine fusions is severely compromised by canola extract com- ponents.

Dialysis of extracts acted as a prepurification step by eliminating some proteins weakly bound to the cation exchanger as a result of precipitation during the dialysis step. However, the dialysis step could be eliminated to simplify and shorten the purification processes. The same separation performance was still achieved in the case of purifying single-mutant T4 lysozyme, but column fouling increased.

Acknowledgment This work is supported by the NSF under Grant No.

BES9522644. We thank Pioneer Hi-Bred International, Inc., Johnston, IA, for providing canola seed. We also thank Dr. Zivko Nikolov and Dr. Ann Kusnadi for their suggestions and help.

Notation AEC anion exchange chromatography

CC canola control CEC cation exchange chromatography CM carboxymethyl Sephadex, weak cation exchanger DSC double-mutant T4 lysozyme (K16/135E) spiked

canola sample HFIEM hollow fiber ion exchange membrane adsorption QAE diethyl[2-hydroxypropyl]aminoethyl Sephadex,

strong anion exchanger SP sulfopropyl Sephadex, strong cation exchanger SSC single-mutant T4 lysozyme (K16E) spiked canola

sample TSC triple-mutant T4 lysozyme (K16/135/147E) spiked

canola sample U1 triple-mutant T4 lysozyme with a fusion tail of

two arginines U2 triple-mutant T4 lysozyme with a fusion tail of

four arginines WTSC wild-type T4 lysozyme spiked canola sample

References and Notes

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Accepted December 4, 1998.

BP980110Y

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