Expression, Purification, and Characterization of Recombinant Nonglycosylated Human Serum Transferrin Containing a C-Terminal Hexahistidine Tag

Protein Expression and Purification 23, 142–150 (2001) doi:10.1006/prep.2001.1480, available online at http://www.idealibrary.com on

Expression, Purification, and Characterization of Recombinant Nonglycosylated Human Serum Transferrin Containing a C-Terminal Hexahistidine Tag Anne B. Mason,*,1 Qing-Yu He,* Ty E. Adams,* Dmitry R. Gumerov,† Igor A. Kaltashov,† Vinh Nguyen,‡ and Ross T. A. MacGillivray‡ *Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405; †Department of Chemistry, University of Massachusetts at Amherst, Lederle Graduate Research Center, Amherst, Massachusetts 01003; and ‡Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received March 20, 2001, and in revised form May 21, 2001; published online August 9, 2001

Attachment of a hexa-His tag is a common strategy in recombinant protein production. The use of such a tag greatly simplifies the purification of the protein from the complex mixture of other proteins in the media or cell extract. We describe the production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at their carboxyl-terminal ends. One of the constructs comprises the entire coding region for hTF (residues 1–679), while the other lacks the final three carboxyl-terminal amino acids. After insertion of the His-tagged hTFs into the pNUT vector, transfection into baby hamster kidney (BHK) cells, and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium. Average maximum expression levels of the His-tagged hTFs were about 40 mg/L compared to an average maximum of 50 mg/L for hTF-NG. The first step of purification involved an anion exchange column. The second step utilized a Poros metal chelate column preloaded with copper from which the His-tagged sample was eluted with a linear imidazole gradient. The His-tagged hTFs were characterized and compared to both recombinant hTF-NG and glycosylated hTF from human serum. The identity of each of the His-tagged hTFs constructs was verified by electrospray mass spectroscopy. In summary, the His-tagged hTF constructs simplify the purification of these metal-binding proteins with minimal

1 To whom correspondence should be addressed. Fax: (802) 8628229. E-mail: [email protected].

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effects on many of their physical properties. The Histagged hTFs share many features common to hTF, including reversible iron binding, reactivity with a monoclonal antibody, and presence as a monomer in solution. 䉷 2001 Academic Press

The transferrins are metal-binding glycoproteins that function in the transport of iron to cells and as bacteriostatic agents in a variety of biological fluids (1, 2). The present-day transferrins have evolved as a result of gene duplication events and consist of two homologous globular lobes termed the N- and C-lobes. Each of these lobes contains a deep cleft capable of binding a metal ion. Crystallographic data for a number of different transferrins show that each ferric ion is directly coordinated to the side chains of two tyrosine residues, one histidine residue, one aspartic acid residue and two oxygens from the synergistic carbonate anion which is anchored by an arginine residue (3–5). The transferrins are abundant in nature, making the purification of the naturally occurring proteins relatively facile. The most compelling reason to produce transferrins by recombinant expression is to allow the mutation of individual amino acid residues to determine their specific contributions to function. An added advantage is the ability to produce homogeneous proteins that can aid in obtaining high-resolution crystal structures. In the case of human serum transferrin 1046-5928/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.

HIS-TAGGED HUMAN SERUM TRANSFERRIN

(hTF),2 avoidance of blood-borne pathogens is also an important consideration. Human serum transferrin is often an essential component of tissue culture medium, especially when culturing human cell lines. Therefore, a concerted effort to develop and optimize expression systems has been made (see below). A remarkable and possibly unprecedented number of different expression systems have been developed. The extensive number of disulfide bonds mean that the production of functional recombinant transferrins is not easily accomplished. Human serum transferrin has 38 cysteine residues, all of which engage in disulfide linkages (there are 8 in the N-lobe and 11 in the C-lobe) (6). This makes expression of a correctly folded protein challenging. In addition, the presence of two carbohydrate attachment sites and a leader sequence that must be properly cleaved, increase the difficulty. In contrast to the challenges faced in producing recombinant transferrins verifying the functionality of the proteins once produced and isolated is relatively straightforward. For example, the ability to bind iron reversibly is easily determined by spectral analysis (7). There have been a number of efforts to produce hTF in a variety of expression systems. Although an initial effort to express hTF in Escherichia coli failed (8), several laboratories have subsequently reported success (9–12). Production of hTF in bacterial systems, however, has yielded extremely limited amounts of functional protein. No convincing demonstration of iron binding has ever been provided by any of the studies. Problems with both proteolysis and inhibition of bacterial growth were encountered. This inhibition may be the result of the well-documented antibacterial activity of the transferrins (1, 2). The most recent study reported a maximum of 60 mg/L of hTF; however, the protein was found in insoluble inclusion bodies and only 3 mg was recovered after renaturation (12). Functional hTF N-lobe has been successfully produced from the methylotrophic yeast, Pichia pastoris, grown in shake flasks at levels up to 50 mg/L in the laboratory of Steinlein et al. (13) and up to 250 mg/L (14) in our laboratory. Recovery of the secreted protein

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following purification was 28 and 70%, respectively. In a subsequent report by Steinlein et al. (15), production levels were increased to 80–100 mg/L and a C-terminal hexahistidine tag was added, to increase both the speed and the recovery of the recombinant protein. The presence of the His tag did not appear to interfere with iron binding by the recombinant N-lobe. Unfortunately, efforts to produce full-length transferrin in this system have not been successful. Production of hTF by insect cells infected with a baculovirus has been reported by two different laboratories (16, 17). In each case a production level of about 20 mg/ L was achieved. The advantage of insect cells is that they can perform posttranslational modifications while sharing the faster growth rates of yeast, bacteria, and filamentous fungi. They can also be grown in suspension cultures and do not require CO2 incubators. The most utilized and best-characterized system to date is a mammalian expression system in which the cDNAs of the relevant proteins are inserted into the pNUT vector and transfected into baby hamster kidney cells. This system has been used to produce large amounts of fully functional recombinant protein and mutated transferrins containing single and double point mutations. Both glycosylated and nonglycosylated human serum transferrins have been expressed (18) and shown by many criteria to be entirely equivalent to native protein in iron binding and interaction with cellular transferrin receptors. Interestingly, neither extensive nor varied glycosylation of the protein produced in the BHK system or the complete absence of glycosylation had an effect on the receptor binding affinity (18). Mutated human serum transferrins have been used to investigate the role of specific residues in function (19, 20). In the present report, we describe production and characterization of human serum transferrin bearing a C-terminal hexa-histidine tag to simplify and accelerate the purification process using the pNUT/BHK system.

MATERIALS AND METHODS 2

Abbreviations used: hTF-Gly, commercially available glycosylated human serum transferrin; hTF-NG, recombinant nonglycosylated human serum transferrin; hTF-His-C1, recombinant human serum transferrin comprised of full-length hTF with a Factor Xa cleavage site and a hexa-His tag attached to the carboxy-terminus of the protein; hTF-His-C2, recombinant human serum transferrin comprised of truncated hTF (missing the final three carboxy-terminal amino acid residues) with a Factor Xa cleavage site and a hexa-His tag attached to the carboxy-terminus of the protein; DMEM-F12, Dulbecco’s modified Eagle’s medium–Ham F-12 nutrient mixture; BHK cells, baby hamster kidney cells; UG, Ultroser G; FBS, fetal bovine serum; BSA, bovine serum albumin; MC, metal chelate; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; TMB, 3,3⬘,5,5⬘-tetramethylbenzidine; ESI, electrospray ionization, TMB, N,N,N⬘,N⬘-tetramethylbenzidine; Pipes, 1,4-piperazinediethanesulfonic acid.

Materials Dulbecco’s modified Eagle’s medium-Ham F-12 nutrient mixture and antibiotic-antimycotic solution (100X) were from Gibco-BRL-Life Technologies (Gaithersburg, MD). Fetal bovine serum was obtained from Atlanta Biologicals (Norcross, GA) and was tested prior to use to assure adequate growth of baby hamster kidney cells. Ultroser G is a serum replacement from BioSepra (Cergy-Pontoise Cedex, France). Ferrous ammonium sulfate was purchased from Sigma and a solution of cobalt (1000 ␮g/mL, AAS standard solution, Specpure)

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was obtained from Alfa Aesar. Cesium iodide was from Aldrich Chemical Co. Corning expanded surface roller bottles, and Dynatech Immunolon 4 Removawells were obtained from Fisher Scientific. The chromatographic resin, Poros 50 HQ, and the metal chelate and QE columns were from PerSeptive Biosystems. The MC column has covalently attached imidodiacetate groups which allows binding of a variety of transition metals through bidentate ligation. The resins Sephacryl S100HR and Sephadex G-75 were from Amersham Pharmacia as was the HiPrep Sephacryl S-200HR (26/60) column. Methotrexate from Cetus (Emeryville, CA) was purchased at a local hospital pharmacy. Centricon 30 microconcentrators, YM-30 ultrafiltration membranes, and a spiral cartridge concentrator (CH2PRS) fitted with an S1Y10 cartridge were from Millipore/Amicon. Human serum transferrin was purchased from Boehringer Mannheim. Rabbit anti-mouse immunoglobulin G was from Southern Biological Associates (Birmingham, AL). Immunopure NHS-LC–biotin and Immunopure avidin–horseradish peroxidase were from Pierce. The TMB Microwell peroxidase substrate system was obtained from Kirkegaard and Perry Laboratories (Gaitherburg, MD). All other chemicals and reagents were analytical grade. A monoclonal antibody that is specific to the N-lobe of hTF was a generous gift from Dade Behring GmbH (Marburg, Germany). Biotinylated diferric hTF was prepared according to the manufacturer’s (Pierce) instructions at a level of approximately two biotin molecules per molecule of transferrin. The biotinylated hTF was stored as a 50% glycerol solution at ⫺20⬚C.

because the crystal structure (21) indicated that the carboxyl-terminal three residues turn into the interior of the hTF C-lobe. It was felt that addition of the His tag to these residues might either disrupt the structure of the C-lobe or be inaccessible, thereby leading to inefficient binding to the metal chelate column. Addition of the Factor Xa cleavage site and His tag to each hTFNG was achieved using a polymerase-chain-reactionbased mutagenesis procedure (22). The mutated transferrins contain the sequences shown below at their C-terminal regions (the lower strand DNA sequences are identical to the oligonucleotide primers used in the PCR-directed mutagenesis). A subclone of the full-length hTF-NG (PstI/XhoI) was used as the template for the PCR. The fragment was then purified and cloned back into the full-length construct. The full-length cDNA was digested with XbaI and XhoI, made blunt ended, and ligated into the SmaI site of pNUT as described in detail previously (23). At each step of the procedure, DNA sequence analysis was performed to verify the identity and correct orientation of the clones. The expression vector pNUT features a mouse metallothionein promoter to regulate expression of a cloned gene and a mutated dihydrofolate reductase which allows for rapid selection with a high concentration of methotrexate. BHK cells were transfected by the calcium phosphate method in 100-mm tissue culture dishes (24). During the transfection and selection periods, the cells were cultured in Dulbecco’s modified Eagle’s medium:nutrient mixture F-12 (Ham) 1:1 supplemented with 10% fetal bovine serum. For selection, 0.44 mM methotrexate was added to the medium within 24 h of the transfection. All surviving cells were taken without a subcloning step because it has previously been determined that subcloning did not lead to higher production of recombinant protein (25). Following the selection process, which takes about 10 days, the transformed cells containing the pNUT plasmids were expanded into flasks for transfer into roller bottles or frozen in 95% FBS/5% DMSO in a liquid nitrogen apparatus. The freezing, passage, and expansion of cells have been described in detail previously (18).

Expression Vectors Two different His-tagged hTF constructs were made. The first, designated hTF-His-C1, was composed of fulllength hTF-NG (residues 1–679) to which a Factor Xa cleavage site and a 6 His tag was added at the carboxyterminal end. The hTF-NG was produced by mutation of the Asn carbohydrate linkage sites at positions 413 and 611 to Asp residues as described previously (18). The second construct, designated hTF-His-C2, was identical except that it contained a truncated hTF-NG (residues 1–676). This truncated form was constructed For hTF-His-C1 Cys Thr 5⬘ CC TGC ACT 3⬘ GG ACG TGA

Phe Arg TTC CGT AAG GCA C-terminus of

Arg Pro Ile AGA CCT ATC TCT GGA TAG C-lobe

For hTF-His-2C Leu Glu Ala Cys Thr 5⬘CTG GAA GCC TGC ACT 3⬘GAC CTT CGG ACG TGA C-terminus of C-lobe

Phe TTC AAG

Ile ATC TAG

Glu GAG CTC Factor

Glu GAG CTC Factor

Gly Arg Ile GGA AGG ATT CCT TCC TAA Xa site

Gly Arg GGA AGG CCT TCC Xa site

Ile ATT TAA

(His)6 (CAT)6 TAA (GTA)6 ATT His tag Stop

(His)6 (CAT)6 (GTA)6 His tag

TAG ATC

TAA ATT Stop

CTC GAG XhoI

TAG ATC

GAG CTC site

CTC GAG XhoI

GAG CTC site

HIS-TAGGED HUMAN SERUM TRANSFERRIN

Production and Purification of hTF-NG, hTF-HisC1, and hTF-His-C2 BHK cells were grown in expanded surface roller bottles (1700 cm2). The culture medium (200 mL/roller bottle) was collected every 2 to 4 days. The first three batches contain DMEM-F12 with 1⫻ antibiotic– antimycotic solution and 10% FBS. Subsequent batches contain the same media with 1% Ultroser G instead of the FBS. The UG contains a small amount of hTF (⬃2–4 mg/L) and other proprietary additives. The presence of UG stimulates protein production to levels that are considerably higher than those found in the presence of FBS. In addition, UG has a lower amount of other protein than FBS, thereby simplifying recombinant protein purification. Iron-nitrilotriacetate and sodium azide (0.02%) were added to the pooled batches of media; 600 ␮L of 25 mM ferrous ammonium sulfate in 0.01 N HCl and 300 ␮L of disodium NTA were premixed and added per liter of medium. This amount was adequate to saturate the recombinant hTF, the glycosylated hTF from the UG, and the bovine TF also from the UG. The collected batches were stored at 4⬚C or frozen prior to purification. The medium was filtered through a plug of cotton to remove cell debris. It was then concentrated and exchanged into 5 mM Tris–HCl buffer, pH 8.0, containing 0.02% sodium azide using a Millipore spiral wound membrane concentrator fitted with a S1Y10 cartridge. Following centrifugation at 6000g for 15 min, the sample was pumped at a flow rate of about 10 mL/ min onto a Poros50 HQ column (2.6 ⫻ 20 cm) equilibrated in 10 mM Tris, pH 8.0. Elution involved a single step of 180 mM Tris–HCl, pH 8.0, and 8-mL fractions were collected. The phenol red and most of the albumin are retained on the column. All fractions containing salmon pink color (the iron loaded TF) were pooled, concentrated using an Amicon-stirred cell fitted with a YM-30 membrane, and exchanged into 10 mM Tris–HCl buffer, pH 8.0, containing 0.02% sodium azide. The glycosylated hTF and the bovine TF from the Ultroser G were separated from the recombinant hTF-NG on a Poros QE/M column (10 ⫻ 100 mm) using a 0–300 mM NaCl gradient over 15 column volumes in 50 mM Tris/ bis-Tris propane buffer, pH 8.0, containing 0.02% NaN3. The column was run on a PerSeptive Biosystems Sprint system allowing simultaneous monitoring of A280, conductivity, and pH. Fractions of 3 mL were collected. Complete separation was monitored by SDS–PAGE, as well as analytical runs over a smaller Poros QE column; multiple passes over the column were usually needed to achieve complete separation. The final purified recombinant diferric hTF-NG was chromatographed on a Sephacryl S-100 HR column (5 ⫻ 80 cm) or a Sephadex G-75 column (2.6 ⫻ 100 cm) at 4⬚C in 100 mM ammonium bicarbonate and stored at ⫺20⬚C.

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Purification of the His-tagged recombinant hTFs followed the same protocol through the large Poros50 HQ chromatography step. The pooled sample from the step elution was concentrated and exchanged into 10 mM potassium phosphate buffer, pH 7.0, containing 500 mM NaCl and 5 mM imidazole. Aliquots of the sample were loaded onto a Poros MC/M column (4.6 ⫻ 100 mm). The MC column was charged with CuSO4 prior to sample application. Two blank runs preceded each sample run. Elution involved a 0–100 mM imidazole gradient over 15 column volumes in the potassium phosphate–NaCl buffer. Fractions of 3 mL were collected. Complete runs which including equilibration and wash segments take 13 min. The His-tagged hTF was pooled, concentrated, and subjected to gel filtration chromatography as described above. The homogeneity of the samples was assessed by SDS–polyacrylamide gel electrophoresis using 8% gels under reducing conditions and visualization with Coomassie blue.

Assay for Recombinant hTF Production The amount of recombinant hTF in the tissue culture medium and at various stages of the purification was determined by an adaptation of a competitive solidphase radioimmunoassay described previously (18), in which biotinylated hTF was substituted for the iodinated hTF. Briefly, removawells were coated with rabbit anti-mouse IgG (1 ␮g/100 ␮L) in 16 mM Na2CO3/34 mM NaHCO3 and incubated overnight at 4⬚C. The wells were washed three times with 200 ␮L of assay buffer (50 mM Tris–HCl, pH 7.4, containing 100 mM NaCl and 0.1% BSA). An appropriate dilution of monoclonal antibody specific to the N-lobe of hTF in a volume of 200 ␮L was added to all wells except those used to determine nonspecific binding. Typically, a 1:5000 dilution of the specific antibody was used. After incubation for 60 min at 37⬚C, the wells were washed three times with 200 ␮L of assay buffer. Biotinylated hTF was added (10 ng/200 ␮L) in the presence or absence of unlabeled standards and samples. A standard curve was generated by competition of biotinylated diferric hTF with unlabeled hTF (16–400 ng/well). Following incubation for 45–60 min, and washing with assay buffer as above, 200 ␮L of avidin–HRP conjugate (1 ␮g/20 mL) was added to all wells and incubation was continued for another 45–60 min at 37⬚C. After washing, the amount of HRP bound was visualized using the TMB substrate system. The TMB–peroxidase substrate was mixed with an equal amount of peroxidase solution and 100 ␮L was added per well. After a 2–3 min incubation at room temperature, the reaction was quenched by addition of 100 ␮L of 1 N phosphoric acid and the A450 of each well was determined using a Molecular Dynamics plate reader.

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Electronic Spectra A Cary 50 Bio spectrophotometer (Palo Alto, CA), was used to scan the iron-containing and iron-free transferrins in the range from 600 to 250 nm. Protein samples were exchanged into 50 mM Hepes buffer, pH 7.4, using Centricon 30 microconcentrators. A baseline of buffer alone was recorded and subtracted from the sample spectra. Determination of Absorption Coefficients Extinction coefficients for the two His-tagged constructs were determined by titration with cobalt(III) as described in detail (26). Removal of iron from the purified samples and exchange into the buffer of choice was accomplished by treatment of the proteins in Centricon 30 microconcentrators as previously described (27). Mass Spectrometry Electrospray mass spectra of all protein samples were obtained using a JMS-700 MStation (JEOL, Tokyo, Japan) two-sector mass spectrometer equipped with a standard ESI source. Solutions of protein in 100 mM ammonium bicarbonate were diluted in H2O/CH3OH/ CH3CO2H (47:50:3, v:v:v) to a final concentration of ca. 5 ␮M and were continuously injected into the source at a rate of 5 ␮L/min. The spray needle potential was kept below 1.9 kV to avoid in-source oxidation of the protein ions. Acceleration voltage was kept at 5 kV and the resolution was set at 1000. All spectra were recorded by scanning the magnet at a rate of 5 s/decade. Prior to data acquisition, the instrument was calibrated in the desired mass range using either Cesium iodide in fast atom bombardment mode or a solution of apo-hTF N-lobe in H2O/CH3OH/CH3CO2H (47:50:3, v:v:v) in ESI mode. Typically, each spectrum was an average of 80– 180 scans.

molecular weights (Mw,app) were plotted versus concentration and extrapolated to infinite dilution. Protein concentrations of 1 and 0.3 g/L were used. RESULTS AND DISCUSSION Production of Recombinant His-Tagged hTF A typical production run for the two His-tagged constructs of hTF is shown in Fig. 1. In this particular run a maximum of 40 to 50 mg/L of each protein was expressed. Various other production runs are consistent with this level of production with a maximum of 30.1 ⫾ 10.2 ␮g/mL (n ⫽ 10) for the hTF-His-C1 construct and 33.7 ⫾ 13 ␮g/mL (n ⫽ 4), for the hTF-HisC2 construct. Somewhat higher production levels on average (49.7 ⫾ 18.6 ␮g/mL, n ⫽ 6) have been obtained for the hTF-NG construct lacking the His tag (unpublished results). The production of recombinant protein while in 10% FBS is quite low (Fig. 1); however, it has been found that the FBS leads to maximum cell density which results in higher production of recombinant hTF once 1% UG is introduced. Only the batches collected after adding UG are used for protein purification. Typically the production levels begin to fall by day 16, which coincides with the sloughing of cells from the roller bottle surface. Not surprisingly, production of recombinant protein is highly correlated with the cell mass. It should be noted that the substitution of biotinylated hTF for radioiodinated hTF in our assays provides several important advantages. First, the cost and the complications of dealing with and disposing of radioactive samples are avoided. Second, we have found that

Analytical Ultracentrifugation Sedimentation equilibrium experiments were performed on a Beckman XL-I analytical ultracentrifuge fitted with the Yphantis-style six-channel centerpieces. Samples of hTF (105 ␮L) in 50 mM Pipes, pH 7.5, containing 150 mM NaCl and 0.05% NaN3 were loaded in parallel to cells containing 130 ␮L of buffer alone. The sample chambers also contained 20 ␮L of FC43 to allow an accurate determination of the boundary. Samples were centrifuged at a speed of 32,100g and a temperature of 20⬚C. Scans were collected at 3-h intervals at a wavelength of 280 nm. Apparent point-average weight ⫺ average molecular weight values (Mw,app) were calculated from plots of ln[c(r)] versus r 2, where c(r) is the concentration of the protein (g/L) at radial position r. To calculate a true molecular weight (M ), weight-average

FIG. 1. Production rate of the His-tagged hTFs secreted by BHK cells in continuous production in expanded surface roller bottles. On the days indicated medium was collected and assayed for recombinant protein.

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TABLE 1 Recovery of Recombinant hTF-His-C1 and hTF-His-C2 from the Supernatant of BHK Cells Containing the Expression Vectorsa Purification step

Volume (ml)

Total A280 units

hTF-His-C1 (mg)

Yield (%)b

Original culture supernant Reduction/exchange Post Poros 50 Metal chelate 1 flowthrough hTF-His-C1 Metal chelate 2 flowthrough hTF-His-C1

2500 370 50 85 180 35 40

4485 1506 238 146 59 133 7.7

65.7(A)b 70.9(A) ⫾ 14.2 60.4(A) ⫾ 4.5 6.7 ⫾ 1.3 42.1(S)b 6.2 ⫾ 1.7 5.5(S)

100.0 108 91.9

Purification step

Volume (ml)

Total A280 units

hTF-His-C2 (mg)

Yield (%)b

Original culture supernant Reduction/exchange Post Poros 50 Metal chelate 1 flowthrough hTF-His-C2 Metal chelate 2 flowthrough hTF-His-C2

2750 275 50 100 150 45 51

5430 1665 282 198 49 189 15.3

76.1(A)b 78.7(A) ⫾ 2.8 66.0(A) ⫾ 5.5 21.2 ⫾ 0.7 35.0(S)b 11.8 ⫾ 3.3 10.9(S)

100.0 103 86.7

64.1c — 72.4c

46.0c — 60.3c

a

Details of the purification procedure are given under Materials and Methods. A refers to the amount of hTF/2N determined by a competitive immunoassay. S refers to the amount of recombinant hTF determined by spectral analysis. c Recovery from first run over the metal chelate column was added to the amount recovered from the rerun of the first flowthrough. b

the biotinylated samples last for up to 2 years under our storage conditions (50% glycerol and ⫺20⬚C). Radioiodinated samples are only usable for a period of 3 months or less due to decay of the radionuclide.

experiments revealed that copper was much more effective than nickel in retaining the His-tagged hTFs. In the case of hTF-His-C1, up to 10 runs could be made without recharging the column with copper. However,

Purification of Recombinant His-Tagged hTF Typical results from the purification protocol are presented in Table 1 and documented visually in the SDS– PAGE in Fig. 2. At the start of the purification protocol, the recombinant protein accounts for about 2% of the total A280 units. The major protein component in the UG is bovine serum albumin which has a copper binding site (28) and thus will also bind to the metal chelate column when it is charged with copper. Therefore, an important part of the purification protocol is elimination of the BSA prior to the MC column which is easily achieved by the Poros 50HQ chromatography step (see Fig. 2, lanes 1 and 2). Following the Poros50 HQ column, the major proteins are the recombinant Histagged hTF, the hTF from the UG and the bovine transferrin also from the UG. The bovine transferrin is not recognized by the monoclonal antibody to hTF used in the assay and therefore is only observed as contaminating A280 (Table 1). Rechromatography of the flowthrough from the first MC column led to the recovery of some additional recombinant protein especially in the case of hTF-His-C2 (see Table 1, Metal chelate 2 flow through and recovered His-tagged protein). With the Poros MC column, the manufacturer recommends the use of Cu2+ since it is the transition metal that is bound most tightly by the resin. Preliminary

FIG. 2. SDS–polyacrylamide gel electrophoresis of the recombinant hTFs at various steps in the purification procedure. Lane 1, tissue culture medium containing hTF-His-C1 prior to the Poros 50 anion exchange column. The major protein band in lane 1 is bovine serum albumin. Lane 2, the same sample after the Poros column which eliminates the BSA; lane 3, the flowthrough from the metal chelate column; lane 4, purified hTF-His6-C1 from the metal chelate column; lane 5, purified hTF-His6-C2 from the metal chelate column; lane 6, recombinant hTF-NG; lane 7, hTF-Gly. The samples are flanked by BioRad low-molecular-weight standards with the molecular weights indicated. Note that samples taken during the purification of the hTF-His-2C look very similar on a gel to those shown in lanes 1–3 for hTF-His6-C1 (results not shown). Approximately 3 ␮g of sample was run in each lane.

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in contrast, we found that less hTF-His-C2 protein was retained with each successive run; i.e., binding capacity was gradually being lost. As indicated in the recovery data above, the ability of the MC column to retain this construct was not as great as for the hTF-His-C1 protein. We believe that this result is consistent with other data indicating that the His tag in the hTF-His-C2 construct is removing the copper from the column (see below). Since hTF can bind many different metals (29) including copper (30), an important control for our experiments was to determine if the MC column retained either apo- or diferric hTF. When 5 mg of either form of hTF was loaded onto the MC column, little or no sample was retained; i.e., neither form of hTF bound to the metal chelate matrix to any appreciable extent (data not shown). The overall protein recovery of the His-tagged hTFs was somewhat better than that found for the untagged hTF-NG. Currently, to achieve complete separation of untagged hTF-NG from hTF-Gly multiple runs are required which lead to a recovery of only about 50%. The long shallow gradient used for the purification results in runs that are 30 min each. We note that recovery of the His-tagged hTFs improved with each production run which was purified, ultimately resulting in the 60– 70% recoveries reported in Table 1. The amount of recombinant hTF expressed by the BHK system compares very favorably with production in other expression systems (see Introduction). Characterization of Recombinant His-Tagged hTFs Following purification, the hTF-His-C1 and hTF-HisC2 were compared to the recombinant hTF-NG by a number of different criteria. The spectral characteristics of the iron form of the three proteins are provided in Table 2 and confirm that the His-tagged constructs bind iron. The visible portion of the spectra is presented in Fig. 3. We attribute observation of a peak at A413 in the His-tagged constructs to the presence of copper bound to the His tag. Evidence for this explanation derives from a spectrum of the apo-protein (data not shown). The same peak was observed in the apo-hTFHis-C2 sample which also displays a faint yellow color. TABLE 2 Summary of Spectral Characteristics for Iron-Loaded hTF and His-Tagged HTF Fe2-hTF–CO3 complexes Protein Fe2hTF-NG Fe2hTF-His-C1 Fe2hTF-His-C2

␭max (nm) ␭min (nm) 464 461 462

403 397 397

Amax/Amin

A280/Amax

A413

1.38 1.19 1.14

27.5 24.6 25.7

No Yes Yes

FIG. 3. Visible spectra of the recombinant diferric hTF-NG and the two His-tagged hTFs. The peak at 413 nm attributed to the binding of copper to the His tag (see Results and Discussion for further details).

The His tag in this construct appears to bind copper with fairly high affinity, since at least some of the copper survives the iron removal protocol involving both low pH and chelators. The spectrum of apo-hTF-HisC1 lacks a peak at A413 and is consistent with complete metal removal from this protein. Extinction coefficients for the recombinant proteins were determined by titration of the apo-proteins with cobalt (III). The results are presented in Table 3. The deviation between the experimental and calculated values is significant only in the case of the hTF-His-C1. This sample failed to give the sharp break point usually found for the cobalt titrations making the determination less precise. We hypothesize that reversible binding of the cobalt by the hexa-His tag in this construct accounts for this result. In contrast, the hexa-His tag in TABLE 3 Experimental and Calculated mM Absorption Coefficients (␧) at 280 nm for Different Transferrins Sample

Experimental ␧a

Calculated ␧b

%Deviationc

hTF (glycosylated) hTF (nonglycosylated) hTF-His-C1 hTF-His-C2

86.68 ⫾ 0.03a n⫽2 85.20 ⫾ 0.25a n⫽3 77.8 84.7

85.12b

1.80

85.12b

0.09

85.12 85.12

8.60 0.20

a The experimental values were determined by titration with cobalt (III), as described in (26) in which the values for glycosylated and nonglycosylated hTF are reported. b The ␧ were calculated from the equation in (32). The value predicted in the reference for transferrin was low (83.37) because there are 19 rather than 5 Cys residues in transferrin. c % dev ⫽ 100 [␧(obs) ⫺ ␧(calc)]/␧(obs).

HIS-TAGGED HUMAN SERUM TRANSFERRIN

the hTF-His-C2 is bound to copper and therefore this construct yields a sharp break point and more precise experimental value. Electrospray mass analysis confirmed the composition of each of the recombinant proteins. The experimentally derived Mr values for each preparation are completely consistent with the mass calculated on the basis of the sequence, as shown in Table 4. The hTFHis-C1 has an addition of 11 amino acids, whereas the hTF-His-C2 has a net difference of 8 amino acids. The results of analysis of the three samples by sedimentation equilibrium ultracentrifugation are also given in Table 4. Although the actual molecular weight estimates yield masses that are lower than the calculated mass in every case with errors of ⫾2–3%, the results show that all three recombinant transferrins exist as monomers in solution and that the correct mass falls within the error of the determination. These results were qualitatively confirmed by chromatography of the His-tagged hTFs on a Sephacryl S-200 HiPrep column where a single peak was observed in each case (data not shown). There is a growing literature documenting the use of hexa-His tags on recombinant proteins to aid in the downstream purification (31). In the present work, separation of the His-tagged recombinant hTFs from the bovine transferrin and small amount of glycosylated human serum transferrin is achieved relatively rapidly and conveniently compared to isolation of recombinant hTF lacking a His tag. This finding is especially important and relevant to production of mutant forms of hTF which may be produced at lower concentrations and which often differ in their chromatographic profiles (20). In summary, substantial amounts of functional recombinant hTF with and without a C-terminal hexaHis tag have been produced in a mammalian cell expression system. The identity of the His-tagged constructs has been confirmed by determination of a precise molecular mass using electrospray mass analysis. The Histagged hTFs share many features common to hTF, including reversible iron binding, reactivity with a monoclonal antibody and presence as a monomer in solution. TABLE 4 Electrospray Mass Analysis and Sedimentation Equilibrium Determinations for Recombinant Human Serum Transferrin with and without a Hexa-His Tag

Protein hTF (Nonglycosylated) hTF-His-C1 hTF-His-C2

Calculated Mr

Experimental Mr

MW sedimentation equilibration

75,413

75,142.0 ⫾ 3.0

73,749 ⫾ 1,641

76,537 76,128

76,536.9 ⫾ 3.8 76,125.0 ⫾ 7.2

74,664 ⫾ 1,995 74,704 ⫾ 1,480

149

The two His-tagged constructs produced differ from each other in a couple of respects. The construct hTFHis-C2 containing the truncated hTF actually removes copper from the metal chelate column, thereby gradually decreasing the capacity of the column to bind protein and requiring recharging of the column with CuSO4. The full-length hTF with the His tag can be purified without recharging the column with copper. We will therefore produce the full-length construct to use in future studies. ACKNOWLEDGMENTS This work was supported by USPHS Grants R01 DK 21739 (A.B.M.) and R01 GM61666 (I.A.K.).

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