Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• transferrins or siderophilins comprise a class of proteins with strikingly similar features.
• X-ray crystallographic analyses of human lactoferrin (Anderson, B.F. ⁇ t &_L_ (1987) Proc. Natl. Acad. Sci. USA £4.:1769-1773) and rabbit serum transferrin (Bailey, S. ⁇ £ ⁇ JL_ (1988) Biochemistry 22:5804-5812) reveal that these proteins consist of two similar lobes connected by a short bridging peptide and that each lobe contains two domains defining a deep cleft containing the binding site for a metal ion and a synergistic anion.
• Chicken ovotransferrin gene has been expressed in transgenic mice (McKnight, G.S. e_£ al. (1983) Cell (Cambridge, MA) 14:335-341) and a fusion protein of part of rat transferrin with galactosidase has been
• This invention pertains to recombinant transferrin, to recombinant transferrin half- molecules comprising at least the metal-binding domains of a single lobe (amino-terminal or carboxy-terminal) of transferrin and to stable cell culture system for expression of the transferrin.
• the recombinant transferrin can be expressed in stable, transformed eukaryotic cells, such as baby hamster kidney cells, to yield essentially homogeneous (monodisperse) preparations of the full or half-molecule forms.
• the invention also pertains to mutant transferrins and transferrin half-molecules which have metal-binding or other properties which are different from the natural (wild-type) form of the transferrin.
• Transferrin half-molecules can be used in metal chelation therapy to treat individuals affected with abnormalities of metal regulation or with metal poisoning.
• transferrin half-molecules especially mutant forms which bind iron with a higher avidity than natural transferrin, can be administered to iron-overloaded individuals, e.g., thalassemics, in order to clear excess toxic iron from their bodies.
• half-molecules, or mutants thereof having altered metal ion selectivities could be used to clear other toxic metals, e.g., lead, mercury, cadmium, copper and zinc from the body.
• Figure 1 shows construction of the hTF/2N expression vector in pNUT.
• a 2.3-kb cDNA encoding human serum transferrin was isolated from a human liver cDNA library and a 1.5-kb Pstl/Hal fragment containing the complete amino-terminal domain coding sequence was cloned into M13mpl8.
• Double translational stop codons and a HindiII recognition sequence were introduced by site-directed mutagenesis, allowing the isolation of a BamHI/Hindlll fragment which, when joined to a BamHI/Hpall fragment, encodes the amino-terminal domain and signal sequence. This fragment was cloned into the eukaryotic expression vector pNUT, giving the vector pNUT-hTF/N2.
• the transferrin cDNA is under the control of the metallothionein promoter (MT-1 pro) and the human growth hormone transcription termination signals (hGH3'); pNUT also contains the SV40 early promoter (SV40) driving expression of a resistant DHFR cDNA (DHFR cDNA) using transcription termination signals from human hepatitis B virus (HBV) .
• Figure 2 shows a Western blot of immuno- precipitates from various baby hamster kidney cell lines. Samples of cell lysates (a) and medium (b) from Zn-induced cell cultures were precipitated with anti-hTF antiserum.
• hGH-pNUT and hTF/N2-pNUT cell lines were selected in 500 ⁇ M MTX and all cell culture was performed in DMEM/10% fetal calf serum.
• Lane 1 BHK cells; lane 2, hGH-pNUT transfected BHK cells; lane 3, hTF/N2- ⁇ NUT transfected BHK cells.
• the positions of molecular weight markers (x 10 ⁇ 3 ) are indicated to the right of the blot, the position of the additional protein band of M r 37,000 is also indicated ( ⁇ 37) to the right of the blot.
• Figure 3 shows the isolation and PAGE analysis of hTF/2N.
• Panel A FPLC isolations on a column of Polyanion SI of recombinant hTF/2N (upper trace) and proteolytically derived hTF/2N (lower trace).
• Panel B NaDodS ⁇ 4-PAGE (5-12% gradient of acrylamide) of molecular weight standards (lane Mr) and 3 ⁇ g of each of peaks a-d from panel A.
• Patent C Urea-PAGE under nonreducing conditions of the FPLC peaks a-d (recombinant hTF/2N species) and peaks e-h (proteolytically derived hTF/2N species) from panel A.
• FPLC fractions were pooled as follows; peak a (fractions 23-27), peak b (28-31), peak c (32-38), peak d (39-45), peak e (28-31), peak f (32-36), peak g (38-44), and peak h (46-51).
• Figure 4 shows titration of the major form recombinant hTF/2N with 10 mM Fe(III)(NTA)2- The amount of protein was 3.68 A28O units in 1.00 mL of 10 mM NaHC ⁇ 3. Visible spectra were run 5-10 minutes after each addition of iron to the magnetically stirred cuvette.
• Figure 5 shows proton magnetic resonance spectra of recombinant hTF/2N.
• the protein sample was 8 mg in 0.1 mL of 0.1 M KC1 in 2 H2 ⁇ .
• Figure 6 shows the 19 F nuclear magnetic resonance spectrum of m-F-Tyr recombinant hTF/2N.
• the protein sample was 6 mg in 0.1 mL of 0.1 M KC1 in H2 ⁇ ; the reference was 0.1 M trifluoroacetic acid in 2 H2 ⁇ .
• Figure 7 shows two separate oligonucleotides used as PCR primers to create the hTF/2C coding sequence.
• An EcoRI restriction fragment including coding sequence for the entire carboxy lobe was used as a template for 25 rounds of PCR amplification.
• Oligonucleotide 1 includes a Smal recognition site and the natural hTF signal sequence at its 5' end and matches the coding sequence for amino acids 334 -341 of hTF at its 3' end.
• Oligonucleotide 2 matches sequence in the 3* untranslated region of the hTF cDNA and introduces a second Smal recognition sequence at this site.
• This invention provides for the production of recombinant transferrin, recombinant transferrin half-molecules and mutant forms of full-length transferrin and transferrin half-molecules which have altered properties, such as improved metal-binding capability, compared to the natural transferrin molecules.
• Recombinant transferrins can be produced in large quantities and in substantially homogeneous (monodisperse) form.
• recombinant half-molecules of human serum transferrin can be produced as an essentially homogeneous preparation substantially free of other human serum proteins.
• half-molecules prepared by proteolysis of the holo-protein are difficult to purify and, in fact, the carboxy-terminal half of human transferrin cannot be satisfactorily prepared by proteolytic means.
• Recombinant techniques also allow the application of mutagenesis to design and produce new forms of transferrin.
• a recombinant transferrin of this invention is produced by transfecting a suitable host cell with a nucleic acid construct encoding the transferrin, culturing the transfected host cell under conditions appropriate for expression and recovering the recombinant transferrin expressed by the cell.
• the amino acid sequences for five transferrins have been reported (Jeltsch, J.-M. and Chambon, P. (1982) Eur. J. Biochem. 122:291-295: MacGillivray, R.T.A. e_£ .al * . (1983) J. Biol. Chem. 2 :3543-3553;
• Transferrin and transferrin half-molecules can be produced by standard techniques of site-directed mutagenesis. See Taylor e_t al.
• mutagenesis can be used to produce mutant transferrins which have metal binding properties that are different from natural transferrin.
• mutants capable of binding iron more avidly than natural transferrin can be produced.
• metal-binding domains can be mutagenized to replace one or more amino acids involved in binding with different amino acids.
• amino acids which are ligands for metal chelation are shown below (the number beside the amino acid indicates the position of the amino acid residue in the primary sequence where the first valine of the mature protein is designated position 1) Amino terminal lobe Carboxy terminal lobe - - 7
• transferrin control binding and these too can be targeted for mutagenesis. These are usually positively charged amino acids such as lysine, histidine or arginine.
• a mutant transferrin half-molecule which binds iron more avidly than natural transferrin can be produced by replacing the lysine residue at position 206 with glutamine (AAG ⁇ CAG) .
• the transferrin-encoding DNA is cloned into a eukaryotic expression vector containing appropriate regulatory elements to direct expression of the DNA.
• a preferred eukaryotic expression vector is the plasmid pNUT described by Palmiter, R.D. ⁇ £ al. (1987) Cell 5fl:435-443. This plasmid contains the metallothionein promoter which includes transcription of the transferrin encoding DNA in the presence of heavy metal and transcription termination signals of human growth hormone.
• pNUT contains dihydrofolate reductase gene under control of the SV40 early promoter with transcription termination signals from human hepatitis B virus to allow selection in cell culture.
• the gene encodes a mutant form of the enzyme which has a 270-fold lower affinity for the competitive inhibitor methotrexate. This allows for the immediate selection of transfected cells in very high concentrations (0.5 mM) of methotrexate and abrogates the need for a recipient cell line that is deficient in dihydrofolate reductase.
• pNUT also contains pUC18 derived sequences which allows it to be amplified in E . coli to provide sufficient amounts of the plasmid for transfection of recipient cells.
• the expression vector containing the DNA encoding the transferrin is incorporated into an appropriate host cell.
• the preferred host cell is a eukaryotic cell which can be transformed with the vector to yield a stable cell line which expresses a functionally active transferrin construct.
• a particularly useful cell is the baby hamster kidney cell. Baby hamster kidney cells can be transfected with a vector carrying the DNA construct encoding a transferrin (such as the pNUT plasmid) to provide a stable cell culture system which expresses and secretes a functionally active transferrin (full or half-molecule) . These cells are well-suited for economical, large scale growth and can be obtained from readily available sources.
• Standard techniques such as calcium phosphate coprecipitation or electroporation can be used to transfect the eukaryotic host cell with the vector.
• the cell is then cultured under conditions appropriate to induce expression of the transferrin.
• baby hamster kidney cells transfected with the pNUT vector are stimulated to express the transferrin construct in the presence of heavy metals.
• Baby hamster kidney cells are preferably cultured in the medium Dulbecco's Modified Eagle's medium-Ham's F-12 nutrient mixture with the serum substitute Ultraser GTM (Gibco) at about 1%.
• the expressed and secreted transferrin can be recovered from the culture medium.
• Standard purification procedures can be employed to yield a substantially homogeneous preparation of the recombinant transferrin.
• the transferrin in the culture medium is saturated with iron and then purified by anion exchange chromatography.
• the recombinant transferrins of the invention can be used to chelate and clear iron or other toxic metals from the body.
• the customary approach to iron chelation in vivo has been to assess a wide variety of naturally-occurring siderophores of microbial origin and synthetic iron chelators for their physiological effects, primarily the ability to bind and clear iron from the body. Many such compounds have been studied with varying abilities to clear iron and often with unacceptable side effects (Pitt, C.G. e_fc al. (1979) J. Pharm. Exp. Therap. 208:12-18).
• the only iron chelator used for clearing excess iron from humans remains deferoxamine, a cyclic peptide from Streptomvces pilosis.
• a preferred transferrin for iron chelation therapy is a mutant transferrin half-molecule which binds iron more avidly than natural transferrin.
• the use of a mutant half-molecule allows for more efficient chelation and removal of the metal.
• a particularly preferred mutant half-molecule is K206Q, described in the Exemplification below, which contains a glutamine rather than a lysine at position 206.
• a transferrin half-molecule is advantageous because unlike the holo-proteins, it passes through the glomeruli of the kidney and is excreted in the urine, so that metal is not only chelated but also cleared from the body.
• the single half-molecules do not bind to transferrin receptors on the membrane of tissue cells and therefore do not deliver iron to these tissues. Further, half-molecules of human transferrin would probably be recognized as "self" by the human body and therefore would not elicit an immunological response.
• mutant half-molecules can be designed to have altered metal ion selectivities.
• the chelators could be used to clear other toxic metals from the body, e.g., lead, mercury, cadmium, copper and zinc.
• the recombinant transferrin is administered to a patient in amounts sufficient to chelate the metal and reduce circulating levels below toxic levels.
• it is administered in a physiologically acceptable vehicle, such as saline, by a parenteral route (typically intravenously) .
• Recombinant full-length human transferrin can be used in nonserum supplements for cell culture media. Transferrin is required for iron uptake by growing cells. The use of recombinant transferrin avoids the risk of contamination (with, e.g., HIV or hepatitis virus) associated with transferrin purified from human serum.
• T4 DNA ligase DNA polymerase I (Klenow fragment) and T4 polynucleotide kinase were purchased from Pharmacia-PL Biochemicals. Restriction endonucleases were purchased from Pharmacia-PL Biochemicals and Bethesda Research Laboratories. Oligodeoxyribo- nucleotides were synthesized on an Applied Biosystems 380A DNA Synthesizer.
• Nitrocellulose filters were obtained from Schleicher and Schuell, 32 P-labeled nucleotides from New England Nuclear, goat anti-human transferrin antiserum from the Sigma Chemical Company, formalin-fixed Staphylococcus aureus cells from Bethesda Research Laboratories, the Protoblot immunoscreening detection system from Promega, the oligonucleotide-directed mutagenesis kit from Amersham, Dulbecco's modified essential medium and fetal bovine serum from Gibco, and anti-human transferrin monoclonal antibody HTF-14 was from the Czechoslovakian Academy of Sciences. All other reagents were analytical grade or purer.
• oligonucleotide coding for the amino-terminal eight amino acids of serum hTF as a hybridization probe.
• the oligonucleotide corresponded to nucleotides 88 to 111 of the hTF cDNA sequence reported by Yang, F. e_£ al. (1984) Proc. Natl. Acad. Sci. USA £:2752-2756).
• the oligonucleotide was end-labeled with T4 polynucleotide kinase and 2p_ ⁇ p (Chaconas, G. and van de Sande, J.H. (1980) Methods Enzymol.
• the eukaryotic expression vector pNUT (Palmiter, R.D. e_fc ali (1987) Cell (Cambridge, MA) lfi:435-443) and baby hamster kidney (BHK) cells were provided by Dr. Richard D. Palmiter (Howard Hughes Medical Institute, University of Washington).
• oligonucleotides were purified on Ci ⁇ reverse-phase columns (Sep-Pak, Waters Associates; Atkinson, T. and Smith, M. (1984) Oligonucleotide Synthesis: A Practical Approach (Gait, M.J., Ed.) pp 35-81, IRL Press, Oxford).
• Plasmid DNA was prepared from E___ coli JM105 and purified by two successive centrifugation steps with cesium chloride density gradients. BHK cells were grown in Dulbecco's modified essential medium (DMEM) with 10% fetal bovine serum to approximately 10 7 cells per 10-cm dish and were subsequently transfected with lO ⁇ g of plasmid by the calcium phosphate co-precipitation technique described by Searle, P.F. et al. (1985) Mol. Cell. Biol. 5_:1480-1489) .
• DMEM Dulbecco's modified essential medium
• DMEM fetal calf serum
• MTX methotrexate
• surviving cells were serially selected to 500 ⁇ M MTX. in some experiments, cells were selected immediately with 500 ⁇ M MTX.
• Large scale roller bottle cultures were initiated by seeding approximately 5 x 10 7 cells into each 850 cm 2 roller bottle containing 100 mL of DMEM-MTX. Cultures were induced at 80% confluency by the addition of ZnS ⁇ 4 to the medium to a final concentration of 0.08 mM. The medium was harvested 40 hours later.
• Immune-precipitation and Western Blotting Immune-precipitation of cell culture medium and cell lysates was performed by the method of Van Oost, B.A. £t & _. (1986) Biochem. Cell Biol. £4:699-705). Precipitates were analyzed by electrophoresis on 12% polyacrylamide gels in the presence of NaDodS ⁇ 4 (Laemmli, U.K. (1970) Nature (London) 127:680-685), followed by blotting onto a nitrocellulose membrane.
• the blot was incubated in PBS containing 0.1 mg/ml gelatin, then treated with goat anti-hTF antiserum (250-fold dilution in PBS), and finally developed with an alkaline phosphatase-conjugated, rabbit anti-goat IgG antibody according to the supplier's instructions.
• Amino Acid Substitution To incorporate 3-fluorotyrosine into the recombinant hTF/2N as a 19 F .NMR probe, the culture medium was supplemented with D,L-m-fluorotyrosine (Sigma Chemical Company) at 16% of the concentration of L-tyrosine in the medium. The cells grew as well on this medium as on the medium lacking D,L-m-fluorotyrosine.
• Fe(III)(NTA)2 was added to saturate all transferrin in the medium. After stirring at room temperature, the solution was dialyzed for 24 hours versus cold running tap water, and then for a few hours versus Milli-Q purified water. Concentrated Tris-HCl buffer, pH 8.4 was added to a final concentration of 5 mM, the preparation was centrifuged to remove any debris, and was loaded onto a column (2.5 x 80 cm) of DEAE-Sephacel (Pharmacia) equilibrated with 10 mM Tris-HCl buffer, pH 8.4.
• the column was then eluted with a linear gradient of NaCl (0 to 0.3 M) in the same buffer. Fractions showing a pink color were analyzed by NaDodS ⁇ 4 ⁇ PAGE, and fractions containing the recombinant protein (Mr 37,000) were pooled. Such fractions also contained bovine transferrin and albumin resulting from the fetal calf serum in the tissue culture medium. After concentration of the pooled fractions to 5 mL on an .Amicon PM-10 membrane, the protein was chromatographed on a column (2.5 x 90 cm) of Sephadex G-75 Superfine (Pharmacia-PL Biochemicals) equilibrated with 100 mM ammonium bicarbonate.
• a second chromatographic step through this column was necessary to resolve completely the hTF/2N from the bovine proteins.
• the A 465 /A 410 was usually ⁇ 1.0, indicating the presence of a contaminating heme-protein (possibly hemopexin) .
• the hTF/2N was finally purified to homogeneity by FPLC on a column (1 x 10 cm) of Polyanion SI (Pharmacia) using a linear gradient of NaCl (O to 0.3 M) in 50 mM Tris-HCl, pH 8.0 over a period of an hour at a flow rate of lml/min. Fractions of 1 mL were collected. Two to four protein bands emerged from the column, depending on the iron-binding status of the protein.
• NaDodS ⁇ 4 ⁇ PAGE was performed with 5% to 12% gradient gels and urea-PAGE was performed according to a modification (Brown-Mason, A. and Woodworth, R.C. (1984) J. Biol. Chem. 259:1866-1873) of the Makey, D.G. and Seal, U.S. (1976) Biochim. Biophvs. Acta 453:250-256 procedure. Electrofocusing was performed on a 0% to 50% sucrose gradient in a 110 mL glass column (LKB) with 0.8% Pharmalyte, pH 5 to 8
• the protein sample in 0.2 mL was diluted with 5 mL of solution withdrawn from the middle of the gradient. The sample was then reinjected into the isodense region of the column and focusing was continued for 24 hours. The gradient was collected from the bottom of the column in 1.5 mL fractions. Individual fractions were analyzed for A28O an ⁇ f° r pH. Fractions with maximum A28O were selected as representing the pis of the apo- and iron-saturated proteins. Iron was readily removed from the iron-protein by incubation in a buffer containing 1 mM NTA, 1 mM EDTA, 0.5 M sodium acetate, pH 4.9.
• the apo-protein was concentrated to a minimum volume on a Centricon 10 (Amicon), then diluted and reconcentrated twice with water and twice with 0.1 N KC1.
• the apo-protein had a tendency to precipitate in pure water, but redissolved readily in 0.1 M KC1.
• the apo-protein was made 10 mM in aHC ⁇ 3 and titrated with a suitable concentration of Fe(NTA)2 while monitoring the absorbance at 465 nm.
• Quantitative Immunoassav of Recombinant hTF/2N A competitive solid state immunoassay was used to assess the concentration of recombinant hTF/2N in the culture fluid and at various stages of the purification (Foster, W.B. st al.
• the expression vector pNUT (Palmiter, R.D. fit al. (1987) fifill (Cambridge, MA) 51:435-443) contains a mouse metallothionein-1/human growth hormone gene fusion that has been shown to direct high levels of human growth hormone in transgenic mice (Palmiter, R.D. et al. (1983) Science (Washington, D.C.) 222:809-814). Important functional features of this vector include a mouse metallothionein-1 promoter to induce cDNA transcription in the presence of heavy metals, pUC18 sequences to allow replication and selection in _E___ coli.
• DHFR dihydrofolate reductase
• the DHFR cDNA encodes a mutant form of the enzyme which has a 270-fold lower affinity for the competitive inhibitor methotrexate (NM) (Simonsen, C.C. and Levinson, A.D. (1983) Proc. Natl. Acad. Sci. USA 80:2495-2499).
• NM methotrexate
• This allows for the immediate selection of transfected cells in very high concentrations (0.5 mM) of MTX and abrogates the need for a recipient cell line that is deficient in DHFR.
• This fragment was inserted into Smal-cut pNUT, thus replacing the human growth hormone gene with a hTF/2N encoding cDNA, but leaving the transcriptional termination signal from the growth hormone gene intact.
• This plasmid was transfected into BHK cells and the resulting transformants were selected in the presence of MTX. To analyze the mRNA transcripts produced by the transfected BHK cells, total RNA was electrophoresed on an agarose gel in the presence of formaldehyde (Maniatis, T. fit al. (1982) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
• the blot was analyzed by using an oligonucleotide to the 3* untranslated region of the hGH gene as a hybridization probe.
• An inducible mRNA of approximately 1.4 kb was detected in the transfected cell line but not in mock-infected BHK cells (data not shown) . This agreed with the predicted size of the hTF/2N mRNA, including the expected hGH 3' untranslated sequence and poly (A) tail.
• Western blot analysis was performed both on cell lysates and the medium of various cell lines ( Figure 2).
• BHK cells BHK cells containing the hGH-pNUT plasmid
• BHK cells containing the hTF/2N-pNUT plasmid were grown in DMEM (BHK cells) or DMEM-MTX (BHK cells containing pNUT vectors).
• DMEM BHK cells
• DMEM-MTX BHK cells containing pNUT vectors
• Bound proteins were eluted by incubation with NaDodS ⁇ 4, electrophoresed on a polyacrylamide gel, and transferred to a nitrocellulose membrane. The membrane was then incubated with goat anti-hTF antiserum and rabbit anti-goat immunoglobulin conjugated to alkaline phosphatase.
• Figure 2 lanes la and lb
• BHK cells with hGH-pNUT plasmid Figure 2, lanes 2a and 2b
• the homogeneity of the hTF/2N product indicates the successful removal of signal sequence as cell lysate and secreted samples comigrate on SDS-PAGE.
• the anti-serum appears to be highly specific for human TF species, since little bovine TF is apparent in the precipitates.
• concentration of hTF/2N in the medium was approximately 10-15 ⁇ g/ml as detected by radioimmunoassay.
• Recombinant hTF/2N was purified by a three-step procedure that led routinely to an 80% yield of the major form of the protein, based on radioimmunoassay.
• the final purification on Polyanion SI led to quantitative resolution of the apo- and iron-saturated forms of both the minor ( ⁇ 5%) and major constituents of the protein ( Figure 3, panel A), as corroborated by urea-PAGE ( Figure 3, panel C) . Note that on urea-PAGE the slowest moving bands are apo-hTF/2N and the faster moving bands are Fe-hTF/2N.
• hTF/2N sequences were determined on an Applied Biosystems 470A protein sequencer. Approximately 200 pmol of each sample was analyzed. ⁇ Twelve sequencer cycles were analyzed. c No residue was identified at cycle 9; however, cysteine residues were not modified prior to the analysis. ⁇ Six sequencer cycles were analyzed.
• a hTF/2N molecule is produced that functions identically with the proteolytically derived species as judged by several independent criteria. This represents the first reported expression in a stable cell culture system of a functionally active form of this important iron transport protein.
• the pNUT based hTF/2N construction described here produces high levels of recombinant protein without the need for a DHFR-deficient cell line or tedious resistance amplification procedures.
• BHK cells are well-suited for economical, large scale growth and we are currently examining their growth characteristics on micro-carrier supports in bioreactor vessels. By using either roller bottles or a fermentor with a capacity of several liters, we can easily produce sufficient recombinant protein even for techniques such as NMR that traditionally have required a high concentration of protein.
• the minor form of recombinant hTF/2N isolated on Polyanion SI migrates more slowly than the major form on urea-PAGE ( Figure 3, panel C), but at the same rate on SDS-PAGE ( Figure 3, panel B) .
• Contamination of apo-hTF/2N with Fe-hTF/2N and vice versa on these gels arises from the method of pooling FPLC fractions, from some loss of bound iron on the urea gel and from binding of contaminating iron during workup of the FPLC samples.
• Identical N-terminal sequences show that the signal peptide has been removed from both minor and major forms of the recombinant protein.
• hTF/2N from human serum (Lineback-Zins, J. and Brew, K. (1980) ⁇ _. Biol. Chem. 255:708-713). the recombinant hTF/2N is non-glycosylated.
• hTF/2N The cause of the difference between major and minor forms of hTF/2N is unknown at present.
• the minor form has never represented more than 5% of the total recombinant protein and is usually less than 1%.
• the goal of isolating a monodisperse recombinant hTF/2N (the major form) has been achieved.
• the coding sequence for human serum transferrin was assembled from restriction enzyme digestion fragments derived from the full-length cDNA clone isolated from a human liver library described above. Since the parental plasmid (pKT-218) of the original clone had a limited number of unique restriction enzyme recognition sites, a series of cloning steps was required to introduce the coding sequence into a convenient vector. This process was initiated by cloning a Hpall/BamHI fragment from the 5' end of the cDNA into the vector pUC 18 (Messing, J. (1983) Meth. Enzympl. 101:20-28).
• the resulting plasmid was digested with BamHI and Hindlll and a BamHI/Hindlll fragment from the human transferrin cDNA was cloned adjacent to the initial fragment.
• the resulting plasmid was then digested with Hindlll and Pstl and a final Hindlll/Pstl fragment from the 3* end of the transferrin cDNA was cloned to complete the assembly of the full-length coding sequence.
• Plasmid DNA was prepared from !___ coli JM105 and purified by two successive centrifugation steps with cesium chloride gradients.
• Baby hamster kidney (BHK) cells were grown in Dulbecco's Modified Eagles' medium-Ham's F-12 nutrient mixture (DMEM-F-12) (Gibco; Sigma) with 10% fetal bovine serum to approximately 10 7 cells per 100 mm dish and were subsequently transfected with 10 ⁇ l of plasmid by the calcium phosphate coprecipitation technique described by Searle fit iL. (1985) Mol. Cell Biol. 5:1480-1489.
• DMEM-F-12 Dulbecco's Modified Eagles' medium-Ham's F-12 nutrient mixture
• the medium was changed to DMEM-F-12 containing 500 ⁇ M ethotrexate to select the plasmid containing cells.
• the cells were serially passaged at approximately 80% confluency with phosphate buffered saline containing EDTA (0.2 gm/1) to five 100-mm dishes, then to five T-175 flasks and finally to five expanded surface roller bottles (200 ml each).
• a serum substitute, Ultraser G (Gibco) at a level of 1% was used in place of fetal calf serum in DMEM-F-12 lacking phenol red.
• the medium is reduced in volume to ⁇ 10 ml and the transferrin is purified by passage over an anion exchange column (Polyanion SI, 1 x 10 cm) as described for the recombinant amino terminal human transferrin half-molecule. See above.
• the isolated recombinant full-length human serum transferrin displays some heterogeneity on this column attributed to variation in the glycosylation pattern.
• the protein is monodisperse on NaDod S ⁇ 4-polyacrylamide gel electrophoresis and has a spectrum and spectral ratios which are comparable to purified human serum transferrin.
• Substitution mutants are designated using the conventional single letter amino acid symbol of the wild type (native) residue, followed by the positional number of the replacement in the primary sequence, (where valine of the mature protein is designated position 1) followed by the symbol for the replacement residue.
• a mutant in which aspartic acid residue at position 63 is replaced by a serine residue would be designated D63S.
• hTF/2N mutants were accomplished by two techniques.
• a D81S substitution was prepared using the method of Nelson, R.M. and Long, G.L. (1989) Analvt. Biochem. lj__Q.:147-151. Briefly, a Hpall/BamHI fragment from the 5* end of the hTF/2N coding sequence was subcloned into pUCl ⁇ and then used as a template for a two step PCR-based mutagenesis procedure. The resulting DNA fragment was then recloned into M13mpl8 and the sequence of the mutant construction was confirmed by dideoxy sequence analysis.
• the fragment was then released from the double stranded form of the sequencing vector by digestion with Xbal and BaroHI and then ligated to a BamHI/Hindlll fragment from the original hTF/2N construction to produce a full length D81S-hTF/2N coding sequence, the fidelity of this splicing was confirmed by restriction digestion analysis and was subsequently cloned into pNUT as before.
• the substitution mutants G65R, D63C, K206Q and H207E were produced by subcloning the entire hTF/2N coding sequence into M13mpl8, which was then used as a template for oligonucleotide-directed mutagenesis (Zoller, M.J. and Smith, M. (1983) Meth.
• mutants include 1) D63S patterned on the naturally occurring mutation found in the C-terminal half of human melanoferrin, b) G65R patterned on the naturally occurring mutant found in the C-terminal half of hTF from a patient in England, c) K206Q based on the wild type mutation in the C-terminal half of ovotransferrin (oTF) from hen's egg white, d) H207E based on the wild type mutation in human lactoferrin (hLTF) and e) D63C as an attempt to change the metal selectivity of the iron binding site. All of these constructions have been expressed in stable transformants of baby hamster kidney cells in 10 to 100 mg amounts of recombinant protein.
• pNUT plasmids have been constructed containing the full length cDNA for oTF and chimeric cDNAs for hTF/2N-oTF/2C and oTF/2N-hTF/2C.
• Characteristics of the site-directed mutants include: the D63S mutant does bind iron (contrary to speculations in the literature) but much less avidly than the wild type protein. For instance, this mutant loses its bound iron on electrophoresis in PAGE gels containing 8 M urea, whereas the wild type retains its bound iron.
• the maximum in the visible spectrum lies at 422 nm in contrast to that or the wild type at 470 nm.
• the G65R mutant binds iron less tightly than does the wild type and has a visible maximum at 470 nm.
• the K206Q mutant binds iron much more avidly than does the wild type, as does its model, OTF/2C. Whereas the red color of the wild type iron protein disappears very rapidly in 0.5 M acetate buffer at pH 4.9, containing 1 mM each of EDTA and NTA, the mutant loses no color at all and requires pH 4 and 1 mM deferoxamine to release its bound iron. The apo-mutant appears to rebind iron more slowly than the wild type protein. The visible maximum lies at 460 nm for this mutant. The full length recombinant hTF runs at the same rate as the serum-derived protein on SDS-PAGE.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Toxicology (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Applications Claiming Priority (3)

Publications (2)

Family

ID=24618532

Family Applications (1)

Country Status (4)

Families Citing this family (6)

Citations (2)

Family Cites Families (1)

• 1992
  • 1992-02-06 WO PCT/US1992/000928 patent/WO1992013550A1/en not_active Application Discontinuation
  • 1992-02-06 JP JP4505865A patent/JPH07502723A/ja active Pending
  • 1992-02-06 CA CA002103583A patent/CA2103583C/en not_active Expired - Lifetime
  • 1992-02-06 EP EP92906503A patent/EP0724633A1/de not_active Ceased

Patent Citations (2)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events