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
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/505—Erythropoietin [EPO]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the glycoprotein hormone erythropoietin regulates the growth and differentiation of red blood cell (erythrocyte) progenitors.
• the hormone is produced in the fetal liver and adult kidney.
• Erythropoietin induces proliferation and differentiation of red blood cell progenitors through interaction with receptors on the surface of erythroid precursor cells.
• Oligonucleotide-directed mutagenesis has been used to prepare structural mutants of erythropoietin, lacking specific sites for glycosylation. Studies indicate that N-linked carbohydrates are important for proper biosynthesis and/or secretion of erythropoietin. These studies also show that glycosylation is important for in vivo, but not in vitro, biological activity. (Dube, S., et al.. J. Biol. Chem. 263:17516-17521 (1988); Yamaguchi, K. , et al.. J. Biol. Chem. 266:20434-20439 (1991); Higuchi, M., et a ., J. Biol. Chem. 267:7703-7709 (1992)).
• amino acids 99-129 were important in the formation of a functional region involved in receptor recognition, either through forming a necessary component of the protein's tertiary structure or through direct participation in receptor binding, or both.
• Preliminary experiments suggested that alterations in localized secondary structure within the 99-129 region resulted in inactivation of erythropoietin. Therefore, a possible structural role for amino acids 99-129 has been postulated.
• amino acids 99-110 (Domain 1) play a critical role in establishing the biologically active conformation of human erythropoietin. (Chern, Y., et al.. Eur. J. Biochem. 202:225-229 (1991)).
• mutant erythropoietin polypeptides are not suitable for elucidating the structure/function relationship that exists between erythropoietin and its cellular receptor.
• suitable erythropoietin antagonists for use, for example, in therapeutic treatment of polycythemias, or over production of erythropoietin.
• the present invention relates to DNA encoding mutated erythropoietin proteins which have altered biological activity, yet retain their secretable properties (i.e., secretable erythropoietin proteins). That is, the present invention relates to DNA encoding secretable erythropoietin proteins which have at least one amino acid residue in Domain 1 which differs from the amino acid residue present in the corresponding position of wildtype erythropoietin and which have altered ability t ⁇ regulate the growth and differentiation of red blood cell progenitors. Domain 1 of the mutants described herein refers to the amino acids which correspond to amino acids 99-110 (SEQ ID NO: 1) of the wildtype recombinant erythropoietin.
• Altered ability is defined as ability different from that of the wildtype recombinant erythropoietin ability to regulate the growth and differentiation of red blood cell progenitors.
• the present invention also relates to the modified secretable mutant erythropoietin proteins encoded by the DNA described above. These modified secretable erythropoietin proteins have altered biological activities. For example, the modified secretable mutant erythropoietin may have decreased biological ability to regulate growth and differentiation of red blood cell progenitor cells. Alternately, a modified secretable mutant erythropoietin protein described herein may exhibit increased heat stability.
• the present invention also relates to methods of modifying or altering the regulating activity of a secretable erythropoietin.
• erythropoietin proteins include at least one amino acid residue different from the amino acid present at the corresponding position in Domain 1 in the wildtype erythropoietin and are referred to as modified secretable human recombinant erythropoietin proteins having altered ability (i.e., decreasing or enhancing ability) to regulate the growth and differentiation of red blood cell progenitors. It is important to note that the ability of erythropoietin to regulate growth and differentiation of red blood cell progenitors depends on the ability of erythropoietin to bind to its cellular receptor. The mutant erythropoietin proteins described herein retain their secretable properties, thus indicating that these mutants also retain their biological conformation and the ability to interact with the erythropoietin receptor.
• this invention relates to modified secretable erythropoietin proteins in which the amino acid sequence of secretable human recombinant erythropoietin is altered at a selected site, or sites, in such a manner that the resulting erythropoietin protein has regulatory ability less than that of the corresponding (unaltered, or wildtype) human recombinant erythropoietin protein (i.e., decreased activity) or regulatory ability greater than that of the corresponding (unaltered, or wildtype) human recombinant erythropoietin protein (i.e., increased activity) .
• Such proteins which regulate red blood cell growth and differentiation to a lesser or greater extent than wildtype recombinant erythropoietin, are referred to, respectively, as modified secretable human recombinant erythropoietin proteins with decreased regulating ability and modified secretable human recombinant erythropoietin proteins with enhanced regulating ability.
• Human recombinant erythropoietin proteins with altered regulating activity differ from the wildtype human recombinant erythropoietin protein in that the amino acid sequences of the erythropoietin proteins with altered activity are different from the amino acid sequence of the wildtype protein at a site, or sites, found to be critical for the growth and differentiation of red blood cell progenitors.
• Alteration includes substitution of a different amino acid, as well as deletion or addition of an amino acid.
• the arginine 103 site is essential for erythropoietin activity.
• replacement of the arginine 103 by another amino acid results in a modified erythropoietin with significantly decreased activity. Modifications at this site, as well as other sites within Domain 1, can similarly be made to enhance regulating activity, as well as to decrease, or reduce regulating ability.
• modified secretable erythropoietin proteins described herein provide useful reagents to further elucidate the structure/function relationship of erythropoietin and its cellular receptor.
• modified secretable erythropoietin proteins with altered regulating ability can also be used for therapeutic purposes.
• modified . erythropoietin proteins with enhanced activity would be a more potent therapeutic, therefore requiring a lower effective dose or less frequent administration to an individual.
• Erythropoietin proteins with decreased activity that still retain their structural integrity and bind to their cognate receptor would be useful to decrease growth and differentiation of red blood cell precursors in certain leukemias and polycythemias.
• an erythropoietin protein that selectively triggers only certain events within the red blood cell precursor cell would be useful in treating various hematological conditions.
• FIG. 1 is a schematic diagram of the in vitro mutagenesis protocol.
• WT wildtype erythropoietin.
• Figure 2 depicts the structure of expression vector pSV-2-erythropoietin.
• Figure 3 is a graphic representation of the specific activities of nine mutant erythropoietin proteins.
• Figure 4 is a graphic representation of the results of monoclonal antibody precipitation of the mutant erythropoietin proteins.
• Figure 5 is a graphic representation of the activity of heat-denatured wildtype erythropoietin as measured by radioimmunoassay (D) and the Krystal bioassay (•) .
• Figure 6A-6B is a graphic representation of the activity of the 103 mutant erythropoietin proteins as measured by radioimmunoassay (D) and the activity of wildtype erythropoietin (•) .
• the present invention is based on the identification of amino acid residues of the erythropoietin polypeptide which are critical for its biological activity and secretable properties. These sites have been precisely defined through oligonucleotide-directed mutagenesis and used to create mutant human recombinant erythropoietin proteins which are altered by one, or more, amino acid substitutions and thus differ from wildtype erythropoietin.
• amino acids 100-109 were studied by alanine scanning utagenesis, as described in detail in Example 1.
• the single-stranded template for the mutagenesis reaction was prepared by growing cultures of bacteria transformed with the Phagemid and infected with a
• the first oligonucleotide was an ampicillin repair oligo designed to convert the vector
• the second oligonucleotide was a mutagenic oligo designed to change a portion of the erythropoietin cDNA sequence.
• mutant second strand was synthesized in vitro using T4 DNA polymerase and ligated.
• This DNA was then transformed into a repair minus strain of E_-_ coli and these cells were grown in the presence of ampicillin.
• the phagemid was then harvested and a second round of transformation was carried out and mutants were selected on ampicillin plates. This results in the production of double stranded phagemid containing both the ampicillin resistance gene and the mutated erythropoietin cDNA.
• Figure 1 shows the region of the erythropoietin cDNA encoding amino acids 96-113 (SEQ ID NO: 2) and the corresponding wildtype erythropoietin DNA sequence encoding amino acids 96-113 (SEQ ID NO: 3).
• the column of numbers on the left hand side of Figure 1 indicates the amino acid substitution. The only amino acid residue substitutions made are as indicated. The remainder of the human recombinant erythropoietin DNA sequence was not altered.
• 100A indicates that amino acid 100, normally a serine residue, was replaced by alanine
• 101A indicates that glycine 101 was replaced by alanine
• so forth SEQ ID NOS: 6-16.
• amino acid 103 was mutated twice.
• the first mutation was the substitution of alanine for arginine 103 (SEQ ID NO: 7) and the second substitution was aspartic acid for arginine (SEQ ID NO: 8) .
• Each mutated erythropoietin cDNA was identified by 5 restriction analysis, using standard laboratory protocols, and its structure was confirmed by DNA sequencing.
• the mutated erythropoietin cDNA was then inserted into the expression vector pSV-2 ( Figure 2) using standard laboratory techniques. (Mulligan, R. C. , et al. Nature
• COS-7 cells were transfected with the pSV-2-erythropoietin constructs.
• the supernatant medium was harvested and the biological activity of the mutant erythropoietin proteins and wildtype erythropoietin was measured by the Krystal bioassay (Krystal, G., EXP. Hematol. 11:649-660 (1983)). Briefly, the bioassay of Krystal measures the
• mice 20 effect of erythropoietin on intact mouse spleen cells. Mice were treated with phenylhydrazine to stimulate production of erythropoietin-responsive red blood cell progenitor cells. After treatment, the spleens were removed, intact spleen cells were carefully isolated and
• radioimmunoassay Incstar, Stillwater, MN
• Specific activities were calculated as international units measured in the Krystal bioassay divided by micrograms as measured as immunoprecipitable protein by RIA.
• Both assays used wildtype recombinant human erythropoietin standardized against the World Health Organization Second International Reference Standard preparation.
• substitution of arginine 103 by alanine (SEQ ID NO: 7) , aspartic acid (SEQ ID NO: 8) , . asparagine (SEQ ID NO: 17) , glutamic acid (SEQ ID NO: 18) , glutamine (SEQ ID NO: 19), histidine (SEQ ID NO: 20), and leucine (SEQ ID NO: 21) essentially eliminated erythropoietin biological activity.
• substitution of lysine for arginine 103 (SEQ ID NO: 22) decreased activity to approximately 10% of wildtype erythropoietin.
• Substitution of alanine for serine 104 decreased activity to approximately 16% of wildtype erythropoietin (SEQ ID NO: 14) .
• Substitution of alanine for leucine 105 reduced the activity to approximately 44 percent of wildtype erythropoietin.
• substitution of alanine for leucine 108 reduced the activity to approximately 37% of wildtype erythropoietin.
• substitution of alanine for serine 100 (SEQ ID NO: 4) and glycine 101 increased the specific activity of the mutant protein.
• mutant erythropoietin proteins described herein provide structurally intact (i.e., with the proper biological conformation) mutant erythropoietin proteins.
• the first monoclonal antibody recognizes an epitope within amino acids 1-26 of erythropoietin.
• the other monoclonals recognize distinct epitopes within amino acids 99-129. It is known that a gross change in the tertiary structure of erythropoietin would result in an inability of one or more of the monoclonal antibodies to recognize the erythropoietin molecule. For example, it has been demonstrated that radio-iodination of erythropoietin in the presence of chloramine-T denatures the molecule, resulting in loss of biological activity and corresponding loss of recognition by monoclonal antibody (data not shown) .
• Figure 4 shows mutant erythropoietin protein precipitated as percent of control of wildtype erythropoietin precipitated using three monoclonal antibodies designated across the horizontal axis, 1-26, 99-129 ⁇ and 99-129/?.
• the three erythropoietin proteins examined were the wildtype erythropoietin, the 103 alanine mutant and the 103 aspartic acid mutant.
• monoclonal 1-26 recognized each of the three recombinant erythropoietin proteins with equal efficiency, indicating that mutation of amino acid 103 to either alanine or aspartic acid did not result in a gross distortion of erythropoietin's conformation.
• monoclonal 99-129 also recognized the wildtype, 103 alanine mutant and 103 aspartic acid mutant with no statistically significant difference among them. This indicates that the conformation within the amino acids 99- 129 is similar among the three recombinant erythropoietin proteins.
• monoclonal 99-129,9 recognized both mutant erythropoietin proteins with approximately half the efficiency as it recognized the wildtype erythropoietin. This is consistent with the subtle structural change introduced by a single amino acid mutation. Taken together, it is reasonable to assume that the inactive point mutants, 103 alanine and 103 aspartic acid, are not grossly denatured.
• erythropoietin analogs with altered side chain properties at this position.
• Arginine was substituted with histidine (R103H) , lysine (R103K) , asparagine (R103N) , gluta ine (R103Q) , leucine (R103L) and glutamic acid (R103E) to generate 6 new altered erythropoietin molecules.
• Culture supernatants of cells transfected with these constructs were tested in the Krystal bioassay and the heat stability assay for biological activity and structural stability, respectively.
• R103K had detectable levels of biological activity in the Krystal bioassay. Its specific activity (in units per microgram erythropoietin protein) was 10.2% ⁇ 1.3% that of wildtype erythropoietin.
• the heat denaturation curve of R103K was essentially identical to that generated for the wildtype protein.
• the heat denaturation curve for R103E was notably different from that of wildtype, and very similar to that of R103D.
• the other 4 mutants had denaturation kinetics intermediate to that of these two proteins. (Data not shown)
• these mutations do not disrupt the structural integrity of the erythropoietin protein, as evidenced by the fact that the mutated protein is secreted. That is, as the data presented herein indicates, these mutant erythropoietin proteins retain their biological conformation. These results also indicate that Domain 1 amino acids 99-110 very likely participate in receptor recognition and activation.
• mutant erythropoietin proteins also demonstrate increased heat stability relative to the wildtype erythropoietin, even though the biological activity of the mutant has been significantly decreased.
• Substitution of alanine at arginine 103 produced erythropoietin mutants with no detectable erythropoietin activity as measured by standard techniques. Mutations at serine 104, leucine 105 and leucine 108 also significantly decreased activity. In a similar manner, other changes at one or more of these critical sites can result in reduction of erythropoietin activity. Conversely, amino acid residues can be introduced at these critical sites to produce modified secretable human recombinant erythropoietin proteins with enhanced biological activity. Conservative substitutions can be made at one or more of the amino acid sites within residues 100-109 of the molecule. For example, alanine and aspartic acid have been used to replace arginine 103.
• substitutions at these critical sites alone or in combination, of amino acids having characteristics different from those of amino acids whose presence at those sites has been shown to eliminate or reduce erythropoietin activity can also be made and their effect on activity assessed as described above.
• substitutions of some, or all, of the amino acids at one, or more, of these critical sites which result in modified secretable erythropoietin proteins with enhanced erythropoietin activity can be made.
• erythropoietin proteins having enhanced activity can be identified.
• arginine 103 is essential for erythropoietin's biological activity. Additionally, serine 104, leucine 105 and leucine 108 also appear to play a significant role in biological activity. Furthermore, these subtle point mutations do not compromise the structural integrity, (i.e., secretability) of the erythropoietin molecule. Thus, it is reasonable to assume that the mutant erythropoietin proteins will be recognized by the erythropoietin cellular receptor in essentially the same manner as the wildtype erythropoietin.
• Modified secretable human recombinant erythropoietin proteins of the present invention can be used for therapy and diagnosis of various hematologic conditions.
• an effective amount of modified secretable recombinant erythropoietin with enhanced activity to regulate the growth and differentiation of red blood cell progenitors can be used therapeutically (in vivo) to treat individuals who are anemic (e.g. as a result of renal disease, chemotherapy, radiation therapy, or AIDS) .
• An effective amount of modified secretable human recombinant erythropoietin protein, as defined herein, is that amount of modified secretable erythropoietin protein sufficient to regulate growth and differentiation of red blood cell progenitor cells.
• modified secretable erythropoietin protein with increased regulatory ability will bind to the erythropoietin receptor and stimulate the growth and differentiation of red blood cell progenitor cells.
• the modified secretable erythropoietin with enhanced activity would be more potent than the wildtype erythropoietin.
• a lower effective dose or less frequent administration to the individual would be required.
• Modified secretable erythropoietin with altered regulating activity can also be used to selectively trigger only certain events regarding the growth and differentiation of red blood cell precursors. For example, it has recently been shown that binding of erythropoietin to its receptor generates two distinct chemical signals in cells, a protein kinase C dependent activation of the proto-oncogene c-myc and a phosphatase mediated signal to c-myb. (Spangler, R. , et al.. J. Biol. Chem. 266:681-684 (1991); Patel, H. R. and Sytkowski, A. J., Abstract 1208, Blood 78(10) Suppl. 1 (1991)).
• a modified secretable erythropoietin can be used to selectively activate either the protein kinase C or the phosphatase pathways.
• modified secretable erythropoietin with decreased activity can be used to treat individuals with various erythroleukemias.
• an effective amount of modified secretable erythropoietin protein with decreased regulatory ability will bind to the erythropoietin cellular receptor.
• the mutant protein upon the mutant erythropoietin protein binding to the receptor, the mutant protein lacks ability to trigger subsequent erythropoietin events.
• mutant erythropoietic because the mutant erythropoietic is bound to the receptor, it prevents wildtype erythropoietic from binding to the receptor (i.e., competitively inhibits the binding of wildtype erythropoietin) .
• the red blood cell progenitors do not proliferate and/or differentiate.
• the mutant erythropoietin proteins of the present invention are secretable, indicating that they retain their structural integrity, and thus fully participate in receptor recognition and binding. The initial interaction of a hormone with its cognate receptor might be expected to result in further conformational changes of the hormone ligand, thereby stabilizing the hormone/receptor complex and allowing the formation of higher ordered complexes.
• erythropoietin protein of the present invention with no detectable erythropoietin activity, binds to its receptor, it is reasonable to assume that the subsequent events triggered by receptor binding will be altered or inhibited. Therefore, it is also reasonable to assume that growth and differentiation of red blood cell progenitor cells will be altered or inhibited, thereby inducing a remission in a red blood cell leukemia.
• modified secretable erythropoietin which retains its structural integrity to bind to the receptor, yet does not activate red blood cell proliferation, would be useful as an antagonist to block such constitutive activation.
• modified secretable erythropoietin proteins with increased stability would provide long- acting erythropoietin antagonists. Modified secretable erythropoietin would be useful to treat various hemoglobinopathies and hemolytic anemias.
• polycythemia vera is characterized by uncontrollable proliferation of red blood cells and is currently treated by chemotherapy, radiation or phlebotomy.
• the increased number of red blood cells increases blood viscosity, leading to a hypertensive condition that can result in a stroke. It is reasonable to predict that an antagonist of erythropoietin, which binds to the receptor and blocks activation, would be a useful, non-invasive treatment.
• hemolytic anemias such as sickle cell anemia and thalassemia
• red blood cells The body responds by increasing the levels of erythropoietin produced to stimulate red blood cell production.
• the red blood cells produced carry defective hemoglobin. It would be useful to use a modified secretable erythropoietin to reduce production of defective erythrocytes while another form of therapy is used to stimulate normal hemoglobin synthesis.
• Modified secretable erythropoietin may be administered to individuals parenterally or orally.
• the modified secretable erythropoietin proteins of this invention can be employed in admixture with conventional pharmaceutically acceptable carriers. Suitable pharmaceutical carriers include, but are not limited to, water, salt solutions and other physiologically compatible solutions.
• the modified secretable erythropoietin proteins of the present invention may be administered alone, or combined with other therapeutic agents.
• modified secretable erythropoietin administered to an individual in a specific case will vary according to the specific modified secretable erythropoietin protein being utilized, the particular compositions formulated, and the mode of application. Dosages for a given individual can be determined using conventional considerations such as the severity of the condition, body weight, age and overall health of the individual.
• Modified secretable erythropoietin can also be used for diagnostic purposes. For example, it can be used in assay procedures for detecting the presence and determining the quantity, if desired, of erythropoietin receptor. A modified secretable erythropoietin with enhanced activity would be useful to increase the sensitivity and decrease the incubation times of such assays. It can also be used in in vitro binding assays to determine the effect of new drugs on the binding of erythropoietin protein to its receptor.
• Modified secretable erythropoietin proteins described herein also provide useful research reagents to further elucidate the role of erythropoietin in erythropoiesis, as well as the structure/function relationship of erythropoietin and its cellular receptor.
• the oligonucleotide-directed mutagenesis used to prepare the modified secretable human recombinant erythropoietin proteins of the present invention was performed using the Altered Sites" In Vitro Mutagenesis System (Promega Corporation of Madison, WI) .
• the Altered Sites" system consists of a unique mutagenesis vector and a simple, straightforward procedure for selection of oligonucleotide-directed mutants.
• the system is based on the use of a second mutagenic oligonucleotide to confer antibiotic resistance to the mutant DNA strand.
• the system employs a phagemid vector, pSELECT * -l, which contains two genes for antibiotic resistance.
• An oligonucleotide is provided which restores ampicillin resistance to the mutant strand during the mutagenesis reaction. This oligonucleotide is annealed to the single-stranded DNA (ssDNA) template at the same time as the mutagenic oligonucleotide and subsequent synthesis and ligation of the mutant strand links the two.
• the DNA is transformed into a repair minus strain 2_. coli. or other suitable host, and the cells are grown in the presence of ampicillin, yielding large numbers of colonies.
• the pSELECT-1 plasmid is a phagemid, defined as a chimeric plasmid containing the origin of a single- stranded DNA bacteriophage. This phagemid produces ssDNA upon infection of the host cells with the helper phage R408 or M13K07.
• the vector contains a multiple cloning site flanked by the SP6 and T7 RNA polymerase promoters and inserted into the lacZ ⁇ -peptide.
• Cloning of a DNA insert into the multiple cloning site results in inactivation of the ⁇ -peptide.
• colonies containing recombinant plasmids are white in a background of blue colonies.
• the SP6 and T7 promoters may be used to generate high specific activity RNA probes from either strand of the insert DNA. These sites also serve as convenient priming sites for sequencing of the insert.
• the pSELECT-1 vector carriers gene sequences for both ampicillin and tetracycline resistance. However, the plasmid is ampicillin sensitive because a frameshift was introduced into this resistance gene by removing the Pst I site. Therefore, propagation of the plasmid and recombinants is performed under tetracycline selection.
• the pSELECT-Control vector provides a convenient white/blue positive control for mutagenesis reactions.
• This vector was derived from the pSELECT-1 vector by removing the Pst I site within the polylinker.
• a lacZ repair oligonucleotide (supplied with the system) may be used to introduce a four base insertion which corrects the defect in the lacZ gene and restores colony color to blue.
• the fraction of blue colonies obtained is an indication of the mutagenesis efficiency.
• lacZ repair oligonucleotide When the lacZ repair oligonucleotide is used in combination with the ampicillin repair oligonucleotide to correct this defect, 80-90% of the ampicillin resistant colonies are blue. When the lacZ repair oligonucleotide is used alone, a mutagenesis efficiency of only 2-5% is seen.
• the mutagenic oligonucleotide must be complementary to the single-stranded target DNA.
• the ssDNA produced by the pSELECT-1 phagemid is complementary to the lacZ coding strand.
• the stability of the complex between the oligonucleotide and the template is determined by the base composition of the oligonucleotide and the conditions under which it is annealed. In general, a 17-20 base oligonucleotide with the mismatch located in the center will be sufficient for single base mutations. This gives 8-10 perfectly matched nucleotides on either side of the mismatch. For mutations involving two or more mismatches, oligonucleotides of 25 bases or longer are needed to allow for 12-15 perfectly matched nucleotides on either side of the mismatch.
• oligonucleotides can be annealed by heating to 70°C for 5 minutes followed by slow cooling to room temperature.
• DNA to be mutated is cloned into the pSELECT-1 vector using the multiple cloning sites.
• the vector DNA is then transformed into competent cells of JM109, or a similar host, and recombinant colonies are selected by plating on LB plates containing 15 ⁇ g/ml tetracycline, 0.5mM IPTG, and 40 ⁇ g/ml X-Gal. After incubation for 24 hours at 37°C, colonies containing recombinant plasmids will appear white in a background of blue colonies.
• helper phage R408 or M13K07 at an m.o.i. (multiplicity of infection) of 10 (i.e., add 10 helper phage particles per cell) .
• helper phages supplied with this system add 40 ⁇ l. Continue shaking for 6 hours to overnight with vigorous agitation.
• phage precipitation solution Promega
• the mutagenesis reaction involves annealing of the ampicillin repair oligonucleotide and the mutagenic oligonucleotide to the ssDNA template, followed by the synthesis of the mutant strand with T4 DNA polymerase.
• the heteroduplex DNA is then transformed into the repair minus E. coli strain DMH71-18 mutS or other suitable strain. Mutants are selected by overnight growth in the presence of ampicillin. Plasmid DNA is the isolated and transformed into the JM109 strain, or other suitable strain. Mutant, ampicillin resistant colonies may be screened by direct sequencing of the plasmid DNA.
• the amount of oligonucleotide required in this reaction may vary depending on the size and amount of the single-stranded DNA template.
• the ampicillin repair oligonucleotide (27 bases long) should be used at a 5:1 oligo:template ratio and the mutagenic oligonucleotide should be used at a 25:1 oligo:template ratio.
• a typical reaction may contain approximately lOOng (0.05 pmol) of ssDNA.
• This procedure is used to isolate pSELECT-1 or pSELECT-Control plasmid DNA from the overnight culture of BMH 71-18 mut S (step B.5, above). A yield of l-3 ⁇ g of plasmid DNA may be expected.
• the yield of plasmid DNA can be determined by electrophoresis on an agarose gel.
• OPTIONAL A heat shock may be performed at this step.
• the Altered Sites mutagenesis procedure generally produces greater than 50% mutants, so colonies may be screened by direct sequencing. A good strategy is to pick 10 colonies and start by sequencing 4 of these. If the mutation is located within 200-300 bases of either end of the DNA insert, the SP6 or T7 sequencing primers may be used for convenient priming of the sequencing reactions.
• EXAMPLE 2 Cell culture and Transfection
• COS-7 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (GIBCO) . Transient expression of cDNAs was performed using a DEAE-Dextran protocol modified by O.lmM chloroquine treatment (Sussman, D.J. & Milman, Mol. Cell Biol. 4:1641-1645 (1984); Ausubel, F.M. , et al., "Current Protocols in Molecular Biology” pp.921-926, John Wiley and Son, New York, (1989)). 3 days before the transfection, COS-7 cells were plated at 2 x 10 5 /10-cm tissue culture dish. 4 ⁇ g DNA were used in each transfection. Medium was collected 3 days after transfection and assayed for erythropoietin activity and protein.
• GIBCO Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
• Wildtype and mutant erythropoietin contained in supernatant medium from COS cell transfections were diluted one- to four-fold with Dulbecco's modified Eagle medium containing 10% fetal bovine serum. After one hour incubation at 37 degrees C with a monoclonal anti-peptide antibody to erythropoietin directed against amino acids 1- 26 or 99-129, an equal volume of Omnisorb (Calbiochem) was added to the samples and the suspension was incubated for one hour at 4 degrees C. The Omnisorb was pelleted by centrifugation at 4000 rpm for 30 seconds.
• the erythropoietin remaining in the supernatant which was not bound by the monoclonal antibody was measured by radioimmunoassay.
• the amount of erythropoietin bound by antibody (as a percent) was calculated by subtracting the amount in the supernatant from 100%, the starting concentration.

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