WO1993008831A1 - Expression d'hemoglobine recombinee et de variants d'hemoglobine recombines dans des levures - Google Patents

Expression d'hemoglobine recombinee et de variants d'hemoglobine recombines dans des levures Download PDF

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Publication number
WO1993008831A1
WO1993008831A1 PCT/US1991/008108 US9108108W WO9308831A1 WO 1993008831 A1 WO1993008831 A1 WO 1993008831A1 US 9108108 W US9108108 W US 9108108W WO 9308831 A1 WO9308831 A1 WO 9308831A1
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globin chain
dna sequence
globin
yeast
variant
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PCT/US1991/008108
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English (en)
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Joseph De Angelo
Nalini M. Motwani
Wajeeh Bajwa
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Strohtech, Inc.
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Priority to PCT/US1991/008108 priority Critical patent/WO1993008831A1/fr
Publication of WO1993008831A1 publication Critical patent/WO1993008831A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention is directed to certain substantially pure hemoglobins comprising certain globin chains.
  • the globin chain may be an alpha-like globin chain or a beta-like globin chain, or variants thereof.
  • the invention is further directed to an expression vector which specifically comprises a DNA sequences encoding a certain globin chain or heme-binding fragment thereof operably linked to a yeast promoter.
  • the invention is also directed to methods for producing certain hemoglobins in yeast.
  • physiological oxygen carriers such as in blood substitute solutions, or as in a plasma expander.
  • Transfusion of a patient with donated blood has a number of disadvantages. Firstly, there may be a
  • a blood substitute is an oxygen carrying solution that also provides the oncotic pressure necessary to maintain blood volume.
  • Two types of substitutes have recently been studied, fluorocarbon emulsions and hemoglobin solutions.
  • Hemoglobin as it exists within the red blood cell is composed of two alpha-like globin chains and two beta-like globin chains, each with a heme residue.
  • One alpha-like globin chain and one beta-like globin chain combine to form a dimer which is very stable.
  • Alpha-like and beta-like globin genes are each a family of related globin genes which are expressed at different stages of development and regulated by oxygen tension, pH, and the development from embryo to fetus to newborn.
  • Two dimers then line up in antiparallel fashion to form tetramers.
  • the binding of dimers to form the tetramers is not as strong as in the case of monomers binding to associate into dimers.
  • the tetramers therefore, have a tendency to fall apart to form dimers and there is always an equilibrium between tetramers, dimers, and monomers.
  • the alpha-like globin genes are clustered together on chromosome 16 and include genes encoding the embryonic zeta-globin chain and the adult alpha-globin chain, present in both the fetus and newborn.
  • the beta-like globin genes reside on chromosome 11 and include genes encoding the embryonic epsilon-globin chain, the fetal gamma-globin chain, and the adult delta-globin and adult beta-globin chains.
  • Two types of gamma-globin chains have been identified, G gamma and A gamma, which differ by the presence of a single glycine or alanine residue,
  • the gamma chain has been found to contain a polymorphic site at position 75, which also can be occupied either by
  • hemoglobins areoleucine or threonine.
  • a variety of hemoglobins may be formed (reviewed in Kutlar et al., 1989, Hemoglobin 13:671-683 and Honig and Adams, Human Hemoglobin Genetics,
  • HbA alpha 2 beta 2
  • HbA 2 alpha 2 delta2
  • HbF alpha 2 gamma 2
  • HbBarts (gamma 4 ), HbH (beta 4 ), and Hb Portland I
  • Hb Gower I (zeta 2 epsilon 2 ), and Hb Gower II (alpha 2 epsilon 2 ).
  • hemoglobin solution contains 45 g of protein. It is estimated that at least 12 million units of blood are used in the U.S. per year. Therefore, the production of 450,000 kg of hemoglobin per year would be required. Secondly, it is important to obtain hemoglobin that is free from
  • hemoglobin is normally a tetramer of 64,000 molecular weight, it can dissociate to form alpha-beta dimers. The dimers are rapidly cleared by the kidneys and the residence time is much too short for cell-free hemoglobin to be useful as a blood substitute. Fourthly, cell-free
  • hemoglobin has too high an oxygen affinity to effectively release oxygen to the tissues due to the absence of 2,3-diphosphoglycerate (2,3-DPG). Efforts to restore 2,3-DPG have been unsuccessful since 2,3-DPG is rapidly eliminated from the circulation.
  • hemoglobin via recombinant DNA systems, chemical modification of hemoglobin, and the production of hemoglobin variants .
  • Human embryonic zeta-globin (Cohen-Sohal, 1982, DNA 1:355-363), human embryonic epsilon-globin (Baralle et al., 1980, Cell 21:621-630), human fetal gamma-globin
  • hemoglobin because of E. coli's inability to remove the N-formyl-methionine by post-translational processing.
  • the amino terminus is known to be critical in determining the oxygen binding properties of human hemoglobin as has been shown in the case of Hb Raleigh (Moo-Penn, et al., 1977, Biochemistry, 16:4872-4879).
  • the hemoglobin produced in bacteria can contain E. coli endotoxins.
  • yeast cells were unable to excise the intervening sequences in both alpha- and beta-globin precursor mRNA (Langford et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1496-1500 and Beggs et al., 1980, Nature (London) 283:835-840). An attempt was also made to secrete beta-globin in
  • GalK-FX-beta-globin sequence a beta-galactosidase secretion signal sequence
  • GalK-FX-beta-globin however remained within the cells under conditions where galactokinase was secreted.
  • dihydrofolate reductase gene has also been disclosed (Lau et al., 1984, Mol. Cell Biol. 4:1469-1475). However, the expression of the globin genes was found to be rather low due to low efficiency of gene transfer.
  • modification examples include crosslinking with polyalkylene glycol (Iwashita, U.S. Patent No. 4,412, 989 and 4,301,144); with polyalkylene oxide (Iwasake, U.S.
  • Patent No. 4,670,417 with a polysaccharide (Nicolau, U.S. Patent Nos. 4,321,259 and 4,473,563); with inositol
  • Hemoglobin has also been chemically modified to decrease the oxygen affinity of isolated hemoglobin.
  • One approach has involved polymerization with pyridoxal
  • heglobin variants include: variants which autopolymerize, variants which prevent the dissociation of the tetramer, variants with lowered intrinsic oxygen affinity, and variants that are stable in alkali. Examples of autopolymerizing
  • hemoglobin variants include Hb Porto Alegre, Hb
  • Hb Porto Alegre is a beta chain variant first reported by Tondo et al. (1974, Biochem. Biophys. Acta 342: 15-20; 1963, Am. J. Human Genet. 15:265-279).
  • the beta-9 serine is replaced by cysteine which is able to form disulfide bonds with other cysteine residues.
  • Hb Porto Alegre forms poly-tetramers. These polymers however do not form in the blood of Hb Porto Alegre carriers.
  • Hb Porto Alegre carriers have a two-fold elevated level of glutathione and three-fold elevated level of glutathione reductase which prevents the polymerization of the Hb Porto Alegre within the red blood cells (Tondo et al., 1982, Biochem. Biophys. Res. Commun. 105:1381-1388). The exact structure of these polymers is not known.
  • the new variant was first reported by Adams et al. (1987, Hemoglobin 11:435- 452). The beta-44 serine is replaced by cysteine in this variant resulting in inter-tetramer disulfide bonds. This variant is believed to form polymers with as many as ten tetramers.
  • Hb Ta-Li is another known polymerizing beta variant.
  • the beta-83 glycine is replaced by cysteine.
  • variants include those with nondissociating tetramers.
  • Hb Rainier a well characterized variant of the beta chain (Greer and Perutz, 1971, Nature New Biology 230:261 and
  • the beta- 145 tyrosine is replaced by cysteine.
  • This cysteine is able to form disulfide crosslinks with beta-93 cysteine which is present in natural beta-globin.
  • This disulfide bond is intra-tetramer, i.e, it is formed between the two beta subunits within a tetramer. This covalent disulfide bond stabilizes the tetramer form and prevents the
  • Hb Rainier has also been found to have a high affinity for oxygen, a reduced Hill coefficient, and only half the alkaline Bohr effect of normal hemoglobin.
  • Hb Motown/Hacettepe is a variant reported to be stable in alkali (Gibb and Rucknagel, 1981, Clinical Research 29:795A and Altay et al., 1976, Biochem. Biophys. Acta 434:1-3).
  • the beta-127 glutamine is replaced by glutamic acid in this variant. This portion of the beta chain is involved in the alphaibeta 1 interface between the monomers forming a dimer.
  • the substituted glutamic acid forms an ionic bond with alpha-31 arginine. This is a stronger bond than that formed between the alpha-31 arginine and the normal beta-127 glutamine and is believed to be responsible for the increased stability of Hb
  • HbF fetal hemoglobin
  • bovine hemoglobin are also in this group of alkali stable variants
  • Hemoglobin 4 (3 & 4) : 275-289 and Bonaventura and
  • Hb Chico where the beta-66 lysine is replaced by threonine (Shih et al., 1987, Hemoglobin 11: 453-464).
  • the P50 of Hb Chico's red blood cells is 38 mm Hg compared with normal red blood cell controls with P50 of 27 mm Hg. All other properties, i.e, Hill coefficient and alkaline Bohr effect are normal.
  • Hb Titusville (alpha-94 aspartate to asparagine) is one of a group of low affinity hemoglobin variants with altered alpha ⁇ beta 2 contacts (Schneider et al., 1975, Biochem. Biophys. Acta 400:365).
  • alpha ⁇ beta 2 interface is stabilized by two different sets of hydrogen bonds between the alpha and beta subunits.
  • One set stabilizes the T-structure which is the low-affinity form and the other stabilizes the R-state which is the high affinity form. It is the shifting back and forth between these two sets of bonds and alternating between the T- and R-states which is responsible for the positive
  • the deoxyhemoglobin is primarily in the T-state. For hemoglobin with one oxygen bound, the amount of R-state molecules increases and therefore binds oxygen with a higher affinity. In hemoglobin with two oxygens bound, there is an even higher proportion of R state molecules. In Hb Titusville, the R-state bonds are disrupted. The alpha-94 aspartate would normally form a non-covalent bond with beta-102 asparagine. Because this bond is disrupted, the equilibrium is pushed in the direction of the T-state and Hb Titusville's oxygen affinity is very low.
  • Hb Beth Israel is another variant affecting the alpha 1 beta 2 interface which destabilizes the high oxygen affinity R-state (Nagel et al., 1976, New Eng. J. Med. 295:125-130).
  • the beta-102 asparagine is replaced by serine.
  • the whole blood of an Hb Beth Israel patient has a P50 of 88 mm Hg as compared with the normal value of 27.
  • the Hill coefficient is biphasic with a value of 1.0 at the high end and 1.8 at the low end. The Bohr effect is normal.
  • a hemolysate of Hb Beth Israel has a P50 of 17 mm Hg and a Hill coefficient of 1.65 at the bottom and 1.29 at the top of the curve as compared to a P50 of 5.6 and a Hill coefficient of 2.72 for normal hemoglobin.
  • yeast With the advent of recombinant DNA technology, efforts have been made to express heterologous DNA in a variety of prokaryotic and eukaryotic systems.
  • One such system is yeast.
  • Yeast has a number of advantages over bacteria and other eukaryotes as a system for the production of polypeptides or proteins encoded by recombinant DNA. Yeast has been used in large scale fermentations for centuries, so the technology for fermenting yeast is well known and a number of yeast hosts are commercially available.
  • yeast can be grown to higher densities than bacteria and many other types of eukaryotic cells, and is readily adaptable to continuous fermentation processing. Since yeast is a eukaryotic organism, yeast may be capable of glycosylating expression products, may exhibit the same codon preferences as higher organisms, and may remove the amino terminal methionine during post-translational processing.
  • heterologous proteins have been expressed in yeast. Examples include interferon (Hitzeman and Leung, U.S. Patent No. 4,775,622, issued October 4, 1988; Hitzeman et al., Canadian Patent No. 1,205,026, issued May 27, 1986; Hitzeman et al., 1981, Nature (London) 293: 717); platelet derived growth factor (Murray et al., U.S. Patent No. 4,801,542, issued January 31, 1989);
  • Heterologous proteins expressed in yeast have been linked to a wide variety of promoters. Examples include operably linking heterologous proteins to SV40 and RSV promoters (Gelfand et al., U.S. Patent No. 4,8710,013, issued September 26, 1989). Additionally, DNA sequences encoding heterologous proteins have been linked to yeast promoters, which are inducible. European Patent
  • GAL1-10 promoter the yeast galactose-induced promoters for galactokinase (GAL1) and UDP-galactose epimerase (GAL10), hereinafter referred to as the GAL1-10 promoter, which is bidirectional.
  • bidirectional yeast promoter is the YPT1/TUB2 intergene sequence which contains overlapping binding sites for the transcription factor BAF1 (Halfter et al., 1989, EMBO J. 8:3029-3037). Broach et al.
  • ADH1 alcohol dehydrogenase transcription
  • TDH3 glyceraldehyde-3-phosphate dehydrogenase gene
  • substantially pure refers to a globin chain that is free of erythrocyte membrane components and E. coli
  • hemoglobins include but are not limited to hemoglobin variants having a lowered oxygen affinity (e.g. HbF Chico (gamma-66 lysine is replaced by threonine); Hb Portland Titusville (zeta-94 aspartate to asparagine); and Hb BovII (N-terminal beta-globin sequence of Met Leu Thr Ala Glu Glu)); a high oxygen affinity variants (e.g. HbA Deer Lodge (beta-2 histidine is replaced with
  • HbA Abruzzo beta-143 histidine is replaced with arginine
  • HbA McKees Rock beta-145 tyrosine is replaced with a termination sequence
  • alkali stable variants e.g. HbA Motown/Hacettepe and a variant in which serine replaces the alpha-104 or zeta-104 cysteine
  • variants which have a lowered oxygen affinity and are stable in alkali e.g. a variant which combines the
  • hemoglobin variant as defined herein is a hemoglobin comprising a least one variant globin chain.
  • variant globin chain refers to a globin whose nucleotide sequence has been altered in such a fashion so as to result in the alteration of the structure or function of the globin, but so that the globin still remains functionally active as defined by the ability to reversibly bind to oxygen.
  • the variant may be naturally occurring or non-naturally occurring.
  • the invention is further directed to yeast cells capable of producing the foregoing hemoglobins.
  • yeast cells contain recombinant DNA vectors which are capable of expressing certain globin chains.
  • hemoglobins may be used in any combination.
  • the invention is also directed to a
  • recombinant DNA vector capable of expressing a globin chain or heme-binding fragment thereof selected from the group including but not limited to a zeta globin, an epsilon globin, a variant globin chain substantially homologous to a human embryonic zeta-globin chain and comprising a serine at the zeta-104 position, a variant globin chain
  • yeast cell substantially homologous to a human fetal gamma-globin chain and comprising a glutamic acid at the gamma-127 position in a yeast cell comprising:
  • a yeast inducible transcriptional promoter which promotes the transcription of the DNA sequence encoding the globin chain or heme-binding fragment thereof;
  • the invention is further directed to making Hb Portland I (zeta 2 gamma 2 ) or II (zeta 2 beta 2 ) comprising the steps of:
  • the first recombinant DNA vector comprises: (i) a yeast inducible transcriptional promoter regulated by galactose; (ii) a DNA sequence located downstream from the hybrid promoter, encoding a human zeta-globin chain; (iii) a URA3 selectable marker or functionally active portion thereof; (iv) a 2 ⁇ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located
  • the second recombinant vector comprises: (i) an inducible promoter or hybrid promoter; (ii) a DNA sequence located downstream from the GAL10 promoter, encoding a human fetal gamma-globin chain or a human adult beta-globin chain; (iii) a LEU2 selectable marker or functionally active portion thereof; (iv) a 2 ⁇ plasmid replication system or functionally active portion thereof; and (v) a transcription termination sequence located downstream from the DNA sequence encoding the fetal gamma-globin or adult beta-globin chain, which comprises the transcription termination region of the alcohol dehydrogenase I gene; and
  • hemoglobin variants Hb Mississippi (beta-44 serine is replaced with cysteine), Hb Motown (beta-127 glutamine is replaced with glutamic acid), and Hb Titusville (alpha-94 aspartate is replaced with asparagine), HbF Porto Alegre (gamma-9 alanine is replaced with cysteine) HbA Porto Alegre (beta-9 alanine is replaced with cysteine) comprising the steps of:
  • the first recombinant DNA vector comprises: (i) a DNA sequence encoding an alpha like- globin chain or variant thereof; (ii) a yeast
  • the transcriptional promoter which promotes the transcription of the DNA sequence encoding the alpha like-globin chain or variant thereof; and (iii) a DNA sequence encoding at least one yeast selectable marker or functionally active portion thereof; and (iv) a yeast replication origin and in which the second recombinant DNA vector comprises (i) a DNA sequence encoding a beta like-globin chain or variant thereof; (ii) a yeast transcriptional promoter which
  • the certain hemoglobins produced by the above methods may be used in applications requiring physiological oxygen carriers such as in blood substitute solutions, or as in a plasma expander. 4. BRIEF DESCRIPTION OF THE FTGURES
  • Figure 1 shows the nucleotide sequences of the embryonic zeta (1A), embryonic epsilon (IB), fetal gamma (1C), adult delta (1D), adult alpha (IE) and adult beta (1F) chains of human hemoglobin.
  • the deduced amino acid sequences are shown underneath.
  • the AUG start codon and the corresponding amino-terminal methionine which is removed by methionine aminopeptidase in a post-translational modification are not shown in the figures.
  • Figure 2 shows a partial restriction map of the plasmid pSP ⁇ C.
  • Figure 3A shows the strategy used to clone the adult beta-globin gene into YEp51.
  • Figure 3B shows the strategy used to clone the Porto Alegre beta-globin gene.
  • Figure 4 shows the restriction map of the plasmid YEpWB51/NAT.
  • Figure 5 shows the strategy for cloning ADH1-terminator into YEpWB51/NAT.
  • Figure 6 shows a map of AAH5.
  • Figure 7 shows the restriction map of the plasmid YEp51T/NAT.
  • Figure 8 shows an autoradiograph of total RNA extracted from yeast strain Sc340 transformed with YEp51 (340g2C) and YEp51T/NAT (340g2B). Total RNA was subjected to electrophoresis on a 1.1% agarose gel, transferred to the Hybond paper and probed with an ApaLI-HindIII fragment
  • RNA (600 bp) of the beta-globin gene from plasmid mp18 ⁇ HS.
  • the level of a control RNA (CYH2) was determined with the plasmid mpl9CYH22 (9.0 kb) which carries the coding region of the CYH2 gene. 20 ⁇ g of the total RNA was loaded into each lane. Sample in each lane is as follows : Lane 1 :
  • 340g2C Lane 2: 340g2B, and Lane 3: 340g2P.
  • ⁇ marks the beta-globin mRNA.
  • the CYH2 mRNA is marked with Cl
  • Figure 9 shows the results of scanning an autoradiograph containing both beta-globin and CYH2 mRNA obtained from a Northern Blot using an LKB gel scanner.
  • the large peak in A (340g2B) represents the beta-globin mRNA and two small peaks at either side of the large peak represent the CYH2 mRNA.
  • Figure 6B shows the results of scanning an autoradiograph containing both Porto Alegre beta-globin mRNA and CYH2 mRNA obtained from a Northern blot using an LKB scanner.
  • the large peak in B (340g2P) represents the Porto Alegre beta-globin mRNA and the two small peaks at either side of the large peak represent the CYH2 mRNA.
  • Figure 10 shows the sequences of and restriction sites present on 51-A-1 (5'-end primer) and 519-A-3 (3'-end primer). These primers were used to synthesize alpha-globin DNA.
  • Figure 11 shows the restriction map of pUT/2A.
  • Figure 12 shows the construction of YEp51T/G.
  • Figure 13 shows the DNA sequence of the gamma globin gene.
  • Figure 14 shows the sequences of and restriction sites present on GAM-5-S (5'-end primer) GAM-3-H (3'-end primer). These primers were used to synthesize gamma-globin DNA.
  • Figure 15 shows the restriction map of plasmid YEp51T/G.
  • Figure 16 shows the strategy used for isolating epsilon cDNA from genomic epsilon DNA.
  • Figure 17 shows the sequences of the primers used to construct the epsilon cDNA:5EPSL-1, INPE-1, INPE-2,
  • Figure 18 shows the restriction map of plasmid
  • Figure 19 show the sequence of and restriction sites present on 5ZETASAC (5'-end primer) and ZETA3HSLS (3'-end primer). These primers were used to synthesize zetaglobin cDNA.
  • Figure 20 shows the restriction map of plasmid
  • Figure 21 shows the sequences of Mu-145Cy, Mu-66Th, and Mu-9Cy.
  • Figure 22 shows a restriction map of YEp51NTl.
  • Figure 23 shows the sequences of and the restriction sites on 5'-end primer, G-5-9CY and the 3'-end primer, GAM-3-H. Site specific mutations are shaded.
  • Figure 24 shows the sequences of and the restriction sites on 5'-end primer, B-G127-5 and the 3'-end primer, Beta-3-H.
  • Figure 25 shows the sequences of and the restriction sites on the 5'-end primer, A-Tit-5 and the 3'-end primer, G10T3H.
  • Figure 26 shows the sequences of and the restriction sites on the 5'-end primer, 51-A3-SL and the 3'-end primer, A-Hin3-3.
  • Figure 27 shows the sequences of and the restriction sites on the 5', B-44C-5, and 3' primers, Beta- 3-H used to synthesize by PCR the Mississippi ⁇ -globin gene. Site specific mutations are shaded.
  • Figure 28 shows the sequences of and the restriction sites on 5'-end primer, A104Ser and the 3'-end primer, G10T3H.
  • Figure 29 shows the sequences of and the restriction sites on 5'-end primer, Z-5-SAL and the 3'-end primer, Z-104S-B.
  • Figure 30 shows the sequences of and the restriction sites on 5'-end primer, Z-BST-5 and the 3'-end primer, Z2-3-H.
  • Figure 31 shows the sequences of and the restriction sites on 5'-end primer, Z-5-SAL and the 3'-end primer, Z-A95-3.
  • Figure 32 shows the sequences of and the restriction sites on 5'-end primer, G2-Mot-5 and the 3'-end primer, GAM-3-H.
  • Figure 33 shows the sequences of and the restriction sites on 5'-end primer, B-Bov2-5 and the 3'-end primer, Beta-3-H.
  • Figure 34 shows the sequences of and the restriction sites on 5'-end primer, B-2ARG-5 and the 3'-end primer, Beta-3-H.
  • Figure 35 shows the sequences of and the restriction sites on 5'-end primer, BN-5-SAL and the 3'-end primer, B-143A-3.
  • Figure 36 shows the sequences of and the restriction sites on 5'-end primer, BN-5-SAL and the 3'-end primer, B-145T-3.
  • Figure 37 shows the sequences of and the restriction sites on 5'-end primer, GAM-5-S and the 3'-end primer, G66T-3.
  • Figure 38 shows a map of mpl ⁇ HS.
  • Figure 39 shows a map of L19 ⁇ At.
  • Figure 40 shows the sequence of TDH3-5'
  • Figure 41 shows the restriction map of plasmid pUC19-H ⁇ At.
  • Figure 42 shows part of the GAL1-10 promoter sequence .
  • Figure 43 shows the sequences of the primers, GAL1-10-5' and GAL1-10-3'.
  • Figure 44 shows the restriction map of plasmid pUC19-GH ⁇ At.
  • Figure 45 shows the restriction map of plasmid pNML-V-G-1.
  • Figure 46 shows the restriction map of plasmid YEpWB51/PORT.
  • the invention is directed to certain substantially pure hemoglobins comprising an alpha-like globin chain or variant thereof and a beta-like globin chain or variant thereof.
  • the alpha-like globin chain may be selected from the group including but not limited to an embryonic zeta-globin chain and an adult alpha-globin chain.
  • the beta-like globin chain may be selected from the group including but not limited to an embryonic epsilon-globin chain, a fetal gamma-globin chain, an adult delta-globin chain, and an adult beta-globin chain.
  • Alpha-like globin and beta-like globin may be mixed with a source of heme to obtain hemoglobin comprising alpha-like globin and beta-like globin.
  • Hemoglobin produced by methods of the present invention may be used in applications requiring physiological oxygen carriers such as in blood substitute solutions, or in a plasma expander.
  • the invention is also directed to recombinant vectors capable of expressing certain globin chains or heme binding fragments thereof in yeast.
  • the invention also relates to methods for expressing the foregoing hemoglobins in yeast where the heme which is produced by the yeast or obtained from an exogenous source is ligated to the globin to form functional hemoglobins in vivo.
  • nucleotide sequence of the genes encoding the human embryonic zeta-globin, the human embryonic epsilon-globin, the human fetal gamma-globin, the human adult delta-globin, the human adult alpha-globin and the human adult beta-globin chains and their derived amino acid sequences are depicted in Figures 1A-F respectively, and are described in the Sequence Description as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO: 6.
  • nucleotide sequences comprising all or portions of the nucleotide sequence depicted in Figures 1A-F which are altered by the substitution of different codons that encode the same or a functionally equivalent amino acid residue thus producing a silent change as well as amino acid sequences comprising all or portions of the amino sequence depicted in Figures 1A, 1B, 1C, 1D, 1E, or 1F which are altered by the
  • genes encoding alpha-like globin and beta-like globin chains may be isolated from hemoglobin
  • the DNA encoding alpha-like globin and/or beta-like globin may be obtained by standard procedures known in the art from cloned DNA (e.g. a DNA "library”), by chemical synthesis, by cDNA cloning or by the cloning of genomic DNA, or fragments thereof, purified from for example human
  • DNA encoding alpha-like or beta-like globin DNA may also be obtained using polymerase chain reaction (PCR) technology (see for example Mullis et al., 1989, U.S. Patent No. 4,800,159).
  • PCR polymerase chain reaction
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions, in addition to coding regions; clones derived from cDNA will contain only exon sequences.
  • a globin gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • DNA fragments are generated, some of which will encode the desired globin gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes.
  • DNAse in the presence of
  • DNA can be physically sheared, as for example, by sonication.
  • the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column
  • identification of the specific DNA fragment containing the globin may be accomplished in a number of ways. For example if an amount of a globin gene or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labelled probe (Benton and Davis, 1977, Science 196:180 and Grunstein and Hogness,1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. If a purified globin-specific probe is
  • nucleic acid fractions enriched in globin sequences may be used as a probe, as an initial selection procedure. It is also possible to identify the appropriate fragment by restriction enzyme digestion (s) and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection on the basis of the properties of the gene, or the physical or chemical properties of its expressed product, as described infra, can be employed after the initial selection.
  • the globin gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization, In vitro translation products of the isolated mRNAs identifies the mRNA, and therefore the complementary DNA fragments that contain the globin sequences.
  • the identified and isolated gene or cDNA can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, cosmids, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322, pUC, pGEM1 ® , or Bluescript ® plasmid derivatives.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc.
  • the gene may be identified and isolated after insertion into a suitable cloning vector, in a "shot gun" approach. Enrichment for a globin gene, for example, by size fractionation or
  • subfractionation of cDNA can be done before insertion into the cloning vector.
  • the globin gene is inserted into a cloning vector which can be used to transform, or infect
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease
  • the cleaved vector and globin gene may be modified by
  • transformation of host cells with recombinant DNA molecules that incorporate an isolated globin gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene.
  • the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
  • globin-containing clone After the globin-containing clone has been identified, grown, and harvested, its DNA insert may be characterized using procedures known in the art.
  • the cloned DNA or cDNA corresponding to the globin gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517), restriction endonuclease mapping (Maniatis et al., 1989,
  • DNA sequence analysis can be performed by any techniques known in the art, including but not limited to chemical methods (Maxam and Gilbert, 1980, Meth. Enzymol.
  • the invention is directed to the following categories of hemoglobin variants: variants which
  • variants in which the tetramer does not dissociate under physiological conditions in vivo variants with lowered intrinsic oxygen affinity, i.e. an oxygen affinity having a P 50 of at least about 10 mm Hg under physiological conditions; variants that are stable in alkali, variants with a higher intrinsic oxygen affinity, i.e. an oxygen affinity having a P 50 of at most about 1 mm
  • poly alpha-like globin or poly beta-like globin may result.
  • Hb Mississippi beta-44 serine is replaced with cysteine
  • Another example includes Hb Porto Alegre.
  • the beta-9 or gamma-9 serine is replaced by cysteine which is able to form disulfide bonds with other cysteine residues.
  • Alkali stable hemoglobin variants are those in which the dimers do not dissociate into monomers in the presence of alkali.
  • An example of a naturally occurring alkali stable mutant is HbA Motown/Hacettepe where beta-127 glutamine is replaced with glutamic acid or alternatively HbF Motown, where gamma-127 glutamine is replaced with glutamic acid. It has been shown that the alpha-104 cysteine cause the hemoglobin to be susceptible to alkali denaturation (Perutz, 1974, Nature 247:371).
  • Examples of non-naturally occurring alkali stable variant include a variant in which serine replaces the alpha-104 or zeta-104 cysteine.
  • HbF Chico gamma-66 lysine is replaced by threonine
  • HbA Titusville alpha-94 aspartate to asparagine
  • bovine hemoglobin has a lower oxygen affinity than human HbA
  • one hemoglobin variant may comprise a (a) a variant globin chain or heme-binding fragment thereof which (i) is substantially homologous to a mammalian adult beta-globin chain, and (ii) comprises an N-terminal beta-globin sequence of Met Leu Thr Ala Glu Glu; (b) a mammalian alpha-like globin chain or heme binding fragment thereof; and (c) heme, and which variant has the ability to bind to oxygen at a low oxygen affinity and is free of erythrocyte membrane components and E. coli
  • substantially homologous refers to the ability of a DNA sequence encoding a first globin chain to hybridize to a DNA sequence encoding a second globin chain under stringent conditions, for example, at about 0.1X SSC at a temperature of about 65°C. For example, if a globin variant is substantially
  • a DNA sequence encoding the globin variant is capable of hybridizing to a DNA sequence encoding the adult beta-globin chain under stringent conditions.
  • the hemoglobin variant may be a a variant having an increased oxygen affinity, a high oxygen affinity variant.
  • examples include but are not limited to HbA Deer Lodge (beta-2 histidine is replaced with arginine) (Labossiere et al., 1972, Clin. Biochem. 5:46-50); HbA Abruzzo (beta-143 histidine is replaced with arginine) (Tentori et al., 1972, Clin. Chim. Acta 38:258-262); and HbA McKees Rock (beta-145 tyrosine is replaced with a termination sequence) (Winslow et al., 1976, J. Clin. Invest. 57:772-781).
  • a variant may be constructed which combines the mutations of HbA Titusville and replacement of the alpha-104 cysteine residue with serine. This may result in the formation of a tetramer with the desirable properties of lowered oxygen affinity and stability in alkali.
  • the globin variants may be produced by various methods known in the art .
  • the manipulations which result in their production can occur at the gene or protein level.
  • the globin may.be altered at the gene level by sitespecific mutagenesis using procedures known in the art .
  • One approach which may be taken involves the use of
  • oligonucleotides to construct variant globins with base substitutions.
  • a short oligonucleotide containing the mutation is synthesized and annealed to the single stranded form of the wild-type globin gene (Zoller and Smith, 1984, DNA 3:479-488).
  • the resulting short heteroduplex can serve as primer for second strand synthesis by DNA polymerase.
  • a single stranded nick is formed which is closed by DNA ligase.
  • two complementary nucleotide to construct variant globins with base substitutions.
  • oligonucleotides are synthesized, each containing the mutant sequence.
  • the duplex that forms after annealing these complementary oligonucleotides can be joined to a larger DNA molecule by DNA ligase provided that the ends of both molecules have complementary single-stranded "sticky" ends.
  • a globin variant may be prepared by
  • fragments of the variant globin are chemically synthesized and these fragments are
  • the resulting variant globin strands may be amplified using procedures known in the art, e.g. PCR technology and subsequently inserted into a cloning vector as described in Section 5.1., supra.
  • site specific mutants may be created by introducing mismatches into the oligonucleotides used to prime the PCR amplification (Jones and Howard, 1990,
  • Manipulations of the globin sequence may be carried out at the protein level. Any of numerous chemical modifications may be carried out by known techniques including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
  • variant globin protein may be chemically synthesized using procedures known in the art, such as commercially available peptide synthesizers and the like. Such standard techniques of polypeptide synthesis can be found described in such publications as Merrifield, 1963, J. Chem. Soc. 85:2149-2154 and
  • the nucleotide sequence coding for a globin chain is inserted into an appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the inserted protein- coding sequence.
  • an appropriate expression vector i.e. a vector which contains the necessary elements for the transcription and translation of the inserted protein- coding sequence.
  • host-vector systems may be utilized to express the DNA sequence encoding the globin chain. These include but are not limited to mammalian cell systems infected with virus (e.g. vaccinia virus,
  • adenovirus etc.
  • insect cell systems infected with virus e.g. baculovirus
  • yeast containing yeast vectors, and bacteria transformed with plasmid DNA, cosmid DNA, or bacteriophage DNA e.g. baculovirus
  • the host cell is a yeast cell.
  • yeast replication origin is required in a recombinant DNA vector capable of
  • nucleotide sequence coding for the alphalike and/or beta-like chain of globin is inserted into a vector which may be expressed in yeast.
  • one DNA sequence encoding one globin chain or variant thereof is inserted into the recombinant DNA vector.
  • the yeast cell is a member of the species Saccharomyces cerevisiae.
  • a vector comprises in addition to the DNA sequence encoding the globin: (a) a yeast transcriptional promoter which promotes the transcription of the DNA sequence encoding the globin chain; (b) a DNA sequence encoding a yeast
  • the first component of the vector, a yeast transcriptional promoter comprises two components: (a) a transcriptional regulatory region which contains a
  • TATA sequence, capping sequence as appropriate, and an RNA polymerase binding sequence, which includes nucleotides upstream from the initiation site for directing the
  • the activator sequence is an upstream activator sequence.
  • the transcriptional regulatory region will preferably be at least 100 base pairs (bp) and will not exceed 3000 base pairs.
  • the regulatory region may begin at least about 200 bp from the initiation codon, usually at least about 300 bp and may begin at 400 bp or farther upstream from the initiation codon.
  • the transcriptional initiation region will be at least about 150 bp, more usually at least about 200 bp, usually not more than about 600 bp, and preferably about 400 bp.
  • the sequence may extend in the downstream direction of transcription from about bp -10 to about bp -25 (relative to transcription initiation at +1).
  • the yeast transcriptional promoter is an inducible promoter.
  • Inducible promoters may be unidirectional or bidirectional. Unidirectional
  • inducible promoters in a preferred embodiment are located upstream from the DNA sequence encoding the globin chain.
  • Unidirectional inducible promoters may include but are not limited to promoters which are regulated by galactose (e.g. UDP-galactose epimerase (GAL10), galactokinase (GAL1)), glucose (e.g. alcohol dehydrogenase II (ADH2 ), and phosphate (e.g. acid phosphatase (PHO5)).
  • the inducible promoter may be a bidirectional promoter.
  • a bidirectional promoter may be located upstream (5' of the ATG start codon) from the DNA sequence encoding a globin chain on the plus strand at one of its ends and upstream from the DNA sequence encoding a globin chain on the minus strand at its other end; and thereby, control the transcription of both.
  • a bidirectional promoter is GAL1-10.
  • the promoter may also be a constitutive promoter.
  • the constitutive promoter is a promoter of glyceraldehyde-3-phosphate dehydrogenase III (TDH3) transcription and is herein after referred to as the TDH3 promoter.
  • TDH3 glyceraldehyde-3-phosphate dehydrogenase III
  • promoters include but are not limited to glyceraldehyde-3- phosphate dehydrogenase II (TDH2), glyceraldehyde-3- phosphate dehydrogenase I (TDH1) , alcohol dehydrogenase I (ADH1), phosphoglycerate kinase (PGK), pyruvate kinase (PYK) , enolase (ENO), and triose phosphate isomerase (TPI).
  • THI glyceraldehyde-3- phosphate dehydrogenase II
  • TDH1 alcohol dehydrogenase I
  • PGK phosphoglycerate kinase
  • PYK pyruvate kinase
  • ENO enolase
  • TPI triose phosphate isomerase
  • the promoter can be a hybrid promoter, in which the sequence containing the transcriptional regulatory region is obtained from one source and the sequence containing the transcription initiation region is obtained from a second source.
  • the sequence containing the transcriptional regulatory region is an upstream activating sequence of a yeast inducible promoter.
  • the inducible promoter can be a unidirectional or a bidirectional promoter.
  • the sequence containing the transcriptional initiation region may be obtained from the transcriptional initiation region of a constitutive promoter.
  • the hybrid promoter comprises a transcriptional regulatory region which is the upstream activation sequence of the
  • the hybrid promoter can regulate the expression of two separate DNA sequences in opposite orientations if the hybrid promoter comprises an upstream activating sequence with
  • a GAL1-10 upstream activating sequence may be flanked on either side by the initiation region of the TDH3 promoter. DNA encoding a globin chain is located downstream from each TDH3 sequence.
  • the ADH2 UAS may be used in place of the GAL1-10 UAS .
  • the transcriptional initiation region of the TDH3 promoter can be substituted by TDH1 . TDH2 , PGK, ENO, TPI, CYC1, or PYK.
  • Another component of the recombinant DNA vector is a sequence encoding a yeast selectable marker.
  • the recombinant DNA vector in one embodiment may contain more than one such sequence.
  • a yeast selectable marker provides for selective pressure for survival of yeast cells expressing the marker.
  • the marker in a preferred aspect, the
  • selectable marker complements a genetic defect in the host strain.
  • URA3 can be used as a selectable marker in a yeast strain which is deficient in the URA3 gene product.
  • sequences may include but are not limited to the LEU2 gene, the URA3 gene, the HIS3 gene, the LYS2 gene, the HIS4 gene, the APE8 gene, the CUP1 gene, and the TRP1 gene .
  • Another example of such a sequence includes the leu2d gene which is a promoter defective LEU2 gene.
  • the leu2d gene is inserted into a multicopy recombinant DNA vector.
  • a yeast cell transformed by a vector comprising the LEU2 or leu2d gene may grow in leucine free media; a yeast cell transformed by a vector comprising the URA3 gene may grow in uracil free media; a yeast cell transformed by a vector comprising the LYS3 gene may grow in lysine free media, a yeast cell transformed by a vector comprising the HIS3 gene may grow in histidine free media, a yeast cell transformed by a vector comprising the ADE8 gene may grow in adenine free media, a yeast cell transformed by a vector comprising the HIS4 gene may grow in histidine free media, a yeast cell transformed by a vector comprising the CUP1 gene may grow in media
  • the recombinant DNA vector may also comprise a DNA sequence encoding a functionally active portion of a yeast
  • selectable marker is a portion of the sequence that encodes a portion of the marker which provides an effective amount of selective pressure for the survival of yeast cells expressing the portion of the marker.
  • the recombinant vector also comprises a yeast replication origin or functionally active portion of the replication origin which effects replication of the vector.
  • Any replication origin useful in yeast may be employed which provides for efficient replication and maintenance (reviewed for example in Kingsman and Kingsman, U.S. Patent No. 4,615,974, issued October 7, 1986).
  • Examples of such replication origins include but are not limited to the 2 ⁇ plasmid replication system, or a functionally active portion thereof and autonomous replicating sequences (ARS).
  • ARS include but are not limited to ARS1 or ARS3.
  • the replication origins may be of high or low copy number, depending on the effect of the construct on the viability of the host.
  • the vector may further comprise centromeric sequences (CEN) which may provide meiotic and mitotic stability. Examples of CEN sequences include but are not limited to CEN3, CEN4, and CEN11.
  • the expression vector may further comprise but does not always require a transcription termination
  • a transcription termination sequence may include the necessary transcription signals for termination and polyadenylation and may be derived from any yeast sequence.
  • the transcription termination sequence is the alcohol dehydrogenase I (ADH1) termination sequence.
  • Other termination sequences suitable for use include but are not limited to those of iso-1-cytochrome c (CYC1), UDP-glucose-4-epimerase (GAL10), phosphoglycerate kinase (PGK). acid phosphatase (PHO5), enolase (ENO), and triose phosphate isomerase (TPI).
  • the transcription termination sequence is at least about 100 bp and should not exceed about 1500 bp. In a preferred embodiment, the transcription termination sequence ranges from about 150 bp to about 1200 bp.
  • inventions may be constructed using recombinant DNA
  • yeast expression vectors and their
  • Section 11 A specific example of a yeast expression vector and its construction, comprising sequences encoding the zeta-globin chain under the control of a the GAL10 promoter is disclosed in Section 10. Specific examples of the expression of hemoglobin by coexpression of plasmids comprising sequences encoding alpha like- and beta like- globin are disclosed in Sections 12-18, 20-24, and 26-28.
  • the expression vectors may be propagated in yeast which may or may not be capable of producing heme.
  • the yeast can be transformed with one or more of the expression vectors using procedures known in the art (e.g. the spheroplast method, (Hinnen et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1929-1933) or the lithium acetate method (Ito et al., 1983, J. Bact. 153:163-168) or through electroporation.
  • Transformants may be selected by the presence of the marker (selectable) gene function in the transformant.
  • a leu2-yeast cell transformed with an expression vector comprising a LEU2 marker gene is selected by virtue of its ability to grow in leucine free media.
  • the transformed yeast cells may be grown in media comprising a nitrogen and carbon source as well as
  • the media should also comprise the inducer.
  • the expression vector comprises DNA
  • hemoglobin may be expressed in the yeast cell transformed with the vector.
  • the heme is produced by the yeast and ligated to the globin to form functional hemoglobins in vivo.
  • the yeast cell may be deficient in components required for heme production, for example 5-aminolevulinic acid. Hemoglobin may still be expressed in such a cell if the required component is added.
  • the protein product of the expressed globin gene may be isolated and purified using standard methods including but not limited to chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. If one globin chain is expressed, the expressed globin chain may be combined with another globin chain and a source of heme to form hemoglobin. If hemoglobin is expressed in the yeast cell, no further steps are necessary.
  • the expressed gene and its product may be analyzed at the genomic level or the protein level using procedures known in the art. For example, hemoglobin gene expression may be analyzed by Southern or Northern
  • the expressed hemoglobin protein may for example be analyzed by Western Blot procedures known in the art and also described herein in Section 6.6., infra.
  • Hemoglobin of large quantity and high purity may be obtained using the methods of the present invention.
  • examples of hemoglobin which may be obtained include but are not limited to HbA (alpha 2 beta 2 ), HbA 2 (alpha 2 delta 2 ),
  • HbF alpha 2 gamma 2
  • HbBarts gamma 4
  • HbH beta 4
  • HbF alpha 2 gamma 2
  • HbBarts gamma 4
  • HbH beta 4
  • Hb Gower II (alpha 2 epsilon 2 ).
  • the hemoglobin will be free of cellular material and other contaminants.
  • alpha-like and/or beta- like globin may be chemically modified using procedures known in the art to increase tetramer stability and/or lower oxygen affinity (see Section 2.1.2., supra for examples of such procedures).
  • a wild-type or variant alpha- like or beta-like globin may be modified.
  • Such chemically modified hemoglobins may also be used in blood substitutes.
  • hemoglobin compositions in addition to being used in blood substitutes, may be used in a blood plasma expander, in a pharmaceutical composition with an acceptable carrier, and with other plasma expanders, or in any application where a physiological oxygen carrier is needed.
  • the pharmaceutical carriers may be such
  • physiologically compatible buffers as Hank's or Ringer's solution, physiological saline, a mixture consisting of saline and glucose, and heparinized sodium-citrate-citric acid-dextrose solution.
  • physiologically compatible buffers as Hank's or Ringer's solution, physiological saline, a mixture consisting of saline and glucose, and heparinized sodium-citrate-citric acid-dextrose solution.
  • the hemoglobin produced by the methods of the present invention can be mixed with
  • colloidal-like plasma substitutes and plasma expanders such as linear polysaccharides (e.g. dextran), hydroxyethyl starch, balanced fluid gelatin, and other plasma proteins.
  • the hemoglobin may be mixed with water soluble, physiologically acceptable, polymeric plasma substitutes, examples of which include polyvinyl alcohol, poly(ethylene oxide), polyvinylpyrrolidone, and ethylene oxide-polypropylene glycol condensates. Techniques and formulations for administering the compositions comprising the hemoglobin generally may be found in Remington's
  • the beta-globin gene from plasmid pSP ⁇ C was modified and cloned into the yeast expression vector, YEp51. ADH1-transcription termination sequences were placed at the end of the beta-globin gene in this plasmid.
  • the modified plasmid was called YEp51T/NAT (for the natural beta-globin gene).
  • Yeast strain Sc340 was transformed with plasmids YEp51T/NAT and YEp51 (control).
  • Total RNA was isolated from yeast strain Sc340 transformed with YEp51 (340g2C), YEp51T/NAT (340g2B). Quantitation of RNA by scanning the autoradiograph showed that mRNA for the natural beta-globin is around 3.0% of total yeast RNA.
  • restriction enzymes Klenow enzyme and T4-DNA ligase were obtained from New England Biolabs
  • HindIII Digestion with this combination of enzymes generated two fragments, a 600 base pair DNA containing the beta-globin gene and a 2700 bp fragment from the plasmid.
  • the 600 bp fragment was isolated from a 0.6% agarose gel. After the band was excised from the gel, the DNA was electroeluted, and ethanol precipitated. The precipitated DNA was spun in an Eppendorf Centrifuge, the supernatant was removed and the DNA pellet was dried under vacuum.
  • the 600 bp fragment was modified by adapter addition before cloning into the plasmid YEp51.
  • the DNA fragment carrying the beta-globin gene isolated from pSPBC was Ncol compatible at the 5'-end while the 3'-end was
  • HindIII compatible These ends had to be modified so that they could be compatible with the restriction sites present in YEp51.
  • a synthetic adapter was used to modify the 5'-end of the isolated fragment. This adapter had a NcoI compatible end at its 3'-end and a SalI compatible end at its 5'-end (see Figure 2). The 3' -end of the isolated fragment did not receive any adapter as the HindIII site was compatible with the HindIII site introduced into the YEp51.
  • the recipient plasmid YEp51 was cleaved with SalI and HindIII restriction enzymes. To insert the isolated fragment containing the beta-globin gene, a three-way ligation was set up (see Figure 3) .
  • the ligation reaction was carried out according to the standard ligation procedures (Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The ligation mixture was
  • the strategy used to insert ADH1 terminator sequences into YEpWB51/NAT is shown in Figure 5.
  • ADH1-transcription termination sequences were isolated from plasmid AAH5 (Ammerer, G., 1983, Methods in Enzymology, 101, pp. 192-201).
  • AAH5 was obtained from Dr. Ben Hall at the University of Washington, Seattle.
  • the plasmid AAH5 was digested with BamHI and HindIII (see
  • FIG. 6 for a map of plasmid AAH5) . Digestion with this combination of enzymes generated three fragments. A 450 base pair (bp) DNA fragment containing the ADH1-transcription termination sequence was isolated from the 0.6% agarose gel. DNA was electroeluted from the gel slice and precipitated with ethanol at -20°C. The precipitated DNA was spun in an Eppendorf Centrifuge for 15 min and the pellet was dried under vacuum. The DNA was suspended in 20 ⁇ l H 2 O. The DNA fragment carrying the ADH1- transcription terminator isolated from AAH5 was BamHI compatible at the 3'-end while the 5'-end was HindIII compatible. These ends were compatible with the
  • the recipient plasmid YEpWB51/NATd _ was cleaved with BclI and HindIII restriction enzymes. As shown in Figure 5, a two-way ligation was set up to insert the isolated fragment.
  • the ligation mixture was transformed into E. coli HB101 cells using standard transformation procedures. Cells were spread on plates containing LB- media with 100 mg/L ampicillin. Plates were incubated overnight at 37°C. Twenty four colonies from the
  • ampicillin plates were picked and a 5 ml culture was inoculated with individual transformants. Cultures were grown overnight at 37°C with vigorous shaking.
  • the plasmid DNA was isolated from 1.5 ml of the overnight culture using standard alkaline miniprep procedures (Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • the plasmid from each transformant was digested with PstI and HindIII restriction enzyme to confirm the presence of a DNA fragment containing the ADHl-terminator.
  • the plasmid carrying the natural beta-globin gene with the ADH1-terminator was called YEp51T/NAT and is shown in Figure 7.
  • the yeast strain Sc340 was obtained from Dr.
  • MATa ura3-52 , leu2, ade1, his3 : GAL10uas-GAL4- URA3+ , MEL+ .
  • the transformants were selected by plating out on minimal media containing 0.67% Bacto yeast nitrogen base without amino acids, 2% glucose, 20 mg/L adenine sulfate, 20 mg/L histidine, and 20 mg/L uracil.
  • the plates were incubated at 28oC for three days and were examined for colony formation.
  • yeast minimal media 0.67% yeast nitrogen base without amino acids
  • glucose 20 mg/L each of adenine, uracil, and histidine.
  • the overnight culture was then used to inoculate 1000 ml of the yeast minimal media containing 2% lactic acid, 3% glycerol and appropriate amino acids.
  • the cultures were inoculated to OD 600 of 0.02. Cultures were grown at 30°C until they reached OD 600 of 0.20 (usually after 48 hours). Induction was initiated by the addition of galactose to a final concentration of 2% in the media.
  • Yeast cells were washed with 150 mM NaCl and the pellet was resuspended in RNA buffer (0.5 M NaCl, 0.2 M Tris-HCl, pH 7.6, 0.1 M EDTA and 1% SDS). Approximately 0.5 g of glass beads (0.45-0.5 mm) were added to the tubes. An equal volume of phenol mixture (phenol: chloroform: isoamyl alcohol 25:24:1, equilibrated with RNA buffer without SDS) was added. Yeast cells were broken by vortexing at maximum speed for 2.5 minutes and the sample was placed on ice for 3 minutes.
  • RNA buffer 0.5 M NaCl, 0.2 M Tris-HCl, pH 7.6, 0.1 M EDTA and 1% SDS.
  • RNA buffer and phenol mixture were added to the cells and tubes were centrifuged. Aqueous phase was transferred to a clean Corex tube and 2.5 volumes of ethanol were added to each tube. RNA was allowed to precipitate at -20°C for 4 to 6 hours. RNA was pelleted by centrifugation and dried under vacuum. RNA pellet was suspended in sterile water.
  • beta-globin Four major steps were involved in the analysis of the expressed beta-globin:
  • the primary antibody is specific for hemoglobin.
  • the secondary antibody is a conjugate of biotin and antibody against IgG of the animal in which the first antibody was raised. Additionally, strepavidin conjugated to horseradish peroxidase was utilized.
  • nitrocellulose membranes wee immersed in Enhanced Chemiluminescent developer ([ECL], Amersham) according to manufacturer's instructions and generated light detected by exposure to X-ray film for 15-60 seconds.
  • Cold disruption buffer 50 mM Tris, 5 mM EDTA, 0.5 mM PMSF, pH 8.0
  • 0.2 ml Cold disruption buffer
  • Ice-cold disruption buffer (1 ml) was added to each sample and the homogenate was transferred to an Eppendorf tube.
  • 200 ⁇ l of homogenate was combined with 200 ⁇ l of freshly prepared standard discontinuous 2X sample buffer (Laemli, 1970, Nature 227:680-685) and the sample was boiled for 10 min.
  • the samples were loaded onto a discontinuous denaturing gel in which the stacking gel was 3.75% acrylamide and the separating gel was 12%-15%.
  • the stacking gel was run at a constant current of 25 mA/cm 2 and the separating gel was run at a current of 33 mA/cm 2 .
  • the transfer unit was filled with transfer buffer (2L methanol, 30.3 g Tris base, 144 g glycine, pH 8.30, in 10L distilled water), 2L of the transfer buffer was put into a shallow pan.
  • Protein was then transferred from the gel to the nitrocellulose paper by applying a voltage of 40V for 1.5 hrs. After transfer was complete, the nitrocellulose sheet was removed and placed into a small, covered shallow pan with 50 ml blocking solution (200 g dried milk per liter PBS) and gently agitated for 1 hr. The blocking solution was discarded and the nitrocellulose was washed three times with PBS containing 0.1% Tween 20. The duration of the washes were 15, 5, and 5 minutes
  • the standard was apo-human beta-globin purified from red blood cell lysate on reverse phase HPLC .
  • the detection limit was less than 1 ng.
  • Total soluble protein was determined with the Bio-Rad Protein Assay Kit according to the manufacturer ' s instructions . Hemoglobin isolated from red blood cell lysate was used as a standard. Insoluble proteins were removed by centrifugation prior to analysis.
  • the alpha-globin gene was isolated using Polymerase Chain Reaction (PCR). The DNA Sequence of this alpha-globin gene was confirmed by sequencing. Results showed that the alpha-globin gene was complete without any deletions or mutations. The resulting plasmid was called pUT/2A.
  • the E. coli strain used for all bacterial transformations was DH5 ⁇ .
  • the genotype of this strain is as follows:
  • the alpha-globin gene was isolated by PCR from plasmid pJW101 (Wilson et al., 1978, Nucleic Acid Research 5: 563-580).
  • the primers used for the PCR, 51-A-1 and 519-A-3, are shown in Figure 10 and are described in the
  • the PCR product was purified by
  • Digested DNA was cleaned by phenol extraction and ethanol precipitation.
  • the yeast expression vector used to clone the alpha-globin gene was prepared by digesting plasmid
  • YEp51UT/NAT with SalI and BamHI YEp51UT/NAT was prepared in the following manner. Specifically, YEpWB51T/NAT was digested with the restriction enzyme KpnI. The linearized plasmid was treated with T4-DNA polymerase to make it a blunt-ended molecule. The URA3 gene was isolated as a 1300 bp SmaI-ClaI fragment from plasmid YEp24. This fragment was also treated with the T4-DNA polymerase to make it blunt-ended. This 1300 bp fragment containing the URA3 gene was ligated to the YEpWB5IT/NAT which was cleaved with KpnI and made blunt-ended.
  • the ligation reaction was carried out according to published procedures (see Section 7.2.3., infra).
  • the ligation mixture was transformed into the E. coli DH5 ⁇ cells using standard transformation procedures (see Section 7.2.3., infra).
  • the cells were spread on plates containing LB-media with 100 mg/l
  • a 7200 bp fragment containing the GAL10 promoter and ADH terminator was gel purified (0.6 % agarose gel in 1X TBE).
  • Transformed cells were plated on LB-media with 100 mg/l ampicillin. Plates were incubated at 37°C overnight. Colonies appearing on these plates were used to inoculate 5.0 ml LB media with 100 mg/l ampicillin and cultures were grown at 37°C overnight. DNA isolated from these cultures was analyzed using restriction enzyme HindII.
  • the reagent kit for DNA sequencing was
  • the primer sequence was 5' CTT CTT TGC GTC CAT CCA 3' and is described in the Sequence Listing as SEQ ID NO :9.
  • the 5' and the 3' ends of the primer were checked for optimal hybridization to ensure minimal non-specific annealing to the template using the HIBIO DNASIS program (Hitachi America, LTD).
  • the sequencing gels were 6.0% and were prepared with Gel-Mix 6 (GIBCO BRL).
  • the sequencing protocol was provided with the Sequetide S-labeled Premix (NEN Research Products).
  • the sequencing gels were fixed in 2 liters fixing solution containing 10% acetic acid and 5% methanol, and were dried for 1 hour at 80°C in a slab dryer by Bio-Rad.
  • Sequencing of the alpha-globin gene in plasmid pUT/2A showed two silent mutations. These silent mutations were both in the wobble position of the mRNA codon and they did not affect the translation of the globin protein.
  • the first mutation was carried over from plasmid pJW101 which was used to create pUT/2A.
  • the second mutation occurred within the plasmid and was two amino acids away from the first one. These mutations might have occurred either during the PCR or was present in the original gene.
  • the gamma-globin gene was obtained from plasmid pJW151 using PCR.
  • the gamma-globin gene was modified by PCR to have a SalI site at the 5'-end and a HindIII site at the 3'-end.
  • the modified gamma-globin gene was cloned into the yeast expression vector YEp5IT/NAT, which contains the ADH1 transcription termination sequence, the GAL10 promoter, and the DNA sequence encoding the beta- globin gene.
  • YEp51T/NAT had been cut with SalI and HindIII to remove the beta-globin gene.
  • the plasmid containing the gamma-globin gene was called YEp51T/G.
  • Yeast strain Sc340 was transformed with YEp51T/G and the transformant was called 340g2G. Following growth of 340g2G and induction by galactose, expressed proteins were analyzed by Western blot analysis. The results from Western blot analysis indicated that gamma-globin was expressed.
  • the E. coli strain used for all bacterial transformations was DH5 ⁇ .
  • the gamma-globin gene was synthesized by PCR using appropriate primers and plasmid pJW151 DNA (Wilson, J.T., et al., Nucleic Acid Research 5:563-581, 1978) as template.
  • the sequence of gamma-globin DNA is shown in Figure 13, and is described as SEQ ID NO: 10.
  • the 5' and 3' primers used for synthesizing the gene, GAM-5-S and GAM-3-H respectively, are shown in Figure 14 and are described as SEQ ID NO: 11 and SEQ ID NO: 12.
  • the PCR product was
  • Plasmid YEp51T/NAT which contains the human beta-globin gene was digested with SalI and HindIII to remove the beta-globin gene.
  • the digested plasmid was electrophoresed in 0.6% agarose gel (in IX TBE). A 7000 bp fragment was electroeluted and ethanol precipitated.
  • a ligation reaction mixture was set up between the gamma-globin obtained by digestion of the PCR product described above and YEp51T/NAT cut with Sail and HindIII (7000 bp). The ligation mixture was used to transform E. coli DH5GC cells using standard transformation procedure and plated on LB plates containing Ampicillin (100 mg/L).
  • Plasmid DNA was isolated from 20 clones and digested with restriction enzyme Pstl. The resulting plasmid was called YEp51T/G ( Figure 15). 8.3. TRANSFORMATION AND GROWTH OF YEAST STRAIN Sc340 CELLS WITH PLASMID YEP51T/G
  • Yeast strain Sc340 cells were transformed with plasmid YEp51T/G (Rose, et al., 1989, Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring
  • the starter culture was grown in SD supplemented with adenine and histidine, and 3% glycerol and 2% lactate as carbon source.
  • the preculture was used to inoculate 2 L of the above media in a Braun Biostat E fermentor.
  • the pH was maintained at 5.5 using a 5% ammonium hydroxide solution.
  • the pO 2 was maintained at
  • the expressed gamma-globin was quantitated by Western Blot analysis using procedures described in Section 6.6., supra. The results indicated that up to 0.05% of the total yeast protein in yeast cell line 340g2G was gamma-globin.
  • PCR was carried out using DNA thermal cycler obtained from Cetus and according to the methods described by Cetus.
  • the genomic clone for human epsilon gene pNEVll was obtained from the Beatson Institute for Cancer Research.
  • the recombinant bacteriophage clones containing beta-type globin genes and flanking sequences were used by scientists at the Beatson Cancer Research Institute.
  • the EcoRI fragment containing epsilon genomic sequences from one of these clones was recloned in pBR322 based plasmid (Montague, Ph.D. Thesis entitled "The Behaviour of Human Globin Gene Recombinants in Mammalian Cells").
  • This plasmid was labeled pNEVll.
  • the DNA from plasmid pNEV11 was isolated and used as a template for PCR.
  • the E. coli strains DH5 ⁇ and NM522 (Invitrogen, Inc.) were used for bacterial transformations,
  • Figure 16 shows the structure of the epsilon globin genome, as well as the strategy used for the
  • the first 92 base exon (exon A) is separated from the second exon (exon B) by 121 bases intron.
  • the second exon is 221 bases and the third exon (exon C) is 120 bases.
  • the second and third exons are separated by 855 bases of intron.
  • the six primers used for the synthesis of epsilon globin cDNA, 5EPSL, INPE-1, INPE-2, INPE-3, INPE-4, and 3-EPH, are described in the Sequence Description as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, and SEQ ID NO: 18, respectively, and are also shown in Figure 17 with pertinent restriction sites.
  • 5EPSL contains the 5' sequences of epsilon cDNA
  • INPE-1 contains a 23 base sequences at the 3' end of the exon A joined to a 15 base sequences at 5' end of the exon 2
  • INPE-2 contains the complementary sequences present in INPE-1
  • INPE-3 contains 3' sequences at exon B joined together with 5* sequences of exon C
  • INPE-4 contains complementary sequences of INPE-3
  • 3-EPH contains 3' end sequences complementary to the coding strand of exon 3 with HindIII site at the 3' end.
  • the genomic clone was PCRed using two outside primers, 5EPSL and 3-EPH. The entire genomic DNA fragment containing 2 kb fragment was obtained with these two primers. This confirmed that the plasmid pNEV11 contains epsilon-globin genomic sequences.
  • the epsilon globin cDNA was cut with Sall/HindIII. Ligation was set between Sall/HindIII cut YEp5iNTl (see Section 11.4., infra for a description of the construction of YEp51NTl).
  • the ligation mixture was transformed in competent E. coli NM522 cells and the DNA was isolated from 24 transformants by alkaline digestion. The DNA samples from the clones were analyzed by
  • Yeast strain Scl041 was transformed with plasmid pYEp51T/ ⁇ 3 (supra, 9.3.) using electroporation.
  • BioRad (Richmond, CA) Gene Pulser with Pulse Controller was used for electroporation.
  • the 0.2-cm cuvettes were obtained from Bio Rad. 40 ⁇ l of yeast cells were
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final
  • the expressed globins were quantitated by Western Blot analysis using procedures described in Section
  • the plasmid p4-7-7 containing zeta globin cDNA was obtained from Dr. Forget's laboratory (Cohen-Solal et al., 1982, DNA 1:255).
  • the yeast expression vector pYES2 was obtained from Invitrogen Corp. (San Diego, CA).
  • the vector contains the GAL1 portion of the divergent
  • GAL1/GAL10 promoter region polylinker for cloning genes, the CYC1 transcription terminator and the URA3 gene for selection in yeast.
  • E. coli strain NM522 was obtained from
  • Competent cells were prepared according to the protocol provided by Invitrogen.
  • the zeta globin cDNA was PCRed using appropriate primers. These primers, 5ZETASAC and
  • ZETA3HSLS are described in the Sequence Description as SEQ ID NO: 19 and SEQ ID NO:20, and are shown in Figure 19 with restriction sites.
  • the pcred DNA was cut with SacI/SphI and cloned into SacI/SphI cut DNA from plasmid pYES2.
  • the DNA was isolated from 24 transformants by alkaline
  • the DNA samples from the clones were analyzed by restriction digestion with SacI/SphI.
  • the plasmid containing zeta globin cDNA was labeled pYES2- ⁇ 2 and is shown in Figure 20.
  • Yeast strain Scl041 was transformed with plasmid pYES2- ⁇ 2 (supra, Section 10.2) using
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final
  • the expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Samples taken after induction had detectable levels of globin (0.13%).
  • mutant globin genes were cloned into yeast expression vector YEp51NT1. This vector contains GAL10 promoter and ADH terminator sequences. The following mutant genes were cloned into this yeast expression vector: i. ⁇ -Motown (127 Gln->Glu)
  • Oligonucleotides used in the Polymerase Chain Reaction were obtained by chemical synthesis on
  • the E. coli strain used for all bacterial transformations was DHS ⁇ . 11.1.1. DNA FRAGMENT ISOLATION
  • Transformed cells were plated on LB-media with 100 mg/L ampicillin. Plates were incubated at 37°C overnight. Colonies appearing on these plates were used to inoculate 5.0 ml LB media with 100 mg/L ampicillin and cultures were grown at 37°C overnight.
  • DNA was isolated from 1.5 ml of the overnight culture using alkaline lysis procedure. Plasmid DNA was analyzed by appropriate restriction enzyme digestion.
  • oligonucleotides were synthesized as a preliminary step in the construction of several globin gene variants.
  • the oligonucleotides to be used in the in vitro mutagenesis procedure with M13 were synthesized and purified.
  • Polyacrylamide gel electrophoresis following kinasing demonstrated that the synthesis was efficient and that the oligonucleotides were ready for use in the M13 system.
  • the [ ⁇ - 32 P]ATP kinased oligonucleotides were analyzed on a 6% acrylamide sequencing gel containing 7M urea.
  • the dye used in this electrophoresis was a mixture of bromphenol blue and xyno-cynol which separate during the procedure, with each dye migrating at different rates .
  • fragments that are approximately 25 bases should migrate with the bromphenol blue dye front, while those of about 90 bases should migrate with the xyno-cynol dye front.
  • the synthetic oligonucleotides ranged in size from 30 to 45 bases which should run between the two dye fronts as was observed.
  • the in vitro mutagenesis kit from Bio Rad provides the necessary components for mutagenesis with the M13 system. Included in this kit are two strains of E. coli to be used in the process.
  • E. coli strain CJ236 contains mutations which result in the incorporation of uracil instead of thymine in DNA.
  • E. coli strain MV1190 is a wild type strain that is used to produce the single stranded DNA following mutagenesis.
  • the E. coli strains that were received in the mutagenesis kit were subcultured on appropriate media according to the genetic markers for selection.
  • the constituents of each type of media as well as a suggested protocol for mutagenesis may be found in the brochure that was received with the kit (New England BioLabs, "M13
  • CJ236 competent cells for use in transfection were prepared by inoculating 100 ml LB broth containing chloramphenicol with 5 ml of an overnight culture of CJ236. The culture was incubated at 37°C in an air shaker until the OD 600 reached 0.8. The cells were centrifuged at 3K rpm for 5 minutes, resuspended in 20 ml 50 mM cold CaCl 2 , and held on ice for 30 minutes. The cells were centrifuged again and resuspended in 4 ml 50 mM CaCl 2 .
  • the CJ236 competent cells were transfected with M13mp19BHS by adding 1 ⁇ l or 5 ⁇ l of DNA to 0.3 ml competent cells. The tubes were held on ice for 40
  • top agar 50°C containing chloramphenicol and 300 ⁇ l of the overnight culture of CJ236. This top agar was poured onto H-medium plates containing chloramphenicol and incubated overnight at
  • the phage was isolated (from those cells which were infected) by touching a toothpick to plaques and
  • Uracil containing DNA was isolated from CJ236 by inoculating 50 ml LB medium containing chloramphenicol with 1.0 ml of an overnight culture of CJ236. The culture was incubated at 37°C with shaking until it reached an OD 600 of 0.3. At this point, the culture was infected with 50 ⁇ l of a -70°C stock culture that was previously infected with phage in order to amplify the production of single stranded DNA. The infected culture was allowed to grow overnight at these conditions. The following day, 30 ml of the culture was centrifuged at 16K rpm for 15 minutes. The supernatant containing the phage particles was transferred to a new tube and centrifuged a second time.
  • the supernatant from this second centrifugation was treated with 150 ⁇ g RNase A at room temperature for 30 minutes.
  • Single stranded DNA was precipitated by adding 7.5 ml of PEG solution (3.5 M ammonium acetate, 20% PEG 8000) and held on ice for 30 minutes. The tube was centrifuged and the supernatant was discarded. The pellet was suspended in 200 ⁇ l of high salt buffer (300 mM NaCl, 100 mM Tris, pH 8.0, 1 mM EDTA), held on ice for 30 minutes, and centrifuged in a
  • the phage was titered on CJ236 and MV1190 to determine whether infection was productive. Following confirmation of productive infection, the DNA was
  • This DNA is the single stranded uracil-containing DNA which was used as a template for the synthesis of the mutagenic strand.
  • the purified oligonucleotides were kinased by treating 5 ⁇ g of each of the six oligonucleotides with T4 polynucleotide kinase and ATP to ensure efficient ligation of the two ends of the newly synthesized DNA strand.
  • the synthesis of the mutagenic strand was carried out by adding 0.25 ⁇ g (0.1 pM) of the
  • T4 DNA Polymerase 1 unit. The reactions were incubated on ice for 5 minutes in order to stabilize the primer by initiation of DNA synthesis under conditions that favor the binding of the primer to the template. The reactions were then incubated at 25°C for 5 minutes and finally at 37°C for 90 minutes. Following the final incubation, 90 ⁇ l of stop buffer (10 mM Tris, pH 8.0, 10 mM EDTA) was added to each reaction and were placed at -20°C until use in the transfection of MV1190.
  • stop buffer 10 mM Tris, pH 8.0, 10 mM EDTA
  • MV1190 cells were transfected with the
  • the clear plaques were picked by inserting a sterile Pasteur pipet into the agar and suspending the plug in 3 ⁇ l LB broth (24 plaques were chosen from each of the plates containing plaques). 100 ⁇ l of an overnight culture of MV1190 was added and the tubes were incubated with shaking overnight at 37°C. Following the incubation period, single-stranded DNA was isolated from the cultures and this DNA was used in sequencing reactions.
  • Dideoxy sequencing was performed to confirm the presence of mutations.
  • the sequencing kit used in this case was obtained from New England Biolabs. Each sequencing reaction was set up using 8 ⁇ l of the single stranded DNA to be sequenced, 1 ⁇ l of the appropriate primer, and 1 ⁇ l 10X sequencing buffer. The primer was annealed to the single stranded template by placing the tubes at 90°C and allowing them to cool to 30°C. 2 ⁇ l of the DNA-primer mixture was used in each individual sequencing reaction along with 2 ⁇ l of the termination mix (50 ⁇ l of the appropriate dNTP's and ddNTP plus 5 ⁇ l [ ⁇ - 32 P] dATP) and 2 ⁇ l of Klenow enzyme diluted to 0.1 units/ ⁇ l.
  • the reaction was incubated at room temperature for 15 minutes and 2 ⁇ l of a chase mixture was added that consisted of a dNTP mixture containing cold dATP and Klenow enzyme. This reaction was incubated again at room temperature for 15 minutes and 4 ⁇ l of dye mix was added to stop the reaction.
  • the samples were denatured by boiling for 2.5 minutes and, placed in an ice water bath, and loaded onto a 6%
  • polyacrylamide sequencing gel containing 7M urea. The gel was run at 55 watts for approximately 4 hours before it was dried under vacuum and placed in an X-ray film cassette for autoradiography.
  • sequencing kit specifically for use with single-stranded DNA was obtained from IBI and a Pharmacia kit was used with T7 DNA polymerase rather than Klenow Enzyme in order to sequence mutants further from the point of primer
  • Yeast shuttle vector YEp51 was modified to have ADH terminator sequences.
  • the ADH terminator was inserted between the GAL10 promoter and the 2 ⁇ present on this vector.
  • plasmid YEp51 was digested with restriction enzyme Bcl1.
  • the linearized DNA molecule was treated with Klenow enzyme and dNTPs to make it blunt ended.
  • a double-stranded oligonucleotide was ligated to the blunt ended plasmid. This oligonucleotide was obtained from BRL and contained sequences for restriction enzyme
  • PEG polyethylene glycol
  • the ADH terminator was obtained from plasmid AAH5 (see Figure 6). Plasmid AAH5 was digested with restriction enzyme BamHI. DNA was blunt-ended with Klenow and dNTPs. Blunt-ended DNA was subjected to phenol
  • DNA was then digested with restriction enzyme NotI and HindIII.
  • NotI and HindIII A 400 bp NotI-HindIII fragment was isolated from a 1.0% agarose gel (IX TBE). DNA was electroeluted from the agarose slice and precipitated with ethanol. This purified DNA fragment was ligated to the above-mentioned plasmid. The ligation mixture was used to transform DH5 ⁇ -cells. Transformed cells were spread on plates containing LB-media with 100 mg/L ampicillin.
  • Plasmid DNA was digested with restriction enzyme NotI and HindIII.
  • the resulting plasmid was called YEp51NTl and is shown in Figure 22 .
  • the vector for cloning the mutated ⁇ -globin gene(s) was prepared by digesting plasmids YEP51T/G (supra, Section 8.4) or YEp51NTl/ ⁇ -P0RT (infra, 11.5.1.) with SalI and HindIII.
  • the vector for cloning the ⁇ -globin gene was prepared by digesting plasmids YEP51T/G (supra, Section 8.4) or YEp51NTl/ ⁇ -P0RT (infra, 11.5.1.) with SalI and HindIII.
  • the vector for cloning the ⁇ -globin gene was prepared by digesting plasmids YEP51T/G (supra, Section 8.4) or YEp51NTl/ ⁇ -P0RT (infra, 11.5.1.) with SalI and HindIII.
  • the vector for cloning the ⁇ -globin gene was prepared by digesting plasmids YEP51
  • the Porto Alegre ⁇ -globin was created by substituting two bases in the natural ⁇ -globin sequence using PCR.
  • the ⁇ -globin gene was obtained as a 450 bp fragment.
  • the 5' and 3' primers used for synthesizing the sequence, respectively, G-5-9CY and GAM-3-H are shown in Figure 23 and are described in the Sequence Description as SEQ ID NO: 22 and SEQ ID NO: 12 respectively.
  • the mutated ⁇ -globin gene obtained by PCR was digested with SalI and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by PCR was ligated to the vector YEp51NTl cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation was performed as described (see Section 11.1., supra) . DNA isolated from the transformed cells was digested with restriction enzyme Pstl. The results obtained from this analysis showed clones that had expected fragments (three fragments when digested with PstI; two fragments from vector without insert). This plasmid was called YEp51NT1/ ⁇ -PORT. 11.5.2. CLONING OF THE MOTOWN (127 Gln->Glu) ⁇ -GLOBIN GENE
  • the Motown ⁇ -globin was created by a base substitution using PCR.
  • the globin gene was isolated as two fragments.
  • the 3'-end of the gene (EcoRI-HindIII) was obtained by PCR.
  • the mutated fragment of the ⁇ -globin gene obtained by PCR was digested with EcoRI and HindIII. This digested DNA fragment (80 bp) was purified by phenol extraction and ethanol precipitation. The 5'-end of the ⁇ -globin gene was isolated from plasmid YEp51T/NAT (supra.
  • Plasmid YEp51T/NAT was digested with restriction enzymes BamHI and HindIII. A 360 bp fragment was isolated.
  • This plasmid was called pNT1/ ⁇ -Mot.
  • the ⁇ -globin gene was isolated as two fragments. The
  • 3'-end of the gene was obtained by PCR using plasmid pl9AlGT as template.
  • the 5'-end of the gene (SalI-HindIII) was obtained as a 450 bp fragment.
  • Primers used for PCR were
  • the template for the PCR was plasmid pJW101.
  • the PCR product was digested with restriction enzymes Sail and
  • HindIII A 300 bp fragment was isolated. This purified 300 bp fragment along with the fragment obtained by PCR were ligated to the vector YEp51NTl/ ⁇ -PORT (See Section 11.5.1., supra, cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation was performed as described (supra, 11.1). DNA isolated from the transformed cells was digested with restriction enzyme HindII. The results obtained from this analysis showed that one clone had expected fragments (six fragments when digested with
  • This plasmid was called pNT1/2ATit.
  • the Mississippi ⁇ -globin was created by substituting two bases in the natural ⁇ -globin gene using
  • the mutated fragment of the ⁇ -globin gene was digested with Accl and HindIII. This digested DNA fragment was purified by phenol extraction and ethanol
  • Plasmid YEp5IT/NAT was digested with restriction enzymes Accl and SalI. A 117 bp fragment was isolated. This purified 117 bp fragment along with the fragment obtained by PCR were ligated to the vector YEp51NT1/ ⁇ -PORT. DNA ligation, E. coli transformation and DNA isolation was performed as described (see Section 11.1., supra). DNA isolated from the transformed cells was digested with restriction enzyme Pstl. The results obtained from this analysis showed that most of the clones analyzed had expected fragments (two fragments when digested with Pstl; three fragments from vector without insert). This plasmid was called pNT1/ ⁇ -Miss.
  • the 104-Ser alpha-globin was created by
  • the alpha-globin gene was isolated as two fragments.
  • the 3'-end of the gene (HindIII-HindIII) was obtained by PCR using plasmid pAlGT (Wilson et al., 1978, Nucl. Acids Res. 5:563-580) as template.
  • the primers used for PCR, A-104Ser (5'-end primer) and G10T3H (3'-end primer), are described in the Sequence Description as SEQ ID NO:32 and SEQ ID NO:28, respectively, and are shown in Figure 28 with restriction sites.
  • the mutated fragment of the alpha-globin gene was digested HindIII. This digested DNA fragment was purified by phenol extraction and ethanol precipitation.
  • the 5'-end of the gene (SalI-HindIII) was obtained by PCR using plasmid pJW101 as template. Primers used for PCR were 51-A3-SL (5'-end primer) and A-Hin3-3 (3'-end primer), are described in the Sequence Description as
  • PCR product was digested with SalI and HindIII. This digested DNA fragment was purified by phenol extraction and ethanol
  • the 104 Ser alpha-globin gene was also cloned in yeast expression vector YEp51UT/NAT. Plasmid
  • Yep51UT/NAT was digested with SalI and BamHI. A 7000 bp fragment was gel purified.
  • the 5'-end of the gene (SalI- HindIII) was obtained by PCR using plasmid pJW101 as template. Primers used for PCR 51-A3-SL (5'-end primer) and A-Hin3-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and are shown in Figure 26 with restriction sites.
  • the PCR product was digested with SalI and HindIII. This digested DNA fragment was purified by phenol extraction and ethanol precipitation.
  • the 3'-end of the gene (Hindlll-HindllI) was obtained by PCR using plasmid pA1GT as template. Primers used for PCR were A-104Ser (5'-end primer) and 519-A-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO:32 and SEQ ID NO: 8, respectively.
  • the mutated fragment of the alpha-globin gene was digested HindIII. This digested DNA fragment was purified by phenol
  • the 104-Ser ⁇ -globin was created by substituting one base in the natural ⁇ -globin gene using
  • the ⁇ -globin gene was isolated as two fragments.
  • the 3'-end of the gene (BstEII-HindIII) was obtained by PCR using plasmid 4p-7-7 as template.
  • Primers used for PCR are Z-BST-5 (5'-end primer) and Z2-3-H (3'-end primer) are shown in Figure 30 and are described in the Sequence Description as SEQ ID NO: 35 and SEQ ID NO: 36 respectively.
  • the mutated fragment of the ⁇ -globin gene was digested with SalI and BstEII. This digested DNA fragment (330 bp) was purified by phenol extraction and ethanol precipitation. The 3'-end of the ⁇ -globin gene obtained by
  • fragments (330 and 100 bp) were ligated to the vector
  • transformed cells was digested with restriction enzyme
  • the 94-Asn ⁇ -globin was created by substituting one base in the natural ⁇ -globin gene using PCR.
  • the ⁇ -globin gene was isolated as two fragments. Both fragments were obtained by PCR.
  • the 5'-end of the gene (SalI-BstEII) was obtained by PCR using plasmid 4p-7-7 as template.
  • the 5' primer used for synthesizing the sequence, Z-5-SAL is described in the Sequence Description as SEQ ID NO: 33 and the 3' primer used for synthesizing the sequence, Z-A95-3, is described in the Sequence Description as SEQ ID NO: 37. Restriction sites on these two primers are shown in Figure 31.
  • the 3'-end of the gene (BstEII-HindIII) was obtained by PCR using plasmid 4p-7-7 as template.
  • the 5' primer used for synthesizing the sequence, Z-BST-5 is described in the Sequence Description as SEQ ID NO: 35 and the 3' primer used for synthesizing the sequence, Z2-3-H, is described in the Sequence Description as SEQ ID NO: 36. Restriction sites on these two primers are shown in Figure 30.
  • a mutated fragment of the ⁇ -globin gene obtained by PCR was digested SalI and BstEII. This digested DNA fragment (330 bp) was purified by phenol extraction and ethanol precipitation. The 3'-end of the ⁇ -globin gene obtained by PCR was digested with restriction enzymes Bst EII and Hind III. A 100 bp fragment was isolated. Purified fragments (330 and 100 bp) were ligated to the vector
  • transformed cells was digested with restriction enzyme
  • the double mutant (Titusville + 104 Ser) alpha- globin was created by substituting one base in the 104 Ser alpha-globin gene using PCR.
  • the alpha-globin gene was isolated as two fragments.
  • the 3'-end of the gene (HindIII -HindIII) was obtained by PCR using plasmid pNT1/ ⁇ 104S as template. Primers used for PCR were A-Tit-5 (5'-end primer) and G10T3H (3'-end primer), are described in the Sequence Description as SEQ ID NO:27 and SEQ ID NO: 28, respectively, and are shown in Figure 25 with restriction sites.
  • the alpha-globin gene fragment containing double mutation was digested HindIII. This digested DNA fragment was purified by phenol extraction and ethanol precipitation.
  • the 5'-end of the alpha-globin gene was obtained from plasmid pJW101 using PCR.
  • the 5'-end of the gene (SalI-HindIII) was obtained by PCR using plasmid pJW101 as template.
  • Primers used for PCR were 51-A3-SL (5'- end primer) and A-Hin3-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and are shown in Figure 26 with restriction sites.
  • the PCR product was digested with SalI and HindIII.
  • This digested DNA fragment was purified by phenol
  • This plasmid was called pNTl/2ATiS.
  • the Motown gamma-globin was created by base substitution in the natural gamma-globin sequence using
  • the gamma-globin gene was obtained as two fragments.
  • the 5 'end of the gene was isolated as SalI-EcoRI fragment (320 bp) from plasmid YEp51T/G.
  • the 3'end of the gene was isolated as SalI-EcoRI fragment (320 bp) from plasmid YEp51T/G.
  • PCR product was digested with restriction enzymes EcoRI and HindIII. Digested fragment was purified by phenol extraction and ethanol precipitation.
  • the purified fragments obtained by PCR and isolated from plasmid was YEp51T/G were ligated to the vector (YEp51NTl cut with SalI and HindIII). DNA ligation, E. coli transformation and DNA isolation was performed as described in Section 11.1., supra. DNA isolated from the transformed cells was digested with restriction enzyme
  • BovII human globin gene 5'-end with four amino acids of the bovine globin gene
  • the 5' primer, B-Bov2-5, used for synthesizing the sequence is described in the Sequence Description as SEQ ID NO:39 and the 3' primer, Beta-3-H, used for synthesizing the sequence is described in the
  • the mutated ⁇ -globin gene obtained by PCR was digested with SalI and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by
  • PCR was ligated to the vector YEp51NT1/ ⁇ -P0RT cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation was performed as described (supra, 11.1.). DNA isolated from the transformed cells was digested with restriction enzyme Pstl. The results obtained from this analysis showed that most of the clones analyzed had expected fragments (two fragments when digested with Pstl; three fragments from vector without insert). This plasmid was called pNT1/ ⁇ -Bov2.
  • the ⁇ -2 Arg beta-globin was created by
  • beta-globin gene was obtained as a 450 bp fragment.
  • Primers used for PCR, B-2ARG-5 (5'-end primer) and Beta-3-H (3'-end primer) are described in the Sequence Description as SEQ ID NO: 40 and SEQ ID NO: 26, respectively, and are shown in Figure 34 with restriction sites.
  • the mutated beta globin gene obtained by PCR was digested with SalI and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by PCR was ligated to the vector (YEp51NTl/ ⁇ -PORT cut with Sal! cut with Hindlll). DNA ligation, E. coli transformation and DNA isolation was performed as described in Materials and Methods section. DNA isolated from the transformed cells was digested with restriction enzyme
  • the 143 Arg beta-globin was created by
  • beta-globin gene was obtained as a 450 bp fragment.
  • Primers used for PCR, BN-5-SAL (5'-end primer) and B-143A-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO: 33 and SEQ ID NO: 41, respectively, and are shown in Figure 35 with restriction sites.
  • the mutated gamma-globin gene obtained by PCR was digested with Sail and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by PCR was ligated to the vector (YEp51NTl/ ⁇ -PORT cut with SalI and HindIII). DNA ligation, E. coli
  • the 145 Term beta-globin was created by replacing amino acid Tyr (amino acid #145) of the human beta-globin with protein termination codon (TAA).
  • TAA protein termination codon
  • the mutated beta-globin gene was obtained as a 450 bp fragment.
  • Primers used for PCR, BN-5-SAL (5'-end primer) and B-145T-3 (3'-end primer), are described in the Sequence Description as SEQ ID NO:33 and SEQ ID NO: 42, respectively, and are shown in Figure 36 with restriction sites.
  • the mutated beta-globin gene obtained by PCR was digested with SalI and HindIII. This digested DNA fragment (450 bp) was purified by phenol extraction and ethanol precipitation. This purified 450 bp fragment obtained by PCR was ligated to the vector (YEp51NT1/ ⁇ -PORT cut with SalI and HindIII). DNA ligation, E. coli
  • the Chico ⁇ -globin was created by substituting one base in the natural ⁇ -globin gene using PCR .
  • the ⁇ -globin gene was isolated as two fragments .
  • the 5 ' -end of the gene (SalI-XcmI) was obtained by PCR using plasmid pJW151 as template.
  • the 5' primer used for synthesizing the sequence, GAM-5-S is described in the Sequence
  • SEQ ID NO: 11 Description as SEQ ID NO: 11 and the 3' primer used for synthesizing the sequence, G66T-3' is described in the Sequence Description as SEQ ID NO: 43. Restriction sites on these two primers are shown in Figure 37.
  • the mutated fragment of the ⁇ -globin gene was digested with SalI and XcmI. This digested DNA fragment (230 bp) was purified by phenol extraction and ethanol precipitation. The 3'-end of the ⁇ -globin gene was
  • Plasmid YEp51NT1/ ⁇ -PORT was digested with restriction enzymes XcmI and
  • HindIII A 220 bp fragment was isolated. This purified 220 bp fragment along with the fragment obtained by PCR were ligated to the vector YEp51NTl cut with SalI and HindIII. DNA ligation, E. coli transformation and DNA isolation were performed as described (supra. 11.1). DNA isolated from the transformed cells was digested with restriction enzyme
  • Yeast strain Sc1114 was transformed with plasmid pNTl/ ⁇ -Mot2 (see Section 11.5.9., supra) using electroporation (see Section 9.4., supra..
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final
  • the expressed globins was quantitated by
  • Yeast strain Sc1115 was transformed with plasmid pNT1/ ⁇ -Bov2 (see Section 11.5.10., supra) using electroporation (see Section 9.4., supra).
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tweenSO, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final
  • the expressed globins was quantitated by
  • Yeast strain Sc340 was transformed with plasmid pNTl/ ⁇ 104S (see Section 11.5.6., supra) using electroporation (see Section 9.4., supra).
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, uracil, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final
  • the expressed globins was quantitated by
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween 80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was adjusted to 7.12 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by Western Blot analysis using procedures described in section 6.6. Globin was detected at a level of 0.6% of soluble protein.
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc Natl Acad Sci USA, 1987, 84:8961).
  • Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer.
  • the suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute.
  • One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm.
  • the cuvette is then removed and the
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • Yeast strain Sc340 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/ ⁇ 143Arg
  • the starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2% .
  • the pH was adjusted to 7.18 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 84:6961).
  • Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer.
  • the suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute.
  • One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm.
  • the cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes.
  • CO carbon monoxide
  • the suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned.
  • the O.D. at 600 nm is measured on the same instrument.
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin .
  • Yeast strain Sc1090 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/ ⁇ 145T (see Section 11.5.13, supra) using electroporation (see Section 9.4., supra).
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was adjusted to 7.04 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by Western Blot analysis using procedures described in section 6.6. Globin was detected at a level of 0.11% of soluble protein.
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 84:8961).
  • Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer.
  • the suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute.
  • One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm.
  • the cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes.
  • CO carbon monoxide
  • the suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D. at 600 nm is measured on the same
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • the expressed alpha and beta globins were separated using an 18% SDS polyacrylamide gel.
  • Phosphate-buffered saline (PBS, 0.9 M NaCl, 0.01 M phosphate, pH 7.6) solution (2 ml) was added to thawed yeast samples (0.2 g wet weight). The samples were centrifuged at 4°C for 10 minutes at 2700 rpm in a Sorvall RT6000B and the supernatant decanted. Cold disruption buffer (50 mM Tris, 5 mM EDTA, 0.5 mM PMSF, pH 8.0) prepared immediately before use (0.2 ml) was added to the pellet, followed by enough ice-cold glass beads to just reach the top surface of the liquid. After vortexing for 30 seconds at maximum speed the samples were placed on ice for 5 minutes. This step was repeated twice. Ice-cold
  • the transfer unit was filled with transfer buffer (2L methanol, 30.3 g Tris base, 144 g glycine in a final volume of 10L, pH 8.3) and 2L of the transfer buffer was put into a shallow pan.
  • transfer buffer (2L methanol, 30.3 g Tris base, 144 g glycine in a final volume of 10L, pH 8.3) and 2L of the transfer buffer was put into a shallow pan.
  • Protein was transferred from the gel to the nitrocellulose paper by applying a voltage of 40V for 1.5 hrs. After transfer was complete, the nitrocellulose was removed and placed in a shallow pan with 50 ml of blocking solution [5% (w/v) BSA in PBS]. The nitrocellulose membrane was incubated for 1 hour with agitation, after which the blocking solution was replaced with washing solution [0.1% Tween 20 (v/v) in PBSI . Three washings of 15, 5 and 5 minutes were carried out. The final wash solution was discarded and 25 ⁇ l of primary antibody in 25 ml of PBS was added to the pan. After incubation for 2 hours, with agitation, the nitrocellulose was washed three times (1 ⁇ 15 and 2 ⁇ 5 minutes).
  • Alpha and beta globins are separated on this gel by molecular weight.
  • Alpha and beta globin were
  • EXAMPLE 10 COEXPRESSION OF ALPHA-GLOBIN AND GAMMA- GLOBIN MOTOWN IN YEAST
  • Yeast strain Se1114 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/ ⁇ -Mot2 (see
  • the starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween 80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2% .
  • the pH was adjusted to 7.09 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by Western Blot analysis using procedures described in section 6.6. Globin was detected at a level of 0.3% of soluble protein.
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1987, 84:8961). Approximately 1.2 ml of
  • suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm. The cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes. The suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D. at 600 nm is measured on the same instrument.
  • CO oxygen-scrubbed carbon monoxide
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • Yeast strain Sc1114 was transformed with plasmids pNT1/ ⁇ 104S (see Section 11.5.6., supra) and
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was adjusted to 6.96 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1987, 84:6961). Approximately 1.2 ml of
  • suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording
  • the difference spectrum will produce a peak around 420 nm and a valley around 435nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • EXAMPLE 12 COEXPRESSION OF ALPHA-GLOBIN AND BETA- GLOBIN 2 ARG IN YEAST
  • Yeast strain Sc1090 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/ ⁇ 2Arg (see
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was adjusted to 6.93 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6.
  • Globin was detected at a level of 0.17% of soluble protein.
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc Natl Acad Sci USA, 1987, 84:8961).
  • Approximately 1.2 ml of suspension of yeast with a final O.D. at 600nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer.
  • the suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute.
  • One ml is then removed and placed in a full length small volume cuvette.
  • This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm. The cuvette is then removed and the
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • Yeast strain Scl012 was cotransformed with plasmids pYES2- ⁇ 2 (see Section 10, supra) and YEp51T/G (see
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, 4ppm aminolevulinic acid (ALV) , and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 4ppm ALV, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was
  • the expressed globins were quantitated by
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1987, 84:8961). Approximately 1.2 ml of
  • suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • a hybrid promoter was constructed by the fusion of the upstream activating sequence of GAL1-10 promoter with the downstream promoter elements of the TDH3 promoter (referred to hereafter as the 3' end of the TDH3 promoter or TDH3-3').
  • the cassette containing the hybrid promoter + beta-globin gene + ADH1 terminator were excised and cloned into the yeast shuttle vector, YEp13.
  • Yeast strain Sc340 was transformed with the resulting plasmid, pNML-V-G-1 and the proteins expressed were analyzed by Western Blot Analysis.
  • restriction enzymes Klenow enzyme and T4-DNA ligase were obtained from New England Biolabs
  • the beta-globin gene was obtained by digestion of plasmid mp18 ⁇ HS with SalI and HindIII (see Figure 3 ⁇ for map of mp18 ⁇ HS). The 600 bp fragment was isolated by electroelution.
  • Plasmid AAH5 was digested with HindIII and BamHl (Ammerer, G., Methods in Enzymology, 101, pp. 192-201, 1963). The resulting 450 bp fragment was isolated by gel electrophoresis. Subsequently, the band containing the 450 bp fragment was precipitated with ethanol and digested with SphI. The 320 bp fragment (HindIII-SphI) containing ADH1 transcription termination sequences was isolated by electroelution.
  • Plasmid pUC19 was cut with SalI and SphI. A three way ligation reaction mixture was set up between the pUC19 fragment, the SalI-HindIII beta-globin fragment, and the HindIII-SphI ADH1 terminator fragment. The ligation was used for transforming competent E.. coli cells (DH5 ⁇ ).
  • Plasmid DNA was isolated from twenty transformants (clones) and analyzed by restriction digestion with SalI-HindIII. The resulting plasmid containing the above two inserts in pUC19 was called L19 ⁇ At, and is shown in Figure
  • the TDH3-3' promoter fragment was synthesized by PCR using appropriate primers and template DNA from plasmid gp491.
  • the primers, TDH3-5' (5'-primer) and TDH3-3' (3'-primer) are shown in Figure 40 and are described in the Sequence Description as SEQ ID NO: 42 and SEQ ID NO: 43.
  • the 180 bp promoter fragment (TDH3-3') synthesized by PCR was digested with ApaLI and SmaI.
  • the plasmid pUC19 was cut with Smal and Sphl.
  • the DNA from plasmid L19 ⁇ At was cut with ApaLI and Sphl and 920 bp fragment was isolated. Three way ligation was set between these three fragments. The transformation of E.
  • GAL1-10 upstream activator sequence (UAS) , which is shown in Figure 42 and described in the Sequence Description as SEQ ID NO : 44, was synthesized by polymerase chain reaction using GAL1-10-5 ' and GAL1-10-3 ' primers and DNA from YEp51 as a template .
  • the sequences of these primers, GAL1-10-5 ' and GAL1-10-3 ' are shown in Figure 43 and are described in the Sequence Description as SEQ ID NO: 45 and SEQ ID NO : 46.
  • the GAL1-10 UAS PCR product was digested with
  • Transformation was carried out using E . coli DH5 ⁇ cells .
  • the DNA isolated from the transformants were screened by restriction enzyme analysis with PvuII, EcoRI, and HindIII to check for the correct insert .
  • pUC19-GH ⁇ At was digested with SacI-SphI to excise the GAL10-UAS + TDH3-3 ' + beta-globin gene + ADH1-terminator cassette from pUC19 which was subsequently blunt-ended. The resulting 1. 43 kb fragment was isolated by electroelution .
  • Plasmid YEp13 obtained from Fred Phantom, Harvard Medical School which contains LEU2 (yeast) and Amp R (E. coli) markers, was digested with BamHI and blunt-ended; the resulting linear DNA was isolated by
  • Ligation was set between the insert and the vector and the ligation mixture was used for transforming competent E. coli cells (DH5 ⁇ ). The transformants were selected on ampicillin plates (100 mg/L). The plasmid DNA was isolated from 24 transformants and analyzed by
  • Strain Sc340 has the following genotype:
  • Yeast strain Sc340 was transformed with plasmid pNML-V-G-1 using the spheroplast procedure (Rose, M. et al., 1989, Methods in Yeast Genetics, Cold Spring
  • the regeneration media contained 1 M sorbitol, 10 mM CaCl 2 , 0.1% yeast nitrogen base, and 2% glucose. The medium was filter sterilized.
  • the plating media was prepared by mixing 182 g sorbitol, 20 g agar, 6.7 g Difco YNB without amino acids, glucose, required amino acids except leucine in 1 L distilled water.
  • the top agar was made by mixing 18.2 g sorbitol, 2 g agar, 0.67 g Difco YNB without amino acids, 2 g glucose and required amino acids in 100 ml distilled water.
  • starter culture cells were grown overnight in minimal media containing 0.67% yeast nitrogen base, 0.5% glucose, and supplemented with uracil, adenine, and histidine.
  • 500 ml of SD media supplemented with 200 ⁇ M ferric citrate and 20 mg/L each of adenine, uracil, and histidine was inoculated with the starter culture to an OD 600 of 0.02.
  • the culture was incubated with shaking (300 rpm) at 30°C, and was induced with 2% galactose for a period of 4 hours before sampling for analysis.
  • beta-globin was quantitated by Western Blot analysis using procedures described in Section 6.6., supra. The results indicated that up to 5.4% of the total yeast protein expressed in transformed Sc340 cells was beta-globin.
  • Yeast strain Sc1041 was cotransformed with plasmids pYES2- ⁇ 2 (see Section 10, supra) and pNM-V-G ⁇ 1
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was adjusted to 7.16 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.04% of soluble protein.
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1987, 84:8961). Approximately 1.2 ml of
  • suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • EXAMPLE 16 COEXPRESSION OF ALPHA-GLOBIN AND BETA- GLOBIN MISSISSIPPI IN YEAST
  • Yeast strain Sc389 was transformed with plasmids pUT/2A (see Section 7, supra) and pNT1/ ⁇ -Miss (see
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was adjusted to 7.10 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1967, 84:8961).
  • Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer.
  • the suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute.
  • One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • Yeast strain Sc1114 was transformed with plasmids pNT1/ ⁇ -Tit (see Section 11.5.3., supra) and
  • the starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2% .
  • the pH was adjusted to 6.9 ⁇ with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.01% of soluble protein.
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1967, 84:6961). Approximately 1.2 ml of
  • suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording
  • hemoglobin If hemoglobin is present the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • Yeast strain Sc1114 was transformed with plasmids pNT1/2ATiS (see Section 11.5.8., supra) and
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was adjusted to 7.10 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml. Samples were collected between four and 51 hours after induction.
  • the expressed globins were quantitated by
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. USA, 1987, 84:8961). Approximately 1.2 ml of
  • suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer. The suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute. One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • starter culture cells were grown in minimal media containing 0.67% yeast nitrogen base, 1% raffinose, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan at 30°C in a shake flask to log phase.
  • the starter culture was used to inoculate 500 ml of media containing 0.67% yeast nitrogen base, 3% glycerol, 2% lactic acid, 0.4% tween80, and supplemented with 20 mg/L each of adenine, histidine, and tryptophan. Incubation was at 30°C with shaking.
  • the culture was induced by adding galactose to a final concentration of 2%.
  • the pH was adjusted to 7.17 with KH(2)PO(4) and hemin was added to a final concentration of 40 ⁇ g/ml.
  • the expressed globins were quantitated by Western Blot analysis using procedures described in Section 6.6. Globin was detected at a level of 0.4% of soluble protein.
  • the whole yeast cell visible carbon monoxide difference spectrum is generated using a procedure adapted from the methods of Springer and Slager (Proc. Natl. Acad. Sci. U.S.A., 1987, 64:8961).
  • Approximately 1.2 ml of suspension of yeast with a final O.D. at 600 nm of about 2.0 is prepared using 0.1 mM PO 4 , pH 7.0, as a buffer.
  • the suspension is then reduced with a small amount of sodium dithionite vortexed, and allowed to sit for one minute.
  • One ml is then removed and placed in a full length small volume cuvette. This suspension is used as the baseline for a scan in a single beam Beckman DU-70 Recording spectrophotometer from 400 to 500 nm.
  • the cuvette is then removed and the suspension is bubbled steadily but not vigorously with oxygen-scrubbed carbon monoxide (CO) for two minutes.
  • CO carbon monoxide
  • the suspension is mixed by gentle rocking for one minute and the spectrum from 400 to 500 nm is scanned. Lastly the O.D. at 600 nm is measured on the same
  • the difference spectrum will produce a peak around 420 nm and a valley around 435 nm. A single peak at 420 nm does not indicate the presence of hemoglobin.
  • the expressed alpha and beta globins were separated using an 18% SDS polyacrylamide gel.
  • Phosphate-buffered saline (PBS, 0.9 M NaCl, 0.01 M phosphate, pH 7.6) solution (2 ml) was added to thawed yeast samples (0.2 g wet weight) .
  • the samples were centrifuged at 4°C for 10 minutes at 2700 rpm in a Sorvall RT6000B and the supernatant decanted.
  • Cold disruption buffer 50 mM Tris, 5 mM EDTA, 0.5 mM PMSF, pH 8.0
  • the transfer unit was filled with transfer buffer (2L methanol, 30.3 g Tris base, 144 g glycine in a final volume of 10L, pH 8.3) and 2L of the transfer buffer was put into a shallow pan.
  • transfer buffer (2L methanol, 30.3 g Tris base, 144 g glycine in a final volume of 10L, pH 8.3) and 2L of the transfer buffer was put into a shallow pan.
  • Protein was transferred from the gel to the nitrocellulose paper by applying a voltage of 40V for 1.5 hrs. After transfer was complete, the nitrocellulose was removed and placed in a shallow pan with 50 ml of blocking solution [5% (w/v) BSA in PBS]. The nitrocellulose membrane was incubated for 1 hour with agitation, after which the blocking solution was replaced with washing solution [0.1% Tween 20 (v/v) in PBS]. Three washings of 15, 5 and 5 minutes were carried out. The final wash solution was discarded and 25 ⁇ l of primary antibody in 25 ml of PBS was added to the pan. After incubation for 2 hours, with agitation, the nitrocellulose was washed three times (1 ⁇ 15 and 2 ⁇ 5 minutes).
  • the wrapped nitrocellulose was then exposed to X-ray film for an appropriate length of time. After development, the X-ray film was scanned using a laser densitometer and the quantity of globin in each sample estimated by comparison with globin standards run on the same gel.
  • Alpha and beta globins are separated on this gel by molecular weight.
  • Alpha and beta globin were
  • EXAMPLE 20 EXPRESSION OF THE PORTO ALEGRE BETA- GLOBIN IN A YEAST EXPRESSION VECTOR CONTAINING THE
  • the natural beta-globin was modified to obtain a Porto Alegre beta-globin gene by replacing a 104 bp AccI-NcoI fragment from the natural beta-globin gene with a synthetic oligonucleotide containing a cysteine as amino acid 9 (instead of a
  • YEpWB51/PORT was
  • yeast strain Sc340 transformed into yeast strain Sc340, a hem1 strain.
  • RNA for the Porto Alegre beta-globin was around 6.0% of total yeast RNA.
  • Western blot analysis indicated that Porto Alegre beta-globin was expressed.
  • T4-DNA ligase were obtained from New England Biolabs
  • YEp51 is shown in Figure 3B.
  • the plasmid pSP ⁇ C (see Figure
  • the 500 bp DNA fragment carrying the natural beta-globin gene fragment isolated from pSP ⁇ C was Accl compatible at the 5'-end while the 3'-end was HindIII compatible.
  • a synthetic oligonucleotide was used to modify the 5'-end of the isolated fragment.
  • This double stranded oligonucleotide (104 bp) contained a codon for cysteine as amino acid 9 instead of a codon for serine and had a AccI compatible end at its 3'-end and a SalI compatible end at it 5 '-end (see Figure 3B).
  • the 3'-end of the isolated fragment did not receive any adapter as the HindIII site was compatible with the HindIII site introduced into the YEp51.
  • the recipient plasmid YEp51 was cleaved with SalI and HindIII restriction enzymes. To insert the isolated fragment containing the beta-globin gene, a three- way ligation was set up (see Figure 3B). The ligation reaction was carried out using the standard ligation procedures (Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The ligation mixture was transformed into the E. coli HB101 cells using standard transformation procedure. Cells were spread on plates containing LB-media with 100 mg/L ampicillin. Plates were incubated overnight at 37°C. Forty eight colonies from the ampicillin plates were picked and a 5 ml culture was inoculated with individual transformant.
  • plasmid DNA was isolated from 1.5 ml of the overnight culture using the quick alkaline plasmid isolation procedure (Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • the plasmid from each transformant was digested with EcoRI to confirm the presence of a DNA fragment containing the Porto Alegre beta-globin gene.
  • the plasmid carrying the Porto Alegre beta-globin gene was called YEpWB51/PORT.
  • the map of plasmid YEpWB51/Port is shown in Figure 46.

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Abstract

L'invention se rapporte à certaines hémoglobines pour ainsi dire pures, qui contiennent certaines chaînes de globine. La chaîne de globine peut être une chaîne de globine du type alpha ou une chaîne de globine du type bêta, ou des variants de ces chaînes. L'invention décrit en outre un vecteur d'expression qui comprend spécifiquement des séquences d'ADN codant pour une certaine chaîne de globine ou pour un fragment de cette chaîne fixant l'hème, qui est relié activement à un promoteur de levure. L'invention se rapporte également à des procédés pour produire certaines hémoglobines dans des levures. Les hémoglobines pour ainsi dire pures de la présente invention et les hémoglobines produites par des procédés décrits dans la présente invention peuvent être utilisées dans des applications nécessitant des vecteurs d'oxygène physiologiques, comme c'est le cas dans des solutions de substituts du sang ou dans des succédanés du plasma sanguin.
PCT/US1991/008108 1991-10-30 1991-10-30 Expression d'hemoglobine recombinee et de variants d'hemoglobine recombines dans des levures WO1993008831A1 (fr)

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US5554638A (en) * 1993-05-24 1996-09-10 Duke University Methods for improving therapeutic effectiveness of agents for the treatment of solid tumors and other disorders
FR2736930A1 (fr) * 1995-07-17 1997-01-24 Biocem Procede de production, par des cellules vegetales, de proteines heminiques, proteines ainsi obtenues et produits contenant ces proteines
US5665869A (en) * 1993-11-15 1997-09-09 Somatogen, Inc. Method for the rapid removal of protoporphyrin from protoporphyrin IX-containing solutions of hemoglobin
US5840851A (en) * 1993-07-23 1998-11-24 Plomer; J. Jeffrey Purification of hemoglobin
US6022849A (en) * 1987-05-16 2000-02-08 Baxter Biotech Technology Saarl Mutant recombinant hemoglobins containing heme pocket mutations
US6114505A (en) * 1995-10-23 2000-09-05 William Marsh Rice University Hemoglobin mutants that reduce heme loss
US6150506A (en) * 1989-05-10 2000-11-21 Baxter Biotech Technology Sarl Modified hemoglobin-like compounds and methods of purifying same
US6160098A (en) * 1995-11-30 2000-12-12 Baxter Biotech Technology Sarl Method for control of functionality during cross-linking of hemoglobins
US6204009B1 (en) 1988-05-16 2001-03-20 BAXTER BIOTECH TECHNOLOGY SàRL Nucleic acids encoding mutant recombinant hemoglobins containing heme pocket mutations
US6337314B1 (en) * 1997-10-08 2002-01-08 Theragem, Inc. Mammalian-derived peptides for the treatment of microbial infections
US6340667B1 (en) 1997-10-08 2002-01-22 Theragem, Inc. Reptilian-derived peptides for the treatment of microbial infections
US6812207B1 (en) 1995-10-23 2004-11-02 William Marsh Rice University Hemoglobin mutants that reduce heme loss
WO2006073119A1 (fr) * 2005-01-06 2006-07-13 Hiroshima-Ken Polypeptide et utilisation de celui-ci

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EP0402300A2 (fr) * 1989-05-10 1990-12-12 Somatogen Inc. Production d'hémoglobine et de ses analogues par des bactéries ou des levures
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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6022849A (en) * 1987-05-16 2000-02-08 Baxter Biotech Technology Saarl Mutant recombinant hemoglobins containing heme pocket mutations
US6204009B1 (en) 1988-05-16 2001-03-20 BAXTER BIOTECH TECHNOLOGY SàRL Nucleic acids encoding mutant recombinant hemoglobins containing heme pocket mutations
US6150506A (en) * 1989-05-10 2000-11-21 Baxter Biotech Technology Sarl Modified hemoglobin-like compounds and methods of purifying same
US5788958A (en) * 1993-05-24 1998-08-04 Duke University Methods for improving therapeutic effectiveness of agents for the treatment of solid tumors and other disorders
US5554638A (en) * 1993-05-24 1996-09-10 Duke University Methods for improving therapeutic effectiveness of agents for the treatment of solid tumors and other disorders
US6020308A (en) * 1993-05-24 2000-02-01 Duke University Methods for improving therapeutic effectiveness of treatment of vascularization disorders
US5840851A (en) * 1993-07-23 1998-11-24 Plomer; J. Jeffrey Purification of hemoglobin
US5665869A (en) * 1993-11-15 1997-09-09 Somatogen, Inc. Method for the rapid removal of protoporphyrin from protoporphyrin IX-containing solutions of hemoglobin
FR2736930A1 (fr) * 1995-07-17 1997-01-24 Biocem Procede de production, par des cellules vegetales, de proteines heminiques, proteines ainsi obtenues et produits contenant ces proteines
WO1997004115A2 (fr) * 1995-07-17 1997-02-06 Biocem Procede de production, par des cellules vegetales, de proteines heminiques, proteines ainsi obtenues et produits contenant ces proteines
WO1997004115A3 (fr) * 1995-07-17 1997-02-27 Biocem Procede de production, par des cellules vegetales, de proteines heminiques, proteines ainsi obtenues et produits contenant ces proteines
US6344600B1 (en) 1995-07-17 2002-02-05 Meristem Therapeutics Method for producing human hemoglobin proteins using plant cells
US6916787B2 (en) 1995-07-17 2005-07-12 Institut National De La Sante Et De Recherche Medicale Method for producing hemin proteins using plant cells, resulting proteins and products containing same
US6114505A (en) * 1995-10-23 2000-09-05 William Marsh Rice University Hemoglobin mutants that reduce heme loss
US6812207B1 (en) 1995-10-23 2004-11-02 William Marsh Rice University Hemoglobin mutants that reduce heme loss
US6160098A (en) * 1995-11-30 2000-12-12 Baxter Biotech Technology Sarl Method for control of functionality during cross-linking of hemoglobins
US6337314B1 (en) * 1997-10-08 2002-01-08 Theragem, Inc. Mammalian-derived peptides for the treatment of microbial infections
US6340667B1 (en) 1997-10-08 2002-01-22 Theragem, Inc. Reptilian-derived peptides for the treatment of microbial infections
WO2006073119A1 (fr) * 2005-01-06 2006-07-13 Hiroshima-Ken Polypeptide et utilisation de celui-ci
JPWO2006073119A1 (ja) * 2005-01-06 2008-06-12 公立大学法人県立広島大学 ポリペプチドおよびその用途

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