Classifications

• • 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

Definitions

• the subject of the present invention is a method for dissociating the extracellular hemoglobin molecule of Annelids, in particular of Arenicola marina, as well as the characterization of the protein chains constituting said molecule.
• the present invention also relates to the characterization of the nucleotide sequences coding for the above-mentioned protein chains, as well as the process for the preparation of these nucleotide sequences.
• Blood is a complex liquid whose main function is to transport oxygen and carbon dioxide, in order to ensure the respiratory processes. It is the hemoglobin molecule, found in red cells, that performs this function.
• the mammalian hemoglobin molecule is formed by the assembly of four functional polypeptide chains similar in pairs (2 chains of globin of type ⁇ and 2 chains of globin of type ⁇ ). Each of these polypeptide chains has the same tertiary structure of a myoglobin molecule (Dickerson and Geis, 1983).
• the heme the active site of hemoglobin, is a tetrapyrrolic protoporphyrin ring, containing at its center a single iron atom.
• the iron atom which fixes oxygen, establishes 6 coordination bonds: four with the nitrogen atoms of porphyrin, one with the proximal histidine F8 and one with the oxygen molecule during the oxygenation of globin.
• Bovine hemoglobin is slightly different from human hemoglobin immunologically, but it more easily transports oxygen to the tissues. However, the risk of viral or spongiform encephalopathy contamination remains high. To be functional, hemoglobin must be in contact with an allosteric effector, 2,3-diphosphoglycerate (2,3-DPG), present only inside red blood cells (Dickerson and Geis, 1983). In addition, without 2,3-DPG and other elements found in red blood cells such as methemoglobin reductase, hemoglobin undergoes an auto-oxidation process and loses its ability to transport oxygen or carbon dioxide. carbon.
• 2,3-DPG 2,3-diphosphoglycerate
• Annelids have been studied extensively for their extracellular hemoglobin (Terwilliger, 1992; Lamy et al., 1996). These extracellular hemoglobin molecules are present in the three classes of Annelids: Polychetes, Oligochaetes and Achetes and even in Vestimentifers. These are giant biopolymers, made up of around 200 polypeptide chains belonging to 6 or 8 different types which are generally grouped into two categories. The first category, with 144 to 192 elements, groups together the so-called "functional" polypeptide chains carrying an active site and capable of reversibly binding oxygen; these are globin-type chains whose masses are between 15 and 18 kDa and which are very similar to the ⁇ and ⁇ type chains of vertebrates.
• the second category comprising 36 to 42 elements, groups together the so-called "structural" polypeptide chains having little or no active site but allowing the assembly of twelfths.
• French patent n ° 2 809 624 relates to the use as a blood substitute of extracellular hemoglobin 'Arenicola marina, an Annélide Polychrise of the intertidal ecosystem, said blood substitute making it possible to eliminate the problems of shortage of donations.
• the global architecture of the hemoglobin of Arenicola marina is known, notably thanks to its fine quaternary study by mass spectrometry (Zal et al., 1997), the primary sequences of the various protein chains which compose it are not known. .
• the object of the present invention is to provide the protein sequences which make up the hemoglobin molecule 'Arenicola marina. Another object of the present invention is to provide the first steps in the in vitro synthesis of extracellular hemoglobin from Arenicola marina in order to develop a blood substitute using biochemical and molecular biology methods. Another object of the present invention consists in providing a process for the preparation of the hemoglobin molecule, possibly simplified, by genetic engineering, in particular in order to increase the stock of this molecule in the context of use as a blood substitute .
• the present invention relates to a method of dissociation of the extracellular hemoglobin molecule of Annelids, in particular of Arenicola marina, making it possible to obtain the protein chains constituting said molecule, said method being characterized in that it comprises a step of placing in the presence of a sample of extracellular hemoglobin from Annelids, in particular from Arenicola marina with at least one dissociating agent, in particular a mixture consisting of dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) or beta-mercaptoethanol and a dissociation buffer, for a time sufficient to separate the protein chains from each other.
• DTT dithiothreitol
• TCEP (2-carboxyethyl) phosphine hydrochloride
• beta-mercaptoethanol a dissociation buffer
• the present invention relates to a process for obtaining the protein chains constituting the extracellular hemoglobin molecule of Annelids, in particular of Arenicola marina, said process being characterized in that it comprises a step of bringing together a sample of extracellular hemoglobin annelids, including d ⁇ Arenicola marina with at least one dissociating agent, and optionally a reducing agent, in particular a mixture of dithiothreitol (DTT) or tris hydrochloride (2-carboxyethyl) phosphine (TCEP) or beta-mercaptoethanol and a dissociation buffer, for a time sufficient to separate the protein chains from each other.
• a reducing agent in particular a mixture of dithiothreitol (DTT) or tris hydrochloride (2-carboxyethyl) phosphine (TCEP) or beta-mercaptoethanol and a dissociation buffer
• extracellular hemoglobin is meant hemoglobin not contained in the cells and dissolved in the blood.
• the expression “dissociation” designates a chemical treatment capable of breaking down weak interactions (hydrophobic, electrostatic, hydrogen ).
• dissociation buffer is meant a liquid containing a buffer for adjusting the pH and containing dissociating agents.
• dissociating agent designates a chemical compound capable of breaking up weak interactions (hydrophobic, electrostatic, hydrogen ).
• Said dissociating agent is chosen in particular from: hydroxide ions, urea or heteropolytungstate ions or guanidinium or SDS (sodium dodecyl sulfate) salts.
• the expression “reduction” designates a chemical treatment capable of breaking up strong interactions such as disulfide bridges.
• the expression “reducing agent” designates a chemical compound capable of breaking up strong interactions such as disulfide bridges.
• the ten protein chains that make up the extracellular hemoglobin molecule at? Arenicola marina include 8 globin-type chains and 2 structure-type chains.
• the extracellular hemoglobin of Arenicola marina with a mass of 3648 ⁇ 24 kDa consists of 198 polypeptide chains belonging to 10 different types grouped into two categories: - the first (156 chains) includes so-called "functional" polypeptide chains carrying an active site capable of reversibly binding oxygen; these are globin-type chains whose masses are between 15 and 18 kDa; these chains are very similar to the ⁇ and ⁇ type chains of vertebrates; and - The second (42 chains) groups together so-called "structure” (or “linker”) polypeptide chains having little or no active site but allowing assembly of the dodecamers; these chains have molecular weights between 22 and 27 kDa.
• the present invention relates to a method for dissociating the extracellular hemoglobin molecule of Arenicola marina, making it possible to obtain the protein chains constituting said molecule, said method being characterized in that it comprises a step of bringing into contact a sample of extracellular hemoglobin from Arenicola marina with a mixture of dithiothreitol (DTT) and dissociation buffer, for approximately one hour to three weeks.
• DTT dithiothreitol
• the dissociation buffer comprises a buffering agent at a concentration of from approximately 0.05 M to approximately 0.1 M, in particular Trisma (tris [hydroxymethyl] aminomethane) , hepes, sodium phosphate, sodium borate, ammonium bicarbonate or ammonium acetate, and 0-10 mM EDTA adjusted to a pH of about 5 to about 12 , and preferably from approximately 7.5 to 12, the whole being in particular adjusted to a pH of approximately from 2 to 12, and preferably from approximately 5 to 12.
• said dissociation buffer comprises EDTA at a concentration of approximately 1 mM adjusted to a pH of approximately 10, in particular with a 2N sodium hydroxide solution.
• the method of the invention is characterized in that the protein chains constituting said molecule are obtained by the reduction of four subunits by a reducing agent, for example in the presence of DTT, said subunits being themselves obtained by bringing together a sample of extracellular hemoglobin from Arenicola marina with different dissociating agents, in particular a dissociation buffer.
• the native molecule dissociates into subunits under the action of non-reducing dissociating agents. There is therefore no rupture of the covalent bonds.
• the subunits are reduced to polypeptide chains (protein chains made up of the assembly of amino acids).
• the 4 subunits mentioned above are therefore: monomers, dimers, trimers and dodecamers.
• the monomers are globin chains.
• Homer and heterodimer dimers are structural chains.
• the trimers are covalent assemblies of three globin chains.
• the dodecamers are made up of 12 protein chains; for example: 3 trimers + 3 monomers, 2 trimers + 6 monomers, 1 trimer + 9 monomers.
• the protein chains can therefore be obtained either in a single step by direct reduction of the extracellular hemoglobin of Arenicola marina, or in two stages, one consisting in the dissociation of the extracellular hemoglobin of Arenicola marina into 4 sub- units and the other in the reduction of said 4 subunits into protein chains.
• the present invention also relates to a dissociation method as defined above, characterized in that the dissociating agents used to obtain the 4 aforementioned subunits are the following: - a dissociation buffer solution comprising: 0.1 M base Trisma (tris [hydroxymethyl] aminomethane) and 0 to 10 mM EDTA adjusted to a pH of approximately 5 to approximately 12, and preferably approximately 7.5 to approximately 12, and - a urea solution of which the concentration is from about 0.1 M to about 8 M, and is in particular equal to 3 M.
• a dissociation buffer solution comprising: 0.1 M base Trisma (tris [hydroxymethyl] aminomethane) and 0 to 10 mM EDTA adjusted to a pH of approximately 5 to approximately 12, and preferably approximately 7.5 to approximately 12, and - a urea solution of which the concentration is from about 0.1 M to about 8 M, and is in particular equal to 3 M.
• the present invention also relates to a dissociation process as defined above, characterized in that the dissociating agents for obtaining the 4 subunits are as follows: - a dissociation buffer solution comprising 0.1 M of Trisma base and 1 mM of EDTA adjusted to pH 10, and - 3M of urea.
• the present invention also relates to a dissociation and DE reduction process as defined above, characterized in that the dissociating and reducing agents used to obtain the protein chains are the following: - a dissociation buffer solution comprising: 0.1 M base Trisma (tris [hydroxymethyl] aminomethane) at a pH of about 8 to about 9, and - a urea solution whose concentration is from about 4 M to about 8 M, and is in particular equal to 8 M, and - 1 to 10% DTT or - a dissociation buffer solution comprising: 0.1 M ammonium bicarbonate at a pH of approximately 8 to approximately 9, and - 1 to 10% DTT
• a dissociation buffer solution comprising: 0.1 M ammonium bicarbonate at a pH of approximately 8 to approximately 9, and - 1 to 10% DTT
• the present invention also relates to a process for preparing pairs of primers from protein chains as obtained according to the process as defined above, said process being characterized in that it comprises the following stages: isolation of each of the protein chains constituting the hemoglobin molecule as obtained according to the process as defined above, - microsequencing of each of the isolated protein chains mentioned above by mass spectrometry and Edman sequencing, in order to obtain a micro sequence corresponding to each of the sequences consisting of 5 to 20 amino acids, and - the determination of the degenerate primers from the above-mentioned micro sequences.
• the first step of isolating the protein chains is in particular carried out by reverse phase liquid chromatography and two-dimensional gel from the above-mentioned mixture comprising the protein chains constituting the hemoglobin molecule as obtained according to the process of dissociation and reduction of l 'invention.
• the expression “microsequence” designates fragments of protein sequences. The aforementioned microsequences can come from both the C- and N-terminal ends but also from internal sequences.
• the protein chains can be obtained by reverse phase liquid chromatography or from 2D gel from purified hemoglobin from Arenicola marina. Each peak or stain was cut and digested with a protease.
• degenerate primers designates nucleotide sequences obtained from fragments of protein sequences. We are talking about degenerate primers due to the degeneracy of the genetic code (several codons for 1 amino acid). The last step in determining degenerate primer pairs corresponds to their synthesis. This step makes it possible to obtain both sense and antisense primers.
• the present invention also relates to pairs of primers as obtained according to the method as defined above, said couples being in particular the following: a) Meaning primer: GAR TGY GGN CCN TTR CAR CG SEQ ID NO: 21) Primer antisense CTC CTC TCC TCT CCT CTT CCT SEQ ID NO: 22) b) Sense primer: TGY GGN ATH CTN CAR CG SEQ ID NO: 23) Antisense primer CTC CTC CTC TCC TCT CCT CTT CCT SEQ ID NO: 22) c) Sense primer : AAR GTI AAR CAN AAC TGG SEQ ID NO: 24) Antisense primer CTC CTC TCC TCT CCT CTT CCT SEQ ID NO: 22) d) Sense primer: TGY TGY AGY ATH GAR GAY CG SEQ ID NO: 25) Antisense primer CTC CTC TCC CT CCT CTT CCT SEQ ID NO: 22) e) Sense primer: AAR GTN
• the present invention also relates to pairs of primers such as obtained according to the method as defined above, said pairs being in particular the following: a) Meaning primer: GAR TGY GGN CCN TTR CAR CG SEQ ID NO: 21 Antisense primer CCA NGC NTC YTT RTC RAA GCA SEQ ID NO: 28 b) Meaning primer: AN TGY GGN CCN CTN CAR CG SEQ ID NO: 29 Antisense primer CCA NGC NTC YTT RTC RAA GCA SEQ ID NO: 28 c) Sense primer: AAR GTI AAR CAN AAC TGG SEQ ID NO: 24 Antisense primer CCA NGC NCC DAT RTC RAA SEQ ID NO: 30 d) Sense primer: TGY TGY AGY ATH GAR GAY CG SEQ ID NO: 25 Antisense primer: CA NGC NYC RCT RTT RAA RCA SEQ ID NO: 31 where: R represents A or G, Y represents C or T, N represents A, G,
• the present invention relates also a process for the preparation of nucleotide sequences coding for the protein chains constituting the hemoglobin molecule of Arenicola marina, from the primers as obtained according to the process as defined above, said process being characterized in that it corresponds to a polymerase chain reaction (PCR) process, comprising a repetition of at least 30 times the cycle consisting of the following steps: * denaturation, by heating, of the single-stranded cDNA coding for one of the chains proteins constituting the hemoglobin molecule of Arenicola marina, so as to denature possible secondary structures and RNA remainders, said cDNA being obtained from mRNA, this step making it possible to obtain strands of cDNA denatured single-stranded strands, * hybridization of the pairs of primers as obtained by the process as defined above with the above-mentioned denatured single-stranded cDNA strands at an adequate temperature, to obtain hybridized primers, and * synthesizing the strand complementary to the c
• the cDNA encoding the aforementioned protein chains is obtained from mRNA, said mRNA being obtained by purification from total RNA extracted from growing juvenile arenicoles, said juvenile arenicoles having a high rate of transcription of the various RNAs messengers. If the above cycle is repeated less than 30 times, DNA amplification is reduced.
• the expression "possible secondary structures” designates anarchic pairings between two cDNA sequences.
• the expression “denaturation by heating of the cDNA” designates the disruption of anarchic pairings between two sequences of cDNA.
• hybridization at an adequate temperature designates the recognition by the primers of their complementary sequences on the template DNA.
• the method for preparing nucleotide sequences according to the invention is characterized in that: - the first step of said method is a step of denaturing the cDNA coding for one of the protein chains constituting the molecule of arenicola marina hemoglobin from approximately 10 seconds to approximately 5 minutes at a temperature of approximately 90 ° C to approximately 110 ° C, - the cycle, repeated approximately 30 to 40 times, comprises the following stages: * a stage of denaturation of the cDNA coding for one of the protein chains constituting the hemoglobin molecule of Arenicola marina from approximately 10 seconds to approximately 5 minutes, at a temperature ranging from approximately 90 ° C to approximately 110 ° C, * a step of hybridization of the pairs of primers of the invention to the aforementioned single-stranded cDNA strands in order to obtain hybridized primers, from approximately 20 seconds to approximately 2 minutes, at a temperature of approximately 50 ° C.
• a step of elongation of the primers hybridized as previously obtained by a polymerase from approximately 20 seconds to approximately 1 minute and 30 seconds, at a temperature ranging from approximately 70 ° C. to approximately 75 ° C.
• - the last stage of the process is a stage of elongation of the primers hybridized as previously obtained by a polymerase from approximately 5 minutes to approximately 15 minutes at a temperature ranging from about 70 ° C to about 75 ° C.
• the PCR reaction of the process of the invention is carried out in particular in the presence of cDNA (5 to 20 ng), sense primer (100 ng) and antisense (100 ng), dNTP (200 ⁇ M final), MgCl 2 (2 mM final), PCR buffer (supplied with the polymerase) (1 X final), Taq polymerase (1 unit) and water (25 ⁇ l qsp).
• the present invention also relates to a preparation process as defined above, characterized in that the pairs of primers used are as defined above.
• a particularly advantageous preparation method according to the invention is a method for preparing nucleotide sequences as defined above, characterized in that the pair of primers used is: (GAR TGY GGN CCN TTR CAR CG; CCA NGC
• the method being characterized in that: - the first step of the method is a step of denaturing the cDNA coding for the protein chains constituting the molecule d arenicola marina hemoglobin, 4 minutes at a temperature equal to 95 ° C, - the cycle, repeated 35 times, comprises the following stages: * a stage of denaturation of the cDNA coding for one of the protein chains constituting the molecule of arenicola marina hemoglobin, for 30 seconds at a temperature equal to 95 ° C., * a step of hybridization of the pairs of primers of the invention to the strands of single-stranded cDNA mentioned above to obtain hybridized primers, of 30 seconds at a temperature equal to 56 ° C, * a step of elongation of the hybridized primers as previously obtained by a polymerase of 40 seconds at a temperature equal to 72 ° C, and - the last step of the process is a step elongation of
• the partial sequence SEQ ID NO: 13 was then completed by 5 'RACE PCR experiments as explained above.
• the nucleotide sequence SEQ ID NO: 13 is a new nucleotide sequence coding for a protein chain corresponding to a globin chain, denoted A2a.
• SEQ ID NO: 13 includes 376 nucleotides.
• the nucleotide sequence SEQ ID NO: 1 (from the initiator codon to the stop codon, ie the transcribed and translated sequence which corresponds to a functional globin monomer) is a new nucleotide sequence coding for a protein chain corresponding to the chain of aforementioned globin, denoted A2a.
• SEQ ID NO: 1 includes 474 nucleotides.
• a particularly advantageous preparation method is a method for preparing nucleotide sequences as defined above, characterized in that the pair of primers used is the following: (AN TGY GGN CCN CTN CAR CG; CCA NGC NTC YTT RTC RAA GCA), N, Y and R being as defined above, said method being characterized in that: - the first step of the process is a step of denaturing the cDNA coding for the protein chains constituting the hemoglobin molecule of Arenicola marina, 4 minutes at a temperature equal to 95 ° C, - the cycle, repeated 35 times, comprises the following stages: * a stage of denaturation of the cDNA coding for one of the protein chains constituting the hemoglobin molecule of Arenicola marina, for 30 seconds at a temperature equal to 95 ° C., * a step of hybridization of the pairs of primers of the invention with the strands of single-stranded cDNA mentioned above to obtain primers hybridized, 30 seconds at a
• the nucleotide sequence SEQ ID NO: 15 is a new nucleotide sequence coding for a protein chain corresponding to a globin chain, denoted A2b.
• SEQ ID NO: 15 includes 288 nucleotides.
• a particularly advantageous preparation method according to the invention is a method for preparing nucleotide sequences as defined above, characterized in that the pair of primers used is the following: (TGY GGN ATH CTN CAR CG; CTC CTC TCC TCT CCT CTT CCT), N, Y and R being as defined above, said process being characterized in that: - the first step of the process is a denaturation step of 4 minutes at a temperature equal to 95 ° C, - the cycle, repeated 35 times, includes the following steps: * a denaturation step of 30 seconds at a temperature equal to 95 ° C, * a hybridization step of 30 seconds at a temperature equal to 53 ° C, * a step of elongation of 40 seconds at a
• the partial sequence SEQ ID NO: 15 was then completed by experiments 5' RACE PCR as explained above.
• the nucleotide sequence SEQ ID NO: 3 (from the initiating codon to the stop codon, ie the transcribed and translated sequence which corresponds to a functional globin monomer) is a new nucleotide sequence coding for a protein chain corresponding to the chain of aforementioned globin, denoted A2b.
• SEQ ID NO: 3 comprises 477 nucleotides.
• a particularly advantageous preparation method according to the invention is a method for preparing nucleotide sequences as defined above, characterized in that the pair of primers used is: (AAR GTI AAR CAN AAC TGG; CCA NGC
• the first step of the process is a step of denaturing the cDNA coding for each of the protein chains constituting the hemoglobin molecule of Arenicola marina, 4 minutes at a temperature equal to 95 ° C.
• the cycle, repeated 35 times includes the following steps: * a step of denaturing the cDNA coding for one of the protein chains constituting the hemoglobin molecule of Arenicola marina, from 1 minute at a temperature equal to 95 ° C., * a step of hybridization of the pairs of primers of the invention to the aforementioned single-stranded cDNA strands to obtain hybridized primers, from 1 minute at a temperature equal to 50 ° C., * a step of elongation of the hybridized primers as obtained previously by a polymerase of 1 minute and 30 seconds at a temperature equal to 72 ° C, and - the last step of the process is a step of e
• the partial sequence SEQ ID NO: 17 was then supplemented with 5 'RACE PCR experiments as explained above.
• the nucleotide sequence SEQ ID NO: 17 is a new nucleotide sequence coding for a protein chain corresponding to a globin chain, noted A1.
• SEQ ID NO: 17 comprises 360 nucleotides.
• the nucleotide sequence SEQ ID NO: 5 (from the initiating codon to the stop codon, ie the transcribed and translated sequence which corresponds to a functional globin monomer) is a new nucleotide sequence coding for a protein chain corresponding to the chain of aforementioned globin, noted Al.
• SEQ ID NO: 5 comprises 474 nucleotides.
• a particularly advantageous preparation process according to the invention is a process for the preparation of nucleotide sequences as defined above, characterized in that the pair of primers used is the following: (TGY TGY AGY ATH GAR GAY CG; CA NGC NYC RCT RTT RAA RCA) OR (TGY TGY AGY ATH GAR GAY CG; CTC CTC TCC
• the first step of the process is a step of denaturation of the cDNA coding for the protein chains constituting the hemoglobin molecule of Arenicola marina, 4 minutes at a temperature equal to 95 ° C
• - the cycle, repeated 35 times comprises the following stages: * a stage of denaturation of the cDNA coding for one of the protein chains constituting the hemoglobin molecule of Arenicola marina, 30 seconds at a temperature equal to 95 ° C, * a step of hybridization of the pairs of primers of the invention to the strands of single-stranded cDNA mentioned above to obtain hybridized primers , from 40 seconds at a temperature equal to 52 ° C, * a step of elongation of the hybridized primers as previously obtained by a polymerase of 30 seconds at a temperature equal to 72 ° C, and - the last step of the process is a elong
• the partial sequence SEQ ID NO: 19 was then completed by 5 'RACE PCR experiments as explained above.
• the nucleotide sequence SEQ ID NO: 19 is a new nucleotide sequence coding for a protein chain corresponding to a globin chain, denoted B2.
• SEQ ID NO: 19 includes 390 nucleotides.
• the nucleotide sequence SEQ ID NO: 7 (from the initiating codon to the stop codon, ie the transcribed and translated sequence which corresponds to a functional globin monomer) is a new nucleotide sequence coding for a protein chain corresponding to the chain of aforementioned globin, denoted B2.
• SEQ ID NO: 7 includes 498 nucleotides.
• a particularly advantageous preparation process is a process for the preparation of nucleotide sequences as defined above, characterized in that the pair of primers used is the following: (AAR GTN ATH TTY GGN AGR GA, - CTC CTC TCC TCT CCT CTT CCT), R, H, N and Y being as defined above, said process being characterized in that: - the first step of the process is a denaturation step of 4 minutes at a temperature equal to 95 ° C, - the cycle, repeated 35 times, includes the following steps: * a denaturation step of 30 seconds at a temperature equal to 95 ° C, * a hybridization step of 40 seconds at a temperature equal to 52 ° C, * an elongation step of 30 seconds at a temperature equal to 72 ° C, and - the last step of the process is an elongation step of 10 minutes at a temperature equal to 72 ° C, in order to obtain a partial reference nucleotide sequence in order to continue the complete determination of this coding
• SEQ ID NO: 9 (from the initiating codon to the stop codon, c (ie the transcribed and translated sequence which corresponds to a functional globin monomer) is a new nucleotide sequence coding for a protein chain corresponding to a globin chain, denoted B1.
• SEQ ID NO: 9 comprises 498 nucleotides.
• a particularly advantageous preparation process is a process for the preparation of nucleotide sequences as defined above, characterized in that the pair of primers used is the following: (GAR CAY CAR TGY GGN GGN GA, CTC CTC TCC TCT CCT CTT CCT), R, N and Y being as defined above, said process being characterized in that: - the first step of the process is a denaturation step of 4 minutes at a temperature equal to 95 ° C.
• the cycle, repeated 35 times includes the following steps: * a denaturation step of 40 seconds at a temperature equal to 95 ° C, * a hybridization step of 1 minute at a temperature equal to 58 ° C, * an elongation step of 1 minute and 10 seconds at a temperature equal to 72 ° C, and - the last step of the process is an elongation step of 10 minutes at a temperature equal to 72 ° C, in order to obtain a partial reference nucleotide sequence to continue the complete determination of this coding sequence, said method comprising an additional 5 ′ RACE PCR step to obtain the nucleotide sequence SEQ ID NO: 11.
• the nucleotide sequence SEQ ID NO: 11 (from the initiating codon at the stop codon, ie the transcribed and translated sequence which corresponds to a functional globin monomer) is a new nucleotide sequence coding for a protein chain corresponding to a linker chain, denoted L1.
• SEQ ID NO: It comprises 771 nucleotides.
• the present invention also relates to protein sequences encoded by one of the nucleotide sequences as obtained according to the method as defined above.
• a preferred protein according to the invention is a protein as defined above, characterized in that it comprises or consists of: - the sequence SEQ ID NO: 2 or SEQ ID NO: 14, - or any sequence derived from the sequence SEQ ID NO: 2 or SEQ ID NO: 14 or of a fragment defined below, in particular by substitution, deletion or addition of one or more amino acids, provided that said derived sequence allows the transport of oxygen, - or any sequence homologous to the sequence SEQ ID NO: 2 or SEQ D NO: 14 or of a fragment defined below, preferably having a homology of at least approximately 75%, in particular of at least approximately 85%, with the sequence SEQ ID NO: 2 or SEQ ID NO: 14, provided that said homologous sequence allows the transport of oxygen, - or any fragment of one of the sequences defined above, provided that said fragment allows the transport of oxygen, in particular any fragment being constituted é of at least about 60 amino acids, and in particular at least about 160 contiguous amino acids in the sequence SEQ ID NO: 2.
• the sequence SEQ ID NO: 2 is a new protein sequence corresponding to an entire chain of globin, denoted A2a.
• the sequence SEQ ID NO: 14 is a new protein sequence corresponding to a fragment of a sequence derived from the globin chain, denoted A2a, represented by the sequence SEQ ID NO: 2.
• the oxygen transport properties of the sequences proteins of the invention can be verified in particular by measuring their absorption spectrum by spectrophotometry typical of oxyhemoglobin.
• a preferred protein according to the invention is a protein as defined above, characterized in that it comprises or consists of: - the sequence SEQ ID NO: 4 or SEQ ID NO: 16, - or any sequence derived from the sequence SEQ ID NO: 4 or SEQ ID NO: 16, or of a fragment defined below, in particular by substitution, deletion or addition of one or more amino acids, provided that said derived sequence allows transport oxygen, - or any sequence homologous to the sequence SEQ ID NO: 4 or SEQ ID NO: 16, or of a fragment defined below, preferably having a homology of at least about 75%, in particular d '' at least about 85%, with the sequence SEQ ID NO: 4 or SEQ ID NO: 16, provided that said homologous sequence allows the transport of oxygen, - or any fragment of one of the sequences defined above, provided that said fragment allows the transport of oxygen, in particular any fragmen t consisting of at least about 60 amino acids, and in particular at least about 160 contiguous amino acids in the sequence SEQ ID NO: 4.
• sequence SEQ ID NO: 4 is a new protein sequence corresponding to an entire chain of globin, denoted A2b.
• sequence SEQ ID NO: 16 is a new protein sequence corresponding to a fragment of a sequence derived from the globin chain, denoted A2b, represented by the sequence SEQ ID NO: 4.
• a preferred protein according to the invention is a protein as defined above, characterized in that it comprises or consists of: - the sequence SEQ ID NO: 6 or SEQ ID NO: 18, - or any sequence derived from the sequence SEQ ID NO: 6 or SEQ ID NO: 18 or of a fragment defined below, in particular by substitution, deletion or addition of one or more amino acids, provided that the said derived sequence allows the transport of oxygen, - or any sequence homologous to the sequence SEQ ID NO: 6 or SEQ ID NO: 18 or of a fragment defined below, preferably having a homology of at least approximately 75%, in particular of at least approximately 85% , with the sequence SEQ ID NO: 6 or SEQ ID NO: 18, provided that said homologous sequence allows the transport of oxygen, - or any fragment of one of the sequences defined above, provided that said fragment allows the transport of oxygen, in particular any fragment consisting of at least approximately 60 amino acids, and in particular of at least approximately 160 contiguous amino acids in the sequence SEQ ID NO: 6.
• sequence SEQ ID NO: 6 is a new protein sequence corresponding to an entire globin chain, noted Al.
• sequence SEQ ID NO: 18 is a new protein sequence corresponding to a fragment of a sequence derived from the globin chain, denoted A1, represented by the sequence SEQ ID NO: 6.
• a preferred protein according to the invention is a protein as defined above, characterized in that it comprises or consists of: - the sequence SEQ ID NO: 8 or SEQ ID NO: 20, - or any sequence derived from the sequence SEQ ID NO: 8 or SEQ ID NO: 20 or of a fragment defined below, in particular by substitution, deletion or addition of one or more amino acids, provided that said derived sequence allows oxygen transport, - or any sequence homologous to the sequence SEQ ID NO: 8 or SEQ ID NO: 20 or of a fragment defined below, preferably having a homology of at least about 75%, in particular at least about 85%, with the sequence SEQ ID NO: 8 or SEQ ID NO: 20, provided that said homologous sequence pe rmits the transport of oxygen, - or any fragment of one of the sequences defined above, provided that said fragment allows the transport of oxygen, in particular any fragment consisting of at least about 60 amino acids, and in particular at least about 160 contiguous amino acids in the sequence SEQ ID NO: 8.
• sequence SEQ ID NO: 8 is a new protein sequence corresponding to an entire chain of globins, denoted B2.
• sequence SEQ ID NO: 20 is a new protein sequence corresponding to a fragment of a sequence derived from the globin chain, denoted B2, represented by the sequence SEQ ID NO: 8.
• a preferred protein according to the invention is a protein as defined above, characterized in that it comprises or consists of: - the sequence SEQ ID NO: 10, - or any sequence derived from the sequence SEQ ID NO: 10 or of a fragment defined below , in particular by substitution, deletion or addition of one or more amino acids, provided that said derived sequence allows the transport of oxygen, - or any sequence homologous to the sequence SEQ ID NO: 10 or of a fragment defined here below, preferably having a homology of at least about 75%, with the sequence SEQ ID NO: 10, provided that said homologous sequence allows the transport of oxygen, - or any fragment of one of the sequences defined above above, under r reserves that said fragment allows the transport of oxygen, in particular any fragment consisting of at least approximately 60 amino acids, and in particular of at least approximately 160 contiguous amino acids in the sequence SEQ ID NO: 10.
• the sequence SEQ ID NO: 10 is a new protein sequence corresponding to a globin chain, denoted B1.
• a preferred protein according to the invention is a protein as defined above, characterized in that it comprises or consists of: - the sequence SEQ ID NO: 12, - or any sequence derived from the sequence SEQ ID NO: 12 or a fragment defined below, in particular by substitution, deletion or addition of one or more amino acids, provided that said sequence derived allows the association of globin chains with each other, - or any sequence homologous to the sequence SEQ ID NO: 12 or of a fragment defined below, preferably having a homology of at least about 75%, with the sequence SEQ ID NO: 12, provided that said homologous sequence allows the association of globin chains with one another, - or any fragment of one of the sequences defined above, provided that said fragment allows the association of chains of globins together, especially everything fragment consisting of at least about 60 amino acids, and in particular at least about 280 contiguous amino acids in the sequence SEQ ID NO:
• sequence SEQ ID NO: 12 is a new protein sequence corresponding to a Linker chain , noted Ll.
• the present invention also relates to nucleotide sequences as obtained according to the method as defined above.
• the present invention also relates to nucleotide sequences coding for a protein as defined above.
• the present invention also relates to a nucleotide sequence as defined above, characterized in that it comprises or consists of: - the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 13 coding respectively for SEQ ID NO: 2 or SEQ ID NO: 14, - or any nucleotide sequence derived, by degeneration of the genetic code, from the sequence SEQ ID NO: 1 or SEQ ID NO: 13, and coding respectively for a protein represented by SEQ ID NO: 2 or SEQ ID NO: 14, - or any nucleotide sequence derived, in particular by substitution, deletion or addition of one or more nucleotides, of the sequence SEQ ID NO: 1 or SEQ ID NO: 13 coding for a protein respectively derived from SEQ ID NO : 2 or SEQ ID NO: 14, - or any nucleotide sequence homologous to SEQ ID NO: 1 or SEQ ID NO: 13, preferably having a homology of at least about 60%, with the sequence SEQ ID NO: 1, - or any fragment
• the stringency conditions correspond to ranges of temperatures between 48 and 60 ° C. and of MgCl 2 concentrations of between 1 and 3 mM.
• the present invention also relates to a nucleotide sequence as defined above, characterized in that it comprises or consists of: - the nucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 15 coding respectively for SEQ ID NO: 4 or SEQ ID NO: 16, - or any nucleotide sequence derived, by degeneration of the genetic code, from the sequence SEQ ID NO: 3 or SEQ ID NO: 15, and coding respectively for a protein represented by SEQ ID NO: 4 or SEQ ID NO: 16, - or any nucleotide sequence derived, in particular by substitution, deletion or addition of one or more nucleotides, of the sequence SEQ ID NO: 3 or SEQ ID NO: 15 coding respectively for a protein derived from SEQ ID NO : 4 or SEQ ID NO: 16, - or any nucleotide sequence homologous to SEQ
• the present invention also relates to a nucleotide sequence as defined above, characterized in that it comprises or consists of: - the nucleotide sequence SEQ ID NO: 5 or SEQ ID NO: 17 coding respectively for SEQ H) NO: 6 or SEQ ID NO: 18, or any nucleotide sequence derived, by degeneration of the genetic code, from the sequence SEQ ID NO: 5 or SEQ ID NO: 17, and coding respectively for a protein represented by SEQ ID NO: 6 or SEQ ID NO: 18, - or any nucleotide sequence derived, in particular by substitution, deletion or addition of one or more nucleotides, from the sequence SEQ ED NO: 5 or SEQ ID NO: 17 coding respectively for a protein derived from SEQ ID NO: 6 or SEQ ID NO: 18, - or any nucleotide sequence homologous to SEQ ID NO: 5 or SEQ ID NO: 17, preferably having a homology d '' at least about 60%, with the sequence SEQ ID NO: 5 or S
• the present invention also relates to a nucleotide sequence as defined above, characterized in that it comprises or consists of: - the nucleotide sequence SEQ ED NO: 7 or SEQ ID NO: 19 coding respectively for SEQ ID NO: 8 or SEQ ED NO: 20, - or any nucleotide sequence derived, by degeneration of the genetic code, from the sequence SEQ ED NO: 7 or SEQ ID NO: 19, and coding respectively for a protein represented by SEQ ED NO: 8 or SEQ ID NO: 20, - or any nucleotide sequence derived, in particular by substitution, deletion or addition of one or more nucleotides, of the sequence SEQ ID NO: 7 or SEQ ED NO: 19 coding respectively for a protein derived from SEQ ID NO : 8 or SEQ ID NO: 20, - or any nucleotide sequence homologous to SEQ ID NO: 7 or SEQ ED NO: 19, preferably having a homology of at least about 60%, with the sequence SEQ ID NO:
• the present invention also relates to a nucleotide sequence as defined above, characterized in that it comprises or consists of: - the nucleotide sequence SEQ ID NO: 9 coding for SEQ ED NO: 10, - or any nucleotide sequence derived , by degeneration of the genetic code, of the sequence SEQ ID NO: 9, and coding for a protein represented by SEQ ID NO: 10, - or any derived nucleotide sequence, in particular by substitution, deletion or addition of one or more nucleotides, of the sequence SEQ ID NO: 9 coding for a protein derived from SEQ ID NO: 10, - or any nucleotide sequence homologous to SEQ ED NO: 9, preferably having a homology of at least about 60%, with the sequence SEQ ED NO: 9, - or any fragment of the nucleotide sequence SEQ ED NO: 9 or of the nucleotide sequences defined above, said fragment preferably consisting of at least about 180 nucleotides, e t
• the present invention also relates to a nucleotide sequence as defined above, characterized in that it comprises or consists of: - the nucleotide sequence SEQ ED NO: 11 coding for SEQ ID NO: 12, - or any nucleotide sequence derived , by degeneration of the genetic code, of the sequence SEQ ID NO: 11, and coding for a protein represented by SEQ ID NO: 12, - or any nucleotide sequence derived, in particular by substitution, deletion or addition of one or more nucleotides, of the sequence SEQ ID NO: 11 coding for a protein derived from SEQ ED NO: 12, - or any nucleotide sequence homologous to SEQ ID NO: 11, preferably having a homology of at least about 60%, with the sequence SEQ ED NO: 11, - or any fragment of the nucleotide sequence SEQ ID NO: 11 or of the nucleotide sequences defined above, said fragment preferably consisting of at least approximately 180 nucleotides, and in particular at least
• the present invention relates to a process for the preparation as defined above, of nucleotide sequences coding for the protein chains constituting the hemoglobin molecule of Annelids, in particular of Arenicola marina, said process being characterized in that it comprises following steps: a step of bringing the above-mentioned hemoglobin molecule into contact with at least one dissociating agent and one reducing agent, in particular a mixture consisting of dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) ) or beta-mercaptoethanol and a dissociation buffer, for a time sufficient to separate the protein chains from one another, allowing the dissociation and then the reduction of said hemoglobin molecule, in order to obtain the protein chains constituting said molecule, - the isolation of the aforementioned protein chains, - microsequencing by mass spectrometry and the sequencing ge of Edman of each of the above-mentioned isolated protein chains, in order
• Figure 1 represents a chromatogram of the hemoglobin of Arenicola marina on a Superose 12-C column.
• the upper curve corresponds to an absorbance of 414 nm and the lower curve to an absorbance of 280 nm. (The collector is programmed to collect between 16 and 18 minutes).
• Figure 2 shows the UV spectrum of functional Arenicola marina hemoglobin (in its oxyhemoglobin form).
• Figure 3 represents the chromatogram (at 414 nm) of the (partially) dissociated HbAm obtained on Superose 12-C and the vertical lines on the chromatogram correspond to the collection windows (corresponding to the recovery of the subunits).
• Figure 4 shows an SDS-PAGE gel obtained for the different fractions collected.
• FIG. 5 represents the chromatogram (at 414 nm) of the (partially) dissociated HbAm obtained on CIM DISK DEAE (anion exchange system) and the vertical lines on the chromatogram correspond to the collection windows.
• the dotted curve indicates the gradient.
• Figure 6 shows an SDS-PAGE gel obtained for the different fractions collected.
• Figure 7 shows the dissociation kinetics of HbAm in the presence of 3M urea.
• the abscissa axis corresponds to the number of days and the ordinate axis corresponds to the percentage of dissociation of the native molecule; the dotted curve corresponds to the dodecamer; the curve with the black squares in the trimer and in the "linker" (structure chain); the curve with the black circles with monomers.
• Figure 8 represents the dissociation kinetics of FfbAm at pH 10.
• the abscissa axis corresponds to the number of days and the ordinate axis corresponds to the percentage of dissociation of the native molecule; the dotted curve corresponds to the dodecamer; the curve with the black canes in the trimer and the "linker"; the curve with the black circles with monomers.
• Figure 9 represents the dissociation kinetics of HbAm in the presence of 3M urea at pH 10.
• the abscissa axis corresponds to the number of days and the ordinate axis corresponds to the percentage of dissociation of the native molecule; the dotted curve corresponds to the dodecamer; the curve with the black squares in the trimer and in the "linker"; the curve with the black circles with monomers.
• Figure 10 shows the monitoring of the reassociation kinetics from the percentage of HbAm (HBL) and Dodecamer (D) and according to the buffer change technique (Centricon or Dialysis).
• the abscissa axis corresponds to the number of days and the ordinate axis corresponds to the percentage of dissociation of the native molecule with the Centricon technique;
• the curve in dotted lines corresponds to HBL with the dialysis technique;
• the curve with the black triangles corresponds to the dodecamer with the Centricon technique;
• the solid line curve corresponds to the dodecamer with the dialysis technique.
• Figure 11 represents the superimposition of the exclusion chromatography chromatograms during reassociation corresponding to different reassociation times.
• Figure 12 shows the HPLC chromatogram obtained after separation of the polypeptide chains by reverse phase on a Symmetry Cl 8 column (Waters).
• the codes (d2, al, a2, b2, c) correspond to the names of the globins as mentioned in the article by Zal et al. (1997).
• the present invention aims to use the extracellular hemoglobin of the marine polychaete Arenicola marina (HbAm) as a blood substitute in vertebrates.
• HbAm marine polychaete Arenicola marina
• synthesis by genetic engineering proves to be an indispensable and necessary path. It is therefore essential to obtain the primary sequences of the protein chains constituting HbAm in order to develop an artificial, functional and stable hemoglobin from the self-assembly properties of this molecule.
• the Aroucole is housed in the horizontal part, its cephalic end oriented towards the blind part. It ingests the sand, extracts the assimilable organic matter from it and then defecates through its caudal end, thus forming mounds of sand twists. Within the mediolittoral stage, the distribution and density of the stands are essentially controlled by the grain size, the concentration of organic matter and the salinity.
• the Aroucole living especially in the areas of swinging of the tides, is brought to undergo variations of oxygen pressure. Its gallery allows it to be in permanent contact with sea water (rich in oxygen), during low tide. 2) Study methods 2.1.
• a low pressure filtration (FPLC) by exclusion is carried out on a column (100 x 3 cm) of Sephacryl S-400 gel (Amersham) (separation range between 20 x 10 3 and 8000 x 10 3 ), in a cold room (4 ° C).
• FPLC low pressure filtration
• Each purification is carried out on 5 ml of sample, eluted with the Arenicola marina salt buffer (10 mM Hepes; 4 mM KC1; 145 mM NaCl; 0.2 mM MgCl 2 adjusted to pH 7.0 with 2N sodium hydroxide).
• the flow rate used for this first purification is 40 rpm and is recovered only the first reddest fraction (containing heme). This fraction is then concentrated using a CentricondOkDa tube retaining the molecules with a weight greater than or equal to 10,000 Da.
• a second purification is then carried out by low pressure filtration (HPLC system, Waters) of 200 ⁇ L aliquot on a column 1 x 30 cm Superose 12-C (Pharmacia, separation range between 5 x 10 3 and 3 x 10 5 Da) at room temperature. The flow rate used is 0.5 ml / min. The samples are stored at 4 ° C and collected in ice.
• the eluate absorbance is monitored at two wavelengths: 280 nm (peak absorbance characteristic of proteins) and 414 nm (peak absorbance characteristic of Theme).
• the fractions containing Theme are isolated using the collector (programmed on a time window corresponding to the retention time of Themoglobin) ( Figure 1).
• the samples are concentrated, assayed, then stored at -40 ° C before use.
• 2.3. Determination of hemoglobins The Drabkin reagent (Sigma), used for the determination, makes it possible to determine the quantity in heme of the solution. Hemoglobin reacts with Drabkin's reagent which contains potassium ferricyanide, potassium cyanide and sodium bicarbonate.
• Hemoglobin is transformed into methemoglobin by the action of ferricyanide.
• the methemoglobins then react with cyanide to form cyanmethemoglobin.
• the absorbance of this derivative at 540 nm is proportional to the amount of heme in the solution.
• the extracellular hemoglobin of Arenicola marina (HBL) contains on average 1 mol of heme for 23,000 g of protein, which allows by simple calculation to obtain the HBL concentration of each sample.
• urea in the presence of a single dissociating reagent: urea, heteropolytungstate ions, salts of guanidinium, SDS or hydroxide ions.
• the agents used act differently on the molecule: - hydroxide ions (OH " ), SDS, guanidinium salts and heteropolytungstate ions destabilize the salt bridges - urea destabilizes hydrophobic interactions Or, the interest is to obtain as quickly and as efficiently as possible the four basic subunits, hence the idea of combining the different dissociating agents and in particular the alkaline pH and urea.
• Ion exchange analyzes The isoelectric point (pHi) of THBL being 4.69 (Vinogradov, 1985), the analysis by ion exchange is carried out on an anionic column CEVI DEAE disk (Interchim). Indeed, HBL is negatively charged for a pH higher than pHi and is therefore fixed on a positively charged resin (DEAE resin). Elution is carried out using ionic strength with a non-linear NaCl gradient from 0 to 1 M (1 M NaCl solution diluted in the dissociation buffer at pH 7.0 and filtered through 0.45 ⁇ m). The dissociation buffer at pH 7 is used as an elution buffer. The flow rate is 4 mL / min.
• trimers must be too close to allow them to be separated by reverse phase. Thus, only the two linkers and the monomers a1 and a2 could be isolated.
• Hemoglobin reassociation 1 Materials and methods The reassociation experiments are carried out on dissociated THbAm according to the protocols cited above ( ⁇ H9, pH10, 3M urea, 4M urea, 3M urea at pH10). Different reassociation buffers are tested in order to obtain optimal reassociation.
• the change of buffer (dissociation buffer ⁇ reassociation buffer) is carried out in two different ways: - The dissociated HbAm is washed 4 times against 4 ml of reassociation buffer on Centricon- 10 (Millipore) at + 4 ° C; - The dissociated HbAm is dialyzed 24 h against MilliQ water (Millipore) (2 2L) then 48 h against the reassociation buffer (3 2L) at + 4 ° C.
• the buffer currently developed consists of 0.1 M Trisma base, 400 mM NaCl, 2.95 mM KCl, 32 mM MgSO 4 , 11 mM CaCl 2 adjusted to pH 7 with concentrated THCl.
• the reassociation is followed according to the same principle as the dissociation ( Figures 10 and 11). A reassociation is observed if the dissociation is of short duration of the order of one minute. This reassociation corresponds to a rearrangement of dissociation intermediaries which are truncated hemoglobins (HBL dissociated from 1 to several twelfths).
• HbAm Reduction of HbAm for the study of different polypeptide chains
• the following protocol can also optionally be envisaged:
• the HbAm (4 mg / ml) is reduced in 100 mM DTT (dithiothreitol) dissolved in the dissociation buffer at pH 8-9 for 1 h at 40 ° C.
• DTT dithiothreitol
• THbAm is washed 4 times on Amicon 30 OOODa (Millipore) of which only the filtrate is recovered (all that is less than 30,000 Da in weight).
• the filtrate is then washed on Amicon 10 OOODa to remove anything less than 10,000 Da.
• Amicon 10 OOODa to remove anything less than 10,000 Da.
• only the monomers between 30,000 Da and 10,000 Da are contained in the sample (range of weights of the globin chains which constitute THbAm).
• Each protein chain (highlighted by a single peak at 280nm) is collected then lyophilized and stored at -40 ° C until the next analyzes. So he has It was possible to separate the following 5 monomers: ai (-15952 kDa), a 2 (-15975 kDa), d 2 (-17033 kDa), b 2 (-16020 kDa) and c (-16664 kDa).
• the two-dimensional gel which is a combination of isoelectric focusing techniques in first dimension and SDS-PAGE in second dimension makes it possible to separate a complex mixture of protein.
• Rock oil is then deposited at the two ends of the support of the electro-focusing strip, and the sample is then deposited in the middle.
• the 17 cm strip is then deposited on the sample, eliminating any air bubbles.
• the strip is then covered with rock oil to prevent evaporation of the sample.
• Active rehydration is then carried out at 50V (20 ° C for 12 hours). Then we focus for two days.
• the strip will then be recovered and placed on a 6-18% acrylamide gel, in particular 10%, 18 cm wide, 20 cm long and 1 mm thick.
• the migration is carried out in a refrigerated enclosure at 10 ° C., for 14 hours at 400 V, 25 mA and 100 W.
• the separation of the protein chains will then be carried out according to their size after having sealed the strip at the top of the gel with the using a 1% agarose solution. Once separated, the protein bands are revealed on the gel by staining with Coomassie blue (Coomassie ® G250).
• the top of the gel is covered with an isobutanol solution saturated with double distilled water.
• the gel is then left for 1 h to polymerize at room temperature, then the top of the gel is rinsed several times with double-distilled water and the whole is placed overnight at 10 ° C.
• the concentration gel solution (acrylamide 0.56 M; methylene bis-acrylamide 6.9 mM; Tris 124 mM; SDS 3.5 mM; TEMED 0.05% (v / v); sodium persulfate 2.2 mM) is poured onto the separation gel and a block making it possible to form the imprint of the strip is introduced into the gel concentration solution.
• the polymerization is complete after 1 h at room temperature.
• steps of discoloration, reduction and alkylation are essential: • successive washes with ammonium hydrogen carbonate (NH HCO) and with acetonitrile (ACN) make it possible to remove the coloring agent present in the gel piece, • the reactions of reduction to dithiothreitol (DTT) and of alkylation to Tiodoacetamide allow the opening and then the blocking of the disulfide bridges formed between two cysteines present in the protein sequence and cysteine-acrylamide bonds.
• NH HCO ammonium hydrogen carbonate
• ACN acetonitrile
• the last step of the method consists in extracting the tryptic peptides from the gel using an extraction solution, composed of acetonitrile and water, added with a little acid.
• Phenyl-Iso-Thio-Cyanate couples to the primary and secondary amino functions of proteins (PTC-Protein).
• the reaction time at 45 ° C is 18 minutes.
• the following peptide bond is weakened, which allows it to be cut in 3 minutes by pure trifluoroacetic acid (TFA) thus generating Tanilino-thiazolinone (ATZ) of the first amino acid (AA) and the protein having lost the 1st AA.
• ATZ-AA is extracted from the reaction medium and converted into an acid medium (TFA 25% in water) into more stable phenyl thio-hydantoin (PTH-AA).
• PTH-AA can therefore be analyzed by HPLC and its nature determined using a standard of PTH-AAs.
• the reaction cycle can be repeated and thus leads to the protein sequence. Edman automated the reaction that bears his name by creating the first protein sequencer in 1967.
• the device is coupled to an HPLC into which it injects PTH-AA. By comparison with a standard spectrum, it is then possible to identify the original amino acid and obtain its quantification. Everything is under the control of a computer which controls the various elements and ensures the acquisition of data and their processing.
• the PCR amplifications of the 5 globins A1, A2a, A2b, B1 and B2, as well as of the linker L1, the nucleotide sequences of which are presented below, began with the design of specific degenerate primers (sense and antisense) of the sub - families A1, A2, Bl and B2. These primers, which allowed the amplification of the five aforementioned globins (A1, A2a, A2b, B1 and B2) and then the cloning and sequencing of the corresponding PCR products, were designed from alignments of protein sequences of globins d 'annelids available in databases.
• the complementary DNA matrices used for the PCR reactions were synthesized from messenger RNA purified from total RNA extracted from Arenicoles, due to the small size of the organisms and their intense growth rate reflecting significant levels of gene expression, including those involved in the synthesis of hemoglobin.
• the complementary DNAs were thus synthesized. These steps used commercial molecular biology kits from Ambion (purification of RNAs), Amersham (purification of mRNA), Promega (RT), Invitrogene (cloning), Abgene (sequencing).
• a second step we developed the PCR reactions, in particular with regard to the determination of the parameters of denaturation time, time and hybridization temperature and elongation time. MgCl 2 concentrations have also been optimized.
• A2a (SEQ ID NO: 2), the pair of primers is used (SEQ ID NO: 19; SEQ ID NO: 20).
• the PCR conditions are as follows: Time and initial temperature of denaturation: 4 min at 95 ° C. Time and temperature of denaturation: 30 s at 95 ° C. ⁇ Time and temperature of hybridization: 30 s at 56 ° C - 35 cycles Time and temperature of elongation: 40 s at 72 ° C Time and temperature of final elongation: 10 min at 72 ° C
• PCR reaction Per reaction: 5-20 ng cDNA 100 ng sense primer 100 ng antisense primer dNTP 200 ⁇ M final MgCl 2 2mM final PCR buffer IX final 1 unit Taq Polymerase Qsp 25 ⁇ L H 2 O Globin A2b
• SEQ ID NO: 4 the pair of primers (SEQ IDNO: 21; SEQ ID NO: 20) is used.
• the PCR conditions are as follows: PCR: 4 min at 95 ° C 30 s at 95 ° C 30 s at 52 ° C 35 cycles 40 s at 72 ° CJ 10 min at 72 ° C
• Linker L1 To obtain the nucleotide sequence SEQ ID NO: 11 coding for the Linker L1 (SEQ ID NO: 12), the pair of primers is used (SEQ ID NO: 25; SEQ ID NO: 20).
• the PCR conditions are as follows: PCR: 4 min at 95 ° C.
• a dodecamer of globin chains is the principal fimctional subunit of the extracellular hemoglobin of Lumbricus tenestris, JBiol Chem, 266 (20): 13091-6, - Vinogradov, SN, Shlom JM and Doyle, M. (1979) Dissociation of the extracellular hemoglobin of Arenicola cristata. Comp. Biochem.

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