Recombinant hemoglobin, nucleic acid encoding same, and preparation method therefor

Abstract

A recombinant hemoglobin, a nucleic acid encoding the recombinant hemoglobin, and a preparation method therefor are provided to address the current problems of complex processes, high production costs, and low yields that exist for improving a hemoglobin having a stable tetrameric structure. The recombinant hemoglobin comprises: two or more units of recombinant hemoglobins, the units of recombinant hemoglobins each comprise four polypeptide chains, and the polypeptide chains are two α chains and two β chains. The amino acid sequences of the four polypeptide chains of each unit of recombinant hemoglobin are linked by means of intra-unit linker peptides to form the unit of recombinant hemoglobin, and two or more of the units are linked by means of inter-unit linker peptides.

Description

Recombinant hemoglobin, nucleic acid encoding same and method of preparation thereof TECHNICAL FIELD

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant hemoglobin, a nucleic acid encoding the same and a method of preparation thereof. BACKGROUND

[0002] Hemoglobin is a protein with oxygen-carrying function in red blood cells, and has a spherical structure. The molecular weight of natural hemoglobin is 64.5KD. Hemoglobin is composed of four polypeptide chains (subunits) which are combined together by non-covalent bonds. The main component in human whole blood is hemoglobin A, which has a structure of α2β2. The α chain contains 141 amino acid residues, and the β chain contains 146 amino acid residues. The α chain and the β chain are combined into a dimer in a relatively tight manner, and two αβ dimers are combined into a hemoglobin molecule in a relatively loose manner.

[0003] Blood products (blood products are defined as any therapeutic material extracted from human blood, including whole blood, labile blood components and plasma-derived medicinal products) can save millions of lives every year, greatly improve life expectancy and improve the quality of life of critically ill patients, and support complex medical and surgical procedures.

[0004] However, the world's blood service is facing great challenges, and needs to provide sufficient blood product supply to meet the needs of patients, while ensuring its quality and safety in the face of known and emerging public health threats. According to statistics in 2002, the amount of blood donation worldwide exceeded 75 million units per year, and only 43 of the 191 member states of the World Health Organization conducted hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV testing on blood donors. Unsafe blood transfusion or injection causes 8-16 million people to be infected with HBV, 2.3-4.7 million people to be infected with HCV, and 8-16 million people to be infected with HIV every year. In addition, the contradiction between the increasing demand for blood and the decreasing amount of blood donation, the hemolysis and even death caused by wrong blood transfusion, the short storage time of human red blood cells, and natural disasters and wars force people to find a substitute for blood, i.e. red blood cell substitutes.

[0005] Hemoglobin-based red blood cell substitutes are biological drugs with oxygen-carrying function based on hemoglobin. Hemoglobin-based red blood cell substitutes can be divided into three categories, i.e. human blood-based red blood cell substitutes, animal blood-based red blood cell substitutes and genetically recombinant hemoglobin-based red blood cell substitutes.

[0006] However, at present, unmodified hemoglobin cannot be used as blood substitute, one of the main reasons is that free hemoglobin tetramer can be degraded into αβ dimer and monomer, which has many side effects. In order to reduce side effects, therefore, stabilizing hemoglobin tetramer is necessary for hemoglobin as blood substitute. At present, the method of chemical modification is often used, such as crosslinking, polymerization and coupling, etc. to increase the molecular weight of hemoglobin. In addition, by using biotechnology, such as site-directed mutagenesis of hemoglobin gene at DNA level, the stability of hemoglobin tetramer can also be improved.

[0007] However, although the chemical modification method is mature, there are still urgent side effects to be solved; (1) due to the difference of chemical reagents used, the difference of methods, the non-specificity of crosslinking reaction, the molecular size, oxygen affinity, subunit synergy, stability and toxic side effects of hemoglobin products are difficult to control, and the final product is a non-uniform mixture; (2) the contamination of pathogenic microorganisms still exists due to the unsolved problem of blood sterilization, which still spreads blood-borne diseases such as mad cow disease and acute and chronic immunogenicity to human; (4) the instability of the final product leads to the rearrangement and combination of crosslinked hemoglobin. Since the free crosslinking agent has cytotoxicity, whether it will fall off from the crosslinked hemoglobin is a problem worthy of attention. Moreover, sometimes the chemical modification also has the complexity of process, which increases the production cost and reduces the production efficiency.

[0008] And sometimes, the site-directed mutagenesis of hemoglobin gene at DNA level also brings blindness, which affects the final function.

[0009] In addition, there is also a method of producing recombinant hemoglobin by co-expression of α and β in cells, but the yield is often low, and after production, the hemoglobin also needs to be crosslinked and chemically modified to increase the stability of tetramer structure.

[0010] It can be seen that there are still deficiencies in the prior art for providing hemoglobin with stable tetramer structure.

[0011] SUMMARY

[0012] The present application provides a kind of recombinant hemoglobin, the nucleic acid of coding the recombinant hemoglobin and preparation method thereof, to solve the problems of complex process, high production cost, large side effect and low yield of hemoglobin with stable tetramer structure at present.

[0013] Therefore, the present application provides the following technical solutions:

[0014] The present application provides a kind of unit recombinant hemoglobin, which is characterized by comprising: at least two polypeptide chains, which include any one or a combination of multiple of α chain and β chain;

[0015] The amino acid sequences of the polypeptide chains of each of the units are connected by the intracellular connecting peptide to form the unit recombinant hemoglobin,

[0016] The units are connected by the intercellular connecting peptide.

[0017] Preferably, the unit recombinant hemoglobin comprises four polypeptide chains, which are two alpha chains and two beta chains respectively.

[0018] The unit recombinant hemoglobin provided by the application also has the following characteristics: the amino acid sequences of the four polypeptide chains are sequentially connected from the N-terminal to the C-terminal by the three intracellular connecting peptides in any order, and the units are sequentially connected from the N-terminal to the C-terminal by the intercellular connecting peptide. That is, in the primary structure, the polypeptides included in the unit recombinant hemoglobin are connected by the intracellular connecting peptide, or the unit recombinant hemoglobins are connected by the intercellular connecting peptide.

[0019] The unit recombinant hemoglobin provided by the application also has the following characteristics: the number of amino acids of the intracellular connecting peptide is greater than or equal to 1, 1-140, 1-120, 1-90, 1-30, 1-35 or 1-40.

[0020] The unit recombinant hemoglobin provided by the application also has the following characteristics: the intracellular connecting peptide for connecting the two alpha chains is a first connecting peptide, the number of amino acids of the first connecting peptide is 1-5, preferably 1, 2 or 3, the intracellular connecting peptide for connecting the two beta chains or connecting one alpha chain and one beta chain is a second connecting peptide, and the number of amino acids of the second connecting peptide is greater than or equal to 3, greater than or equal to 5, 5-140, 5-130, 5-120, 5-90, 5-85, 5-30, 5-35, 5-40, 10-30, 10-35 or 10-40.

[0021] The unit recombinant hemoglobin provided by the application also has the following characteristics: the amino acid of the first connecting peptide is 1 glycine.

[0022] The recombinant hemoglobin provided by the present invention also has the following characteristics: the second linker peptide contains any one or more major amino acid residues of G, S, T and A, and the percentage of the number of the major amino acid residues relative to the total number of amino acid residues in the second linker peptide is at least greater than 45%, 50%, 60%, 70%, 80% or 90%. More preferably, the major amino acid residues contain any one or more amino acids of G and S, and the total amount of G and S contains is at least greater than 40%, greater than 45%, greater than 60%, greater than 70% or greater than 80% of the total number of amino acids in the second linker peptide.

[0023] The recombinant hemoglobin provided by the present invention also has the following feature: the second linker peptide contains at least a flexible unit.

[0024] The recombinant hemoglobin provided by the present invention also has the following characteristics: the second linker peptide further contains hydrophilic and hydrophobic units composed of alternating hydrophilic and hydrophobic amino acids. In this case, from the N-terminus to the C-terminus, the amino acid sequence of the second linker peptide is: flexible unit—hydrophilic and hydrophobic units composed of alternating hydrophilic and hydrophobic amino acids—flexible unit.

[0025] The recombinant hemoglobin provided by this invention also has the following characteristic: the structure of the flexible unit is (GGGGS)a, preferably, a is 1-4.

[0026] More preferably, the flexible unit is selected from:

[0027] (1) GGGGS;

[0028] (2) GGGGSGGGGS;

[0029] (3)GGGGSGGGGSGGGGSGGGGS.

[0030] The recombinant hemoglobin provided by this invention also has the following characteristic: the structure of the affinity / relativity unit is any one or more of (RADA)b, (KLDL)c, (LK)d, and (LD)f, preferably: b is 1-5, c is 1-5, d is 1-10, and f is 1-10.

[0031] More preferably, the affinity unit is selected from:

[0032] (1) RADARADARADARADA;

[0033] (2) KLDLKLDLKLDL;

[0034] (3)LKLKLKLKLKLK;

[0035] (4)LDLDLDLDLDLD.

[0036] The recombinant hemoglobin unit provided by this invention also has the following characteristic: the amino acid sequence of the second linker is any one of SEQ ID NO: 1-6, 10-12, 21, and 22, or has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence homology with any one of SEQ ID NO: 1-6, 10-12, 21, and 22.

[0037] Preferably, the amino acid sequences of all the second linker peptides are all the same, partially the same, or all different.

[0038] The recombinant hemoglobin provided by this invention also has the following feature: the amino acid sequence of the four polypeptide chains from the N-terminus to the C-terminus is any one of the following: α chain-α chain-β chain-β chain, β chain-β chain-α chain-α chain, β chain-α chain-α chain-β chain, α chain-β chain-α chain-β chain, and β chain-α chain-β chain-α chain.

[0039] The recombinant hemoglobin provided by this invention also has the following characteristics: the species from which the α-chain originates is not particularly limited, but preferably from any one or more of humans, pigs, or cattle; similarly, the species from which the β-chain originates is not particularly limited, but preferably from any one or more of humans, pigs, or cattle; and / or

[0040] The amino acid sequence of the α chain is any one or more of SEQ ID NO: 7, 13 and 14, or has at least 80%, 85%, 90%, 95%, 97 or 99% sequence homology with any one or more of SEQ ID NO: 7, 13 and 14, respectively.

[0041] The amino acid sequence of the β chain is any one or more of SEQ ID NO: 8, 15 and 16, or has a sequence homology percentage of at least 80%, 85%, 90%, 95%, 97 or 99% with any one or more of SEQ ID NO: 8, 15 and 16, respectively.

[0042] The recombinant hemoglobin provided by the present invention also has the feature of further comprising heme bound to each of the polypeptide chains.

[0043] The present invention also provides a recombinant hemoglobin, characterized in that it comprises: two or more of the aforementioned recombinant hemoglobin units, wherein the two or more units are linked together by inter-unit linking peptides.

[0044] The recombinant hemoglobin provided by this invention also has the following characteristics:

[0045] (1) The number of amino acids in the inter-unit linking peptide is greater than or equal to 0, 0-100, 0-90, or 0-85, and / or

[0046] The inter-unit linker peptide contains a combination of any one or more amino acid residues from G, S, A, and T, and / or the component linker peptide contains a combination of any one or more amino acid residues from E and D:

[0047] Preferably, the percentage of any one or more amino acid residues of G, S, T and A in the inter-unit linker peptide is at least greater than 45%, 50%, 60%, 70%, 80% or 90% of the total number of amino acid residues in the inter-unit linker peptide. Further, the amino acid sequence of the inter-unit linker peptide is any one of SEQ ID NO: 17-20, or has at least 80%, 85%, 90%, 95%, 97 or 99% sequence homology with any one or more of SEQ ID NO: 17-20.

[0048] (2) The number of units is 2, 3 or 4.

[0049] The present invention also provides a nucleic acid, characterized in that it comprises: nucleotides encoding the aforementioned recombinant hemoglobin.

[0050] The present invention also provides a carrier, characterized in that it comprises the aforementioned nucleic acid.

[0051] The present invention also provides a host cell, characterized in that it comprises: the aforementioned nucleic acid and / or the aforementioned vector.

[0052] Preferably, the host cell is a prokaryotic or eukaryotic cell. Further, the host cell is selected from yeast cells. More specifically, the yeast cells are selected from one or more combinations of Saccharomyces cerevisiae and Kluyveromyces genus. In another preferred embodiment, the Kluyveromyces genus is selected from one or more combinations of Kluyveromyces lactis, Kluyveromyces marx, and Kluyveromyces dob.

[0053] The application of the aforementioned nucleic acid, vector, or host cell of the present invention in the preparation of the aforementioned recombinant hemoglobin.

[0054] The present invention also provides an in vitro cell-free protein synthesis system, characterized in that it comprises: a cell extract; and an mRNA or DNA template encoding the aforementioned recombinant hemoglobin. Preferably, the cell extract is the yeast cell extract, and further, it is derived from any combination of one or more of the following: Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces var. kusnezoffii. More preferably, the yeast cell extract is derived from Kluyveromyces var. kusnezoffii, more preferably Kluyveromyces lactis, and even more preferably from Kluyveromyces kusnezoffii.

[0055] Furthermore, it also includes one or more of the following components:

[0056] Amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, energy supply system, polyethylene glycol, and aqueous solvent.

[0057] The present invention also provides the aforementioned method for in vitro cell-free synthesis of recombinant hemoglobin, characterized in that it includes: providing a cell-free in vitro protein synthesis system;

[0058] The recombinant hemoglobin is obtained by adding an mRNA or DNA template encoding the aforementioned recombinant hemoglobin to the cell-free protein synthesis system and performing an in vitro synthesis reaction. Preferably, the volume ratio of the DNA template to the cell-free in vitro protein synthesis system is 1:10 to 1:50, more preferably 1:20 to 1:40; most preferably 1:25 to 1:35, and particularly preferably 1:30.

[0059] The in vitro cell-free synthesis method provided by the present invention also has the following features: simultaneously adding heme to the cell-free synthesis system, adding heme during or after the in vitro synthesis reaction to generate recombinant hemoglobin bound with the heme, preferably, the added heme contains ferric iron.

[0060] Preferably, the added heme contains ferric iron.

[0061] The in vitro cell-free synthesis method provided by the present invention also has the following characteristics: wherein the cell-free in vitro protein synthesis system comprises: yeast cell extract, amino acid mixture, dNTP, RNA polymerase, DNA polymerase, energy supply system, polyethylene glycol, and aqueous solvent; furthermore: the yeast cell extract is derived from any one or more combinations of the following group: Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces var. kulariiflora.

[0062] Preferably, the yeast cell extract is derived from: Kluyveromyces, more preferably Kluyveromyces lactis; or the yeast extract accounts for 50-80% of the entire cell-free in vitro protein synthesis system.

[0063] The present invention also provides the use of the aforementioned recombinant hemoglobin as a blood substitute, and for use in cancer, stroke, hemorrhagic shock, acute mountain sickness (AMS), peripheral artery disease (PAD), and Parkinson's disease (PD).

[0064] Invention Function and Effect

[0065] The recombinant hemoglobin, the nucleic acid encoding the recombinant hemoglobin, and the preparation method thereof provided by the present invention have at least the following advantages:

[0066] (1) Recombinant hemoglobin connects four polypeptide chains in the primary structure via linking peptides.

[0067] The tandem linkage allows the α and β chains to interact more easily during the in vivo or in vitro synthesis and expression of recombinant hemoglobin, forming monomeric structures with similar functions to tetramer hemoglobin. Furthermore, due to the primary structural linkage, this structure is more stable, less prone to dissociation, and can form stable tetramers. The tandem α and β chains also have a one-to-one ratio, avoiding the possibility of insufficient matching between separate α and β chains leading to structures with varying quantities. This results in higher yields, easier production, and eliminates the need for additional chemical cross-linking, avoiding the side effects of chemical modification. The process is simpler and less costly. Moreover, tandem linkage of multiple recombinant hemoglobin units also yields stable recombinant hemoglobin.

[0068] (2) The present invention uses the nucleic acid of recombinant hemoglobin obtained by tandemly linking multiple groups of four polypeptide chains in the primary structure with the encoding linking peptide as a template, and can be directly produced in vitro in a cell-free synthesis method, which is fast and low cost. Attached Figure Description

[0069] Figure 1 and Figure 2 show the different experimental results of the effect of adding Hemin to the reaction solution for the in vitro synthesis of EGFP protein (PC);

[0070] Figure 3 shows the photos after Hemin was added to the reaction solution of 16 scHemoglobin (monohemoglobin) in the in vitro synthesis reaction, and after expression and purification, and then replaced with PBS solution.

[0071] Figure 4 shows the results after adding 20 μM Hemin to the reaction solution of the six recombinant hemoglobins synthesized in vitro, expressing and purifying them, and then replacing them with PBS solution.

[0072] Figure 5 shows the results of SDS-PAGE electrophoresis detection after normal expression and purification of the in vitro synthesis reaction system without the addition of DNA template but with the addition of Hemin.

[0073] Figure 6 shows the spectroscopic properties of hemoglobin under oxygen-carrying and anaerobic conditions as revealed in Reference 1.

[0074] Figure 7-29 shows the spectroscopic characteristics of the 23 proteins in Example 4 after binding to heme, as detected by reference 1. Detailed Implementation

[0075] The specific embodiments of the present invention will be described below with reference to the accompanying drawings. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on the technical concept of the present invention and existing technologies, and are not limited to the specific descriptions of the embodiments of the present invention.

[0076] Unless otherwise specified, the methods used in the embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0077] The definitions of terms used herein are intended to incorporate the generally accepted prior art definitions of each term in the field of biotechnology. Examples are provided where appropriate. Unless otherwise limited, either individually or as part of a larger group, these definitions apply to the terminology used throughout this specification.

[0078] In this article, “recombinant hemoglobin” refers to hemoglobin molecules and / or their variants with a molecular size of at least about 65 kDa, synthesized by any standard molecular biology technique, rather than isolated or purified from any animal or human source.

[0079] In this article, a “variant” refers to a polypeptide or polynucleotide sequence that differs from the reference polypeptide or polynucleotide sequence but retains its essential properties. Typically, variants are very similar to the reference polypeptide or polynucleotide sequence overall and are identical in many regions.

[0080] The variant may, for example, comprise an amino acid sequence of a parent polypeptide sequence having at least one conserved amino acid substitution; or, the variant may comprise an amino acid sequence of a parent polypeptide sequence having at least one non-conserved amino acid substitution, wherein the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant, and the non-conserved amino acid substitution can enhance the biological activity of the variant, such that the biological activity of the variant is increased compared to the parent polypeptide.

[0081] When a reference polypeptide or polynucleotide sequence is used, the term "percentage of sequence homology" refers to a comparison between a polynucleotide and a polypeptide, and is determined by comparing two best-aligned sequences within a comparison window, where a portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (excluding additions or deletions) used for the best alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue is present to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the longer sequence within the comparison window, and multiplying the result by 100 to produce the percentage of sequence homology. Homology is evaluated using any of a variety of sequence comparison algorithms and procedures known in the art. Such algorithms and procedures include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW. In some implementations, a basic local alignment search tool (“BLAST”) well known in the art is used to evaluate protein and nucleic acid sequence homology (see, for example, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402).

[0082] In this article, "Heme" refers to heme iron, an iron porphyrin compound that is a cofactor of hemoglobin, as well as a cofactor of myoglobin, cytochrome, peroxidase, catalase, etc.

[0083] In this article, "active structure" refers to the ability of the prepared recombinant hemoglobin to carry oxygen, and to be prepared as or used as a blood substitute. For example, the protein before it binds to heme. As long as it can bind to heme and has the function of carrying oxygen, it can be prepared as a blood substitute. The protein before it binds to heme is considered to have an active structure.

[0084] 1. Unitary recombinant hemoglobin

[0085] The recombinant hemoglobin provided by this invention comprises four polypeptide chains, namely two α chains and two β chains. The amino acid sequences of the four polypeptide chains are linked together by intra-unit linking peptides to form the recombinant hemoglobin. The order and position of the amino acid sequences of the four polypeptide chains linked by the intra-unit linking peptides are not particularly specified and can be arbitrary. That is, the four polypeptide chains are linked together at the primary structure by one or more intra-unit linking peptides.

[0086] In a preferred embodiment, the three intra-unit linking peptides connect the amino acid sequences of the four polypeptide chains sequentially from the N-terminus to the C-terminus in any order, preferably in any of the following orders: α chain-α chain-β chain-β chain, β chain-β chain-α chain-α chain, β chain-α chain-α chain-β chain, α chain-β chain-α chain-β chain, and β chain-α chain-β chain-α chain: "-" indicates the linking peptide. The amino acid sequences of the three linking peptides may all be the same, or only two may be the same, or all three may be different.

[0087] The number of amino acids in the linker peptide within the unit is designed to ensure that the recombinant hemoglobin forms a functional active structure, and secondly, to maintain a certain level of stability. Preferably, the number of amino acids in the linker peptide is greater than or equal to 1, 1-140, 1-120, 1-90, 1-30, 1-35, or 1-40.

[0088] In one example, the intra-unit linking peptides include two types:

[0089] One type is used to connect two α chains, called the first linker peptide. Preferably, the number of amino acids in the first linker peptide is 1-5, more preferably 1, 2 or 3.

[0090] Another type is used to link two β chains or one α chain and one β chain, called a second linker peptide. Preferably, the number of amino acids in the second linker peptide is any one of the following: greater than or equal to 3, greater than or equal to 5, 5-140, 5-130, 5-120, 5-90, 5-85, 5-30, 5-35, 5-40, 10-30, 10-35, and 10-40 amino acids.

[0091] In one example, the first linker peptide has one glycine amino acid.

[0092] In one example, the second linker peptide contains one or more major amino acid residues of G, S, T, and A, and the number of said major amino acid residues accounts for at least 50%, 60%, 70%, 80%, or 90% of the total number of amino acid residues in the second linker peptide. More preferably, the major amino acid residues contain one or more amino acids of G and S, and the total number of said major amino acid residues accounts for at least 40%, 45%, 60%, 70%, or 80% of the total number of amino acids in the second linker peptide.

[0093] The amino acid sequence of the second linker is, for example, any one of SEQ ID NO: 1-6, 10-12, 21, and 22, or has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence homology with any one of SEQ ID NO: 1-6, 10-12, 21, and 22.

[0094] All of the second linker peptides may have identical, partially identical, or completely different amino acid sequences.

[0095] In one example, the second linker peptide contains at least a flexible unit.

[0096] In one example, the flexible unit has a structure of (GGGGS)a, preferably, a is 1-4. More preferably, the flexible unit is selected from:

[0097] (1) GGGGS;

[0098] (2) GGGGSGGGGS;

[0099] (3)GGGGSGGGGSGGGGSGGGGS.

[0100] In one example, the second linker peptide also contains hydrophilic and hydrophobic units that alternate between hydrophilic and hydrophobic amino acids. In this case, from the N-terminus to the C-terminus, the amino acid sequence of the second linker peptide is: flexible unit — hydrophilic and hydrophobic units that alternate between hydrophilic and hydrophobic amino acids — flexible unit.

[0101] The alternation of hydrophilic and hydrophobic amino acids in this article refers to the following different situations:

[0102] (1) Alternating between a hydrophilic amino acid and a hydrophobic amino acid;

[0103] (2) A group of hydrophilic amino acids consisting of one or more consecutive hydrophilic amino acids alternates with a group of hydrophobic amino acids consisting of one or more consecutive hydrophobic amino acids. In this case, the amino acids in each unit can be the same or different. For example, the hydrophilic amino acids in each group of hydrophilic amino acids can be the same or different. The amino acid composition between different hydrophilic groups or between different hydrophobic groups can be the same or different, for example.

[0104] In one example, the structure of the affinity unit is any one or more of (RADA)b, (KLDL)c, (LK)d, and (LD)f, preferably: b is 1-5, c is 1-5, d is 1-10, and f is 1-10.

[0105] More preferably, the affinity unit is selected from:

[0106] (1) RADARADARADARADA;

[0107] (2) KLDLKLDLKLDL;

[0108] (3)LKLKLKLKLKLK;

[0109] (4)LDLDLDLDLDLD.

[0110] In one example, the amino acid sequence of the second linker is any one of SEQ ID NO: 1-6, 10-12, or has a sequence homology of at least 85%, 90%, 95%, 97%, 98%, or 99% with any one of SEQ ID NO: 1-6, 10-12.

[0111] Preferably, the amino acid sequences of all the second linker peptides are all the same, partially the same, or all different.

[0112] In one example, the amino acid sequence of the α chain is SEQ ID NO: 7 or has a sequence homology percentage of at least 80%, 85%, 90%, 95%, or 99% with SEQ ID NO: 7; and the amino acid sequence of the β chain is SEQ ID NO: 8 or has a sequence homology percentage of at least 80%, 85%, 90%, 95%, or 99% with SEQ ID NO: 8.

[0113] 2. Recombinant hemoglobin

[0114] The recombinant hemoglobin provided by the present invention comprises two or more of the aforementioned recombinant hemoglobin units, which are linked together by inter-unit linking peptides.

[0115] In one example, the inter-unit linking peptide connects the units sequentially from the N-terminus to the C-terminus.

[0116] In one example, the number of amino acids in the inter-unit linking peptide is greater than or equal to 0, 0-100, 0-90, or 0-85.

[0117] In one example, the interunit linker peptide contains a combination of any one or more amino acid residues from G, S, A, and T.

[0118] In one example, the component linker peptide contains a combination of any one or more amino acid residues from E and D.

[0119] Preferably, the percentage of any one or more amino acid residues of G, S, T and A in the inter-unit linker peptide is at least 45%, 50%, 60%, 70%, 80% or 90% of the total number of amino acid residues in the inter-unit linker peptide.

[0120] In one example, the amino acid sequence of the interunit linking peptide is any one of SEQ ID NO: 17-20, or has at least 80%, 85%, 90%, 95%, 97% or 99% sequence homology with any one or more of SEQ ID NO: 17-20, respectively.

[0121] There is no particular limitation on the number of units; it can be 2, 3, or 4, or even more.

[0122] 3. The present invention also provides a nucleic acid comprising: a nucleotide sequence encoding the aforementioned unit recombinant hemoglobin or recombinant hemoglobin.

[0123] The present invention also provides a carrier comprising the above-described nucleic acid.

[0124] The present invention also provides a host cell comprising the above-mentioned nucleic acid and / or the above-mentioned vector. The host cell is derived from prokaryotic or eukaryotic cells, and further, the cell is selected from one or more combinations of the following groups: *Escherichia coli* cells, human cells, Chinese hamster cells, ovarian cells, insect cells, wheat germ cells, rabbit reticulocytes, and yeast cells.

[0125] Furthermore, the host cell is selected from yeast cells, and even further, the yeast cell is selected from one or more combinations of Saccharomyces cerevisiae and Kluyveromyces genus yeasts. In another preferred embodiment, the Kluyveromyces genus yeast is selected from one or more combinations of Kluyveromyces lactis, Kluyveromyces marx, and Kluyveromyces dob.

[0126] The nucleic acid, vector, or host cell provided by this invention can be used in the preparation of recombinant hemoglobin.

[0127] The present invention also provides an in vitro cell-free protein synthesis system, comprising: cell extract and mRNA or DNA template encoding the aforementioned recombinant hemoglobin.

[0128] In one example, the cell extract is a yeast cell extract, further, derived from any combination of one or more of the following: Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces var. kluyveromyces; preferably, the yeast cell extract is derived from Kluyveromyces var. kluyveromyces, more preferably Kluyveromyces lactis, and even more preferably, from Kluyveromyces var. kluyveromyces.

[0129] In one example, the in vitro cell-free synthesis system also includes one or more of the following components: a mixture of amino acids, dNTPs, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent.

[0130] 4. In vitro cell-free synthesis methods for recombinant hemoglobin, including:

[0131] Provides a cell-free in vitro protein synthesis system;

[0132] The single-chain polypeptide was synthesized in vitro by adding an mRNA or DNA template encoding the aforementioned recombinant hemoglobin to a cell-free protein synthesis system.

[0133] The volume ratio of DNA template to cell-free in vitro protein synthesis system is 1:10-1:50, preferably 1:20-1:40; most preferably 1:25-1:35, and particularly preferably 1:30.

[0134] In one example, the cell-free in vitro protein synthesis system includes: yeast cell extract, a mixture of amino acids, dNTPs, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent.

[0135] In one example, the yeast cell extract is derived from any one or more of the following group: Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces kluyveromyces; preferably, the yeast cell extract is derived from any one of Kluyveromyces kluyveromyces, more preferably Kluyveromyces lactis, Kluyveromyces marx, and Kluyveromyces dob.

[0136] In one example, yeast extract comprised 50-80% of the entire cell-free in vitro protein synthesis system.

[0137] In vitro protein synthesis refers to the synthesis of proteins in a cell-free in vitro synthesis system, including at least the translation process. This includes, but is not limited to, IVT (in vitro translation), IVTT (in vitro transcription-translation), and IVDTT (in vitro replication-transcription-translation). In this invention, the IVTT reaction is preferred. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as a D2P system, D-to-P system, or DNA-to-Protein system; and the corresponding in vitro protein synthesis methods are also referred to as D2P methods, D-to-P methods, or DNA-to-Protein methods.

[0138] In a preferred embodiment, in the in vitro cell-free synthesis method of the present invention, the technical elements such as the in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, and various detection methods can each be independently selected from suitable embodiments or implementation methods from the following documents, including but not limited to CN111484998A, CN106978349A, CN108535489A, CN108690139A, and CN108949801. Documents including A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, and CN110964736A. Unless otherwise provided, these documents and their references are cited in their entirety and for all purposes.

[0139] The present invention synthesizes the aforementioned recombinant hemoglobin using the aforementioned in vitro cell-free synthesis method, which has a short cycle, is easy to operate, has low cost, and can obtain stable hemoglobin with the aforementioned active structure.

[0140] Furthermore, in vitro cell-free synthesis methods also include:

[0141] Simultaneously, heme is added to the cell-free synthesis system, during or after the in vitro synthesis reaction to generate recombinant hemoglobin bound with the heme. Preferably, the added heme contains ferric iron.

[0142] The following specific experimental examples further illustrate the content of this invention.

[0143] In the following embodiments:

[0144] (1) The IVTT reaction system consisted of: 22 mM 4-hydroxyethylpiperazine ethanesulfonic acid at pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM a mixture of nucleoside triphosphates (adenine triphosphate, guanine triphosphate, cytosine triphosphate, and uracil triphosphate), 0.08-0.24 mM a mixture of amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine), 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphate kinase, and 0.027-0.054 mg / mL creatine phosphate kinase. T7 RNA polymerase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, and finally 50-80% by volume yeast cell extract.

[0145] The yeast cell extract was obtained from Kluyveromyces oryzae, specifically, in this embodiment, Kluyveromyces lactis was used.

[0146] (2) The transformation of each target plasmid is carried out as follows:

[0147] Add 1 μl of the target plasmid to 20 μl of DH5α, place on ice for 30 min, heat shock at 42℃ for 45 s, place on ice for 2 min, add 500 μl of culture medium, and incubate at 37℃ with shaking for 1 h (200 rpm). Transfer 100 μl to an LB agar plate (containing antibiotic), incubate upside down at 37℃ for 12–16 h, and then store at 4℃.

[0148] (4) The transformed plasmid was extracted and amplified:

[0149] Amplification system: random primers with a final concentration of 20-30 μM, plasmid template of 0.05-0.15 μg / mL, dNTPs of 0.5-1 mM, 2×BSA, 1×phi29 reaction buffer (components: 50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, pH 7.5).

[0150] Taking a 10ml amplification system as an example:

[0151] Mix 10 ml of amplification system, a final plasmid concentration of 4 ng / μl, and 5 μl of 0.05-0.1 mg / mL phi29 DNA polymerase. Incubate at 37°C and 30 rpm for 2 hours, then perform gel electrophoresis (1% agarose gel) for identification. The resulting product is used as the DNA template.

[0152] In the following embodiments, unless otherwise specified, the constructed structures are all in the N-end to C-end manner.

[0153] Example 1 Construction of Recombinant Hemoglobin

[0154] Different intra-unit linkers were designed to tandem the four subunits of hemoglobin, i.e., the four polypeptide chains, into a single scHemoglobin (unit). Different inter-unit linkers were also designed to tandem two units, resulting in recombinant hemoglobins numbered HEM027, HEM028, HEM031, HEM034, HEM035, and HEM044. The corresponding subunit tandem configurations are shown in Table 1, as are the inter-unit linkers used. In this embodiment, the amino acid sequence of the α chain is SEQ ID NO: 7, and the amino acid sequence of the β chain is SEQ ID NO: 8.

[0155] Recombinant hemoglobin genes containing different linker peptides were synthesized using gene synthesis methods. These genes were then directly inserted into our optimized plasmids (D2P1.08t, specifically described in Figure 1 of patents 2022111062129 or 2023104761829), with insertion sites at BamHI and HindIII. Plasmids containing the target gene fragments (HEM027, HEM028, HEM041, HEM034, HEM035, HEM044, and HEM048) were obtained, and transformed and amplified to obtain DNA templates encoding the structures shown in Table 1.

[0156] Add 15 ng / μL DNA template to the IVTT reaction system. Mix well and incubate at 25–30°C for 3 hours. After the reaction is complete, collect the reaction solution.

[0157] The reaction solution after the IVTT reaction was purified and eluted by adding nickel magnetic beads. The eluent was then subjected to SDS-PAGE electrophoresis to detect the protein bands. The bands showed that the sizes of the obtained proteins were consistent with the theoretical molecular weights.

[0158] Example 2: Expression and purification of scHemoglobin (unit recombinant hemoglobin)

[0159] Single-chain scHemoglobins were constructed individually. Six scHemoglobins were constructed, as shown in Table 2. In this embodiment, the amino acid sequence of the α chain is SEQ ID NO: 7, and the amino acid sequence of the β chain is SEQ ID NO: 8.

[0160] Following the same method as in Example 1, after obtaining the DNA template, the IVTT reaction system was used for reaction and purification without the addition of heme. The obtained protein was verified by SDS-PAGE electrophoresis, and the results showed that scHemoglobin could be correctly expressed.

[0161] Example 3: Co-expression and purification with Hemin

[0162] Because natural hemoglobin needs to bind with heme to perform its oxygen-carrying function, the artificial hemoglobin we produce also needs to bind with heme to function properly. However, heme binds to ferrous ions (Fe2+), which are very unstable and easily oxidized to ferric ions (Fe3+) in the air. Ferrous ions can only exist stably when heme binds to hemoglobin. Therefore, it is currently difficult to purchase large quantities of heme that is not bound to hemoglobin on the market. Commercially available heme is generally not readily available, has long lead times, and is expensive.

[0163] We attempted to use Hemin (heme chloride) as the cofactor of scHemoglobin. Hemin is heme with ferric iron.

[0164] First, the potential effect of Hemin concentration in the IVTT reaction system was tested:

[0165] For each unit in Table 2, hemoglobin fused with EGFP protein was expressed using the aforementioned IVTT method. Different concentrations of Hemin were added to the synthesized IVTT reaction solution to test the expression level of EGFP protein and to determine whether the addition of Hemin affected the expression level of PC. The results showed that a final concentration of 3.3 μM Hemin did not affect the IVTT expression level of PC, as shown in Figure 1. Subsequently, the concentration of Hemin was increased, and the test was continued, as shown in Figure 2. A final concentration of 99 μM Hemin did not affect the IVTT expression of PC.

[0166] In other words, the amount of hemin added has no effect on IVTT response expression, and there is no particular limit to the amount added. It is mainly higher than the expected production of hemoglobin to ensure that all expressed hemoglobin is in the hemin-bound state.

[0167] Then, the constructed structures shown in Tables 1, 2 and 3 were expressed and synthesized. Hemin with a final concentration of 20 μM was added to the synthesized IVTT reaction solution for expression and purification. The results obtained for each construction were subjected to gel electrophoresis. The gel electrophoresis results were consistent with the molecular weight, indicating that the target protein was obtained.

[0168] Furthermore, after replacing the purified proteins corresponding to each construct with PBS solution, they appeared blood red (as shown in Figure 3, the images corresponding to the constructed structures after binding with heme are as follows: First row, from left to right, images corresponding to the constructed structures HM001, HM002, HM003, HM004, HM005 and HEM006 after binding with heme; Second row, from left to right, images corresponding to the constructed structures HM007, HM008, HM009, HEM010, HM013, HM014, HEM015 and HEM022; Third row, from left to right, images corresponding to HEM023, HEM024 and HEM022; As shown in Figure 4, the images corresponding to the constructed structures after binding with heme are as follows: First row, from left to right, images corresponding to HM027, HM028, HM031, HM034, HM035, HEM044; Second row, HEM048).

[0169] Because hemoglobin itself is colorless, and the blood-red color comes from heme, to prevent the final product from containing free heme after purification, we conducted the following control experiment (HEM-NC): During the IVTT reaction, no DNA template was added, but Hemin was added for normal expression and purification. The resulting protein eluent was also buffer-replaced, ultimately yielding PBS buffer. SDS-PAGE electrophoresis and color observation were performed, and no scHemoglobi bands were observed (results shown in Figure 5). Furthermore, after replacement with PBS solution, the solution was colorless and transparent, without the blood-red color. This indicates that the blood-red sample we previously obtained was the color produced only after the protein binds to Hemin.

[0170] Example 4 Oxygen-carrying experiment

[0171] Oxygen-carrying experiments were performed on the proteins obtained from the various constructions in Tables 1, 2, and 3, which were purified according to Example 3, as detailed below:

[0172] The spectroscopic properties of hemoglobin under oxygen-carrying and anaerobic conditions are shown in Figure 9 (Reference 1: Patel, Mira P et al. “Development and validation of an oxygen dissociation assay, a screening platform for discovering, and characterizing hemoglobin-oxygen affinity modifiers.” Drug design, development and therapy 2018, vol. 121599-1607):

[0173] As shown in Figure 6, under oxygen-carrying conditions, hemoglobin exhibits peaks at 415, 541, and 577 nm. Figure 6 clearly shows that the absorbance of hemoglobin at 415 nm under oxygen-carrying conditions is higher than that of hemoglobin at 430 nm under anaerobic conditions. Under anaerobic conditions, hemoglobin exhibits peaks at 430 nm and 555 nm. Figure 6 clearly shows that the absorbance of hemoglobin at 430 nm under anaerobic conditions is higher than that of hemoglobin at 430 nm under oxygen-carrying conditions.

[0174] The conversion of hemoglobin from an oxygen-carrying state to an anaerobic state is catalyzed by sodium dithionite: upon adding sodium dithionite to oxygen-carrying hemoglobin, the hemoglobin is reduced and loses oxygen, becoming deoxygenated, resulting in a conversion of its spectroscopic properties. Based on this, the proteins bound to heme obtained according to the constructions in Tables 2 and 3 of Example 3 were subjected to spectroscopic detection, and the results are shown in Figure 7-29:

[0175] Among them, the protein concentrations of HEM031 and HEM035 were too high during the spectral scan, and some peaks were at the upper limit of the instrument's reading, so there were fluctuations in the spectral peaks, but the overall peak shape was as expected.

[0176] As can be seen from Figures 7-29, the optical properties of the 23 proteins bound to heme obtained in Example 3 are consistent with the schematic diagram in Figure 9. That is, it is confirmed that the present invention can synthesize functional hemoglobin by linking the amino acid sequences of the four polypeptide chains with linking peptides. Furthermore, recombinant hemoglobin obtained by further tandem synthesis of multiple unit hemoglobins can also yield functional hemoglobin.

[0177] Example 5 Stability Test

[0178] Protein stability is determined by the Tm value. Tm is measured using the Unchained Uncle Protein Stability Analyzer.

[0179] The intrinsic fluorescence of proteins originates from amino acids containing benzene rings (tryptophan, tyrosine, and phenylalanine), which emit photons that can be absorbed (i.e., fluorescence) when excited by ultraviolet light. The intensity of the emitted fluorescence varies depending on the environment of these aromatic amino acids within the protein. Therefore, the trend of intrinsic fluorescence can be used to indicate conformational changes in the tertiary structure of proteins, thereby analyzing protein conformational stability.

[0180] By detecting changes in fluorescence signals during the heating process, a thermal denaturation curve can be obtained, which describes one or more phase transitions during protein denaturation. The midpoint of this transition (Tm) can be used to describe the protein's stability. The higher the Tm, the higher the protein stability.

[0181] The Tm value of the same hemoglobin used in the oxygen-carrying experiment in Example 4 was tested. The average Tm value was not much different from that of the control group bovine hemoglobin, indicating that the stability of the modified hemoglobin maintained the level of the original protein.

[0182] Example 6 P50 Test

[0183] The protein tested in Example 4 was subjected to a P50 test.

[0184] P50 Experimental Principle:

[0185] The measurement principle of the portable oxygen emission function detection system is based on the detection of the optical properties of hemoglobin using dual-wavelength spectrophotometry, and the measurement of oxygen partial pressure (unit: mmHg) using a Clark oxygen electrode. The signals from these two channels are fed back to the computer to plot the oxygen balance curve.

[0186] The absorption spectra of oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) differ significantly, and this difference can be used to observe the oxygen saturation of hemoglobin. A beam of polychromatic light passes through a sample cell, is split into monochromatic light, and then reaches a photomultiplier tube detector. For measuring hemoglobin samples, 570 nm is selected as the reference wavelength, and 560 nm as the measurement wavelength. During the deoxygenation of HbO2 into Hb, the absorbance at the reference wavelength remains essentially unchanged, while the absorbance at the measurement wavelength changes significantly. This change is collected, recorded, and used to plot the Y-axis of a curve.

[0187] The device used in this invention is a portable oxygen emission function detector (Bloodox-2019).

[0188] The device uses a Clark oxygen electrode to directly measure the partial pressure of oxygen, a method that offers high accuracy and repeatability. Under normal atmospheric pressure (760 mmHg), the partial pressure of oxygen in a fully saturated sample is 150 mmHg. This saturation point is used for full-range calibration before plotting the curve.

[0189] When the oxygen on hemoglobin is replaced by an inert gas, such as nitrogen, hemoglobin undergoes the following reaction:

[0190] Specific testing process:

[0191] 1. Before starting the machine, check whether the nitrogen cylinder, air cylinder and their pipelines are twisted or bent, whether the waste liquid bottle is properly installed, and whether the power supply is connected.

[0192] 2. Install the oxygen-permeable membrane. Rinse the surface of the oxygen electrode with distilled water and shake off excess water to ensure it is free of any adhering substances. With the electrode facing upwards, apply a drop of saturated potassium chloride solution to the electrode surface, cover it with the oxygen-permeable membrane, and secure it using an auxiliary device. Adjust the oxygen-permeable membrane to ensure it is flat and tightly adhered to the front end of the electrode. Minimize wrinkles on the oxygen-permeable membrane and avoid air bubbles between the membrane and the electrode.

[0193] 3. After slightly moistening the sample cell with distilled water, slowly push the electrode in, keeping the O-ring fixed in the electrode groove to ensure a proper seal.

[0194] 4. Ensure the oxygen-permeable membrane is installed correctly, the sample cell contains a magnetic stir bar, and the air and nitrogen compression bottles are open with the outlet pressure within the range of 0.1-0.2 MPa. Then, press the power switch on the back of the main unit.

[0195] 5. Activate the oxygen-permeable membrane. Aspirate 4 ml of distilled water into the sample cell. In the BL10 software, select "Control" → "Preparation," and in the opened window, set N2 20 min, Air 10 min, and Repeat 3. Press the Start button to begin. After air is introduced, the PO2 value will increase; after nitrogen is introduced, the PO2 value will decrease. After the cycle is complete, read the PO2 value on the touchscreen. If the PO2 value is around 6 or below 6, the equipment is operating well.

[0196] 6. Sample Preparation. Prepare 10% vitamin C solution, Tween 20, fetal bovine serum, 10 mg / ml hemoglobin sample, buffer, and 1M NaOH. In an EP tube, add 500 μl of serum, 500 μl of hemoglobin sample, 2.9 ml of buffer, and 68 μl of 10% vitamin C. Adjust the pH to neutral by adding an appropriate amount of NaOH, then add 4 μl of Tween 20 and mix well. Wait for the reaction to proceed for 5 minutes. It is recommended that the added sample contain 3-6 mg of hemoglobin.

[0197] 7. After the oxygen permeable membrane is activated, check the settings interface: PO2 150, Temperature 37.0℃, N 28ml / min, Air 6ml / min. If not, adjust accordingly.

[0198] 8. Empty the distilled water from the sample cell and draw in the sample. Press the "Ready" button on the touchscreen, observe whether there is gas in the sample cell, and wait for the temperature and PO2 to stabilize for 10 minutes.

[0199] 9. Once Ready and stable, turn on the light source. Click the ">" button on the touchscreen, adjust the value of A to between 1.5 and 2.5, adjust the value of B to be close to the value of A, and at the same time, adjust the value of lg(A / B) to be close to 0 (0.00XXXX). Adjust PO2 to 150.

[0200] 10. Open the data acquisition software BL10 on your computer. Click Option to open a new window, select SaveFilePath, and set the Operation Data Path, Folder Name (recommended to choose \YYYYMMDD\), and Format (recommended to choose TXT) to save the results. Click Apply and OK to save the settings.

[0201] 11. Click Experiments to open a new window. Enter the PO2Saturated value as the starting point for the oxygen dissociation curve. MaximumCount is the time limit for curve acquisition. For example, when MaximumCount = 1800, the curve will automatically stop being plotted after the real-time Count value reaches 1800 (i.e., 1800 seconds). You can manually stop the acquisition at any time by clicking the Stop button on the software acquisition interface before the Count value reaches the set MaximumCount value. Enter the sample number Sample (required), and enter the experimental group StudyNo and Comment as needed. These two fields are optional.

[0202] 12. Press the Start button to begin the deoxygenation process. At this time, the N2 button on the touch screen will be automatically activated, and the gas introduced into the sample cell will be automatically converted from air to nitrogen. The software will record the deoxygenation curve in real time.

[0203] 13. When the curve plotting is nearing completion, if the PO2 value changes very slowly or stabilizes at a certain value, manually stop the plot by pressing the Stop button on the software. Alternatively, you can wait for the real-time Count value to reach the set value MaximumCount and then stop automatically.

[0204] 14. After the curve is drawn, turn off the LampPower button on the host control panel.

[0205] 15. After the curve is drawn, turn off the LampPower button on the host control panel.

[0206] 16. Aspirate approximately 4 ml of buffer solution and retain it in the sample reservoir.

[0207] Note: If the sample cell needs to be removed from the slot during the above process and the device sounds an alarm, you need to turn off LampPower on the control panel, or click RESET to restore the parameter settings (step 7). Before starting to collect curves, double-check the settings of each parameter.

[0208] Test results showed that the P50 of the aforementioned single recombinant hemoglobin increased from approximately 13 mmHg to approximately 30 mmHg; the P50 of the aforementioned recombinant hemoglobin (multiple recombinant hemoglobin units tandem) also increased from approximately 13 mmHg to approximately 30 mmHg. This meets the usage requirements.

[0209] The sequences mentioned above in this article are summarized in Table 4.

Claims

1. A recombinant hemoglobin, characterized in that, include: Two or more units of recombinant hemoglobin, The recombinant hemoglobin unit comprises at least two polypeptide chains, wherein the polypeptide chains are any one or more combinations of α chains and β chains; The recombinant hemoglobin unit is formed by linking the amino acid sequences of the polypeptide chains of each unit recombinant hemoglobin unit with intra-unit linking peptides. Two or more units are linked together by inter-unit linking peptides; Preferably, the unit recombinant hemoglobin comprises four polypeptide chains, namely two α chains and two β chains.

2. The recombinant hemoglobin according to claim 1, characterized in that: in, The intra-unit linking peptides connect the amino acid sequences of the four polypeptide chains sequentially from the N-terminus to the C-terminus in any order, and the inter-unit linking peptides connect each unit sequentially from the N-terminus to the C-terminus.

3. The recombinant hemoglobin according to claim 1 or 2, characterized in that: in, The number of amino acids in the linking peptide within the unit is greater than or equal to 1, 1-140, 1-120, 1-90, 1-30, 1-35, or 1-40.

4. The recombinant hemoglobin according to claim 3, characterized in that: The intra-unit linker peptide used to connect two α chains is called the first linker peptide. The number of amino acids in the first linker peptide is 1-5, preferably 1, 2, or 3. The intra-unit linker peptide used to link two β chains or one α chain and one β chain is a second linker peptide, and the number of amino acids in the second linker peptide is greater than or equal to 3, greater than or equal to 5, 5-140, 5-130, 5-120, 5-90, 5-85, 5-30, 5-35, 5-40, 10-30, 10-35 or 10-40 amino acids.

5. The recombinant hemoglobin according to claim 4, characterized in that: The first linker peptide has one glycine amino acid.

6. The recombinant hemoglobin according to claim 4 or 5, characterized in that: The second linker peptide contains any one or more major amino acid residues of G, S, T and A, and the number of the major amino acid residues accounts for at least 45%, 50%, 60%, 70%, 80% or 90% of the total number of amino acid residues in the second linker peptide. More preferably, the major amino acid residues contain any one or more amino acids of G and S, and the total number of G and S accounts for at least 40% or 45% or 60% or 70% or 80% of the total number of amino acids in the second linker peptide.

7. The recombinant hemoglobin according to claim 4 or 5, characterized in that: The second linker peptide contains at least a flexible unit.

8. The recombinant hemoglobin according to claim 7, characterized in that: The second linker peptide also contains hydrophilic and hydrophobic units composed of alternating hydrophilic and hydrophobic amino acids. In this case, from the N-terminus to the C-terminus, the amino acid sequence of the second linker peptide is: flexible single... The unit is a flexible unit composed of alternating hydrophilic and hydrophobic amino acids.

9. The recombinant hemoglobin according to claim 7 or 8, characterized in that: The structure of the flexible unit is (GGGGS)a. Preferably, a is 1-4. More preferably, the flexible unit is selected from: (1) GGGGS; (2) GGGGSGGGGS; (3)GGGGSGGGGSGGGGSGGGGS.

10. The recombinant hemoglobin according to claim 8 or 9, characterized in that: The structure of the affinity unit is any one or more of (RADA)b, (KLDL)c, (LK)d, and (LD)f. Preferably: b is 1-5, c is 1-5, d is 1-10, f is 1-10. More preferably, the affinity unit is selected from: (1) RADARADARADARADA; (2) KLDLKLDLKLDL; (3)LKLKLKLKLKLK; (4)LDLDLDLDLDLD.

11. The recombinant hemoglobin according to any one of claims 4-10, characterized in that: in, The amino acid sequence of the second linker is any one of SEQ ID NO: 1-6, 10-12, 21, and 22, or has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence homology with any one of SEQ ID NO: 1-6, 10-12, 21, and 22. Preferably, the amino acid sequences of all the second linker peptides are all the same, partially the same, or all different.

12. The recombinant hemoglobin according to any one of claims 1-11, characterized in that: The amino acid sequences of the four polypeptide chains, from the N-terminus to the C-terminus, are linked in any of the following order: α chain - α chain - β chain - β chain, β chain - β chain - α chain - α chain, β chain - α chain - α chain - β chain, α chain - β chain - α chain - β chain and β chain - α chain - β chain - α chain.

13. The recombinant hemoglobin according to any one of claims 1-12, characterized in that: in, The α chain is derived from any one or more of humans, pigs, or cattle; and the β chain is derived from any one or more of humans, pigs, or cattle; and / or The amino acid sequence of the α chain is any one or more of SEQ ID NO: 7, 13 and 14, or has at least 80%, 85%, 90%, 95%, 97 or 99% sequence homology with any one or more of SEQ ID NO: 7, 13 and 14, respectively. The amino acid sequence of the β chain is any one or more of SEQ ID NO: 8, 15, and 16, or has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence homology with any one or more of SEQ ID NO: 8, 15, and 16, respectively. Compare.

14. The recombinant hemoglobin according to any one of claims 1-11, characterized in that, Also includes: Heme that is bound to each of the aforementioned polypeptide chains.

15. The recombinant hemoglobin according to any one of claims 1-14, characterized in that: in, The number of amino acids in the inter-unit linking peptide is greater than or equal to 0, 0-100, 0-90, or 0-85, and / or The inter-unit linker peptide contains a combination of any one or more amino acid residues from G, S, A, and T, and / or the component linker peptide contains a combination of any one or more amino acid residues from E and D. Preferably, the percentage of any one or more amino acid residues from G, S, T, and A in the inter-unit linker peptide relative to the total number of amino acid residues in the inter-unit linker peptide is at least greater than 45%, 50%, 60%, 70%, 80%, or 90%. Further, the amino acid sequence of the inter-unit linker peptide is any one of SEQ ID NO: 17-20, or has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence homology with any one or more of SEQ ID NO: 17-20, respectively. and / or The number of units is 2, 3, or 4.

16. A nucleic acid, characterized in that, include: Nucleotides encoding the recombinant hemoglobin according to any one of claims 1-15.

17. A carrier, characterized in that, include: The nucleic acid vector according to claim 16.

18. A host cell, characterized in that, include: The nucleic acid of claim 16 and / or the vector of claim 17, Preferably, the host cell is selected from prokaryotic cells or eukaryotic cells; further, the host cell is selected from yeast cells; even further, the yeast cell is selected from one or more combinations of Saccharomyces cerevisiae and Kluyveromyces genus yeasts; in another preferred embodiment, the Kluyveromyces genus yeast is selected from one or more combinations of Kluyveromyces lactis, Kluyveromyces marx, and Kluyveromyces dob.

19. The use of the nucleic acid of claim 16, the vector of claim 17, or the host cell of claim 18 in the preparation of the recombinant hemoglobin of any one of claims 1-15.

20. An in vitro cell-free protein synthesis system, characterized in that, include: Cell extracts; as well as The mRNA or DNA template encoding the recombinant hemoglobin according to any one of claims 1-15; preferably, the cell extract is the yeast cell extract, further, derived from any one of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces var. kusnezoffii. Or a combination of multiple; preferably, the yeast cell extract is derived from: Kluyveromyces, more preferably Kluyveromyces lactis, and even more preferably from Kluyveromyces; Furthermore, it also includes one or more of the following components: Amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, energy supply system, polyethylene glycol, and aqueous solvent.

21. The method for in vitro cell-free synthesis of recombinant hemoglobin according to any one of claims 1-15, characterized in that, include: Provides a cell-free in vitro protein synthesis system; The recombinant hemoglobin is obtained by adding an mRNA or DNA template encoding the recombinant hemoglobin according to any one of claims 1-15 to the cell-free protein synthesis system and performing an in vitro synthesis reaction. Preferably, the volume ratio of the DNA template to the cell-free in vitro protein synthesis system is 1:10 to 1:50, more preferably 1:20 to 1:40; most preferably 1:25 to 1:35, and particularly preferably 1:

30.

22. The in vitro cell-free synthesis method according to claim 21, characterized in that, Also includes: Simultaneously, heme is added to the cell-free synthesis system, during or after the in vitro synthesis reaction to generate recombinant hemoglobin bound with the heme. Preferably, the added heme contains ferric iron.

23. The in vitro cell-free synthesis method according to claim 21 or 22, characterized in that: in, The cell-free in vitro protein synthesis system includes: yeast cell extract, amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, energy supply system, polyethylene glycol, and aqueous solvent; Furthermore: The yeast cell extract is derived from any one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces var. sacchari; Best location: The yeast cell extract is derived from: Kluyveromyces, more preferably Kluyveromyces lactis; or Yeast extract accounts for 50-80% of the entire cell-free in vitro protein synthesis system.

24. The use of recombinant hemoglobin according to any one of claims 1-15 as a blood substitute.

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