Recombinant hemoglobin

Abstract

The present invention provides a recombinant hemoglobin, a nucleic acid encoding the same, and a method for producing the same, in order to improve the stability of hemoglobin having a tetrameric structure and solve problems such as complicated processes, high production costs, and low yields. The recombinant hemoglobin comprises four polypeptide chains, each of which is composed of two α chains and two β chains. The amino acid sequences of the four polypeptide chains are connected by a linker peptide, and preferably, the linker peptide sequentially connects the amino acid sequences of the four polypeptide chains in any order from the N-terminus to the C-terminus.

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology, and specifically relates to recombinant hemoglobin, a nucleic acid encoding the recombinant hemoglobin, and a method for producing the same. [Background technology]

[0002] Hemoglobin is a protein that transports oxygen within spherical red blood cells. The molecular weight of native hemoglobin is 64.5 kD. Hemoglobin is composed of four polypeptide chains (subunits) bound by non-covalent interactions. The main component of human whole blood is hemoglobin A, which has an α2β2 structure, in which the α chain contains 141 amino acid residues and the β chain contains 146 amino acid residues. The α and β chains are tightly bound to form a dimer, and two αβ dimers are loosely bound to form a hemoglobin molecule.

[0003] Blood products (defined as any therapeutic substance extracted from human blood, including whole blood, labile blood components, and plasma-derived medicines) save millions of lives each year, significantly extend the life expectancy of patients at risk of death, improve quality of life, and support complex medical and surgical procedures.

[0004] However, in the face of known and emerging public health threats, blood supply services worldwide face significant challenges in ensuring the quality and safety of blood products and the supply of sufficient blood products to meet patient needs. According to 2002 statistics, more than 750 million units of blood are donated worldwide each year, yet only 43 of the World Health Organization's blood donors (with a total of 191 member states) were tested for hepatitis B virus (HBV), hepatitis C virus (HCV), and AIDS virus (HIV). Unsafe transfusions or injections result in 80,000–160,000 HBV infections, 23,000–47,000 HCV infections, and 80,000–160,000 HIV infections each year. Furthermore, the discrepancy between increasing demand for blood and declining donations, hemolysis and even death due to incorrect transfusions, the short shelf life of human red blood cells, natural disasters, and wars have forced people to seek alternatives to blood.

[0005] Hemoglobin-based red blood cell substitutes are hemoglobin-based biological medicines with oxygen-carrying function. They can be classified into three categories: human blood-based red blood cell substitutes, animal blood-based red blood cell substitutes, and recombinant hemoglobin-based red blood cell substitutes.

[0006] However, currently, unmodified hemoglobin cannot be used as a blood substitute. One of the main reasons is that free hemoglobin tetramers decompose into αβ dimers and monomers, which can cause numerous side effects. To reduce side effects, stabilizing the hemoglobin tetramer is a prerequisite for using hemoglobin as a blood substitute. Currently, chemical modification methods such as crosslinking, polymerization, and coupling are commonly used to increase the molecular weight of hemoglobin. Chemically modified products can also be achieved using biotechnology, such as site-directed mutagenesis of the hemoglobin gene at the DNA level.

[0007] However, although chemical modification methods are relatively mature, they still have some side effects that must be addressed urgently. (1) Due to the different chemical reagents and methods used and the nonspecific nature of the crosslinking reaction, it is difficult to control the molecular size, oxygen affinity, subunit synergy, stability, and toxic side effects of the hemoglobin product, resulting in a heterogeneous mixture of the final product. (2) Because the issue of blood disinfection has not been resolved, contamination with pathogenic microorganisms still exists, posing a risk of blood-borne diseases such as mad cow disease and acute and chronic immunogenicity in humans. (4) The instability of the final product leads to rearrangement of crosslinked hemoglobin. Because free crosslinkers are cytotoxic, whether they are released from crosslinked hemoglobin is a notable issue. Furthermore, chemical modification can involve complex processes, increasing production costs and reducing production efficiency.

[0008] However, site-specific mutation of the hemoglobin gene at the DNA level can be blind and affect the final function.

[0009] Alternatively, recombinant hemoglobin can be produced by co-expressing α and β hemoglobin in cells, but this often results in very low yields and requires cross-linking chemical modification to increase the stability of the tetrameric structure after production.

[0010] It is recognized that the prior art has deficiencies for providing hemoglobin with a stable tetrameric structure. Summary of the Invention

[0011] The present invention provides recombinant hemoglobin, a nucleic acid encoding the same, and a method for producing the same, in order to improve the stability of hemoglobin having a tetrameric structure and solve problems such as complicated processes, high production costs, significant side effects, and low yields.

[0012] To this end, the present invention provides the following technical solutions:

[0013] The present invention provides a recombinant hemoglobin comprising four polypeptide chains, each of which is two α chains and two β chains, wherein the amino acid sequences of the four polypeptide chains are connected by a linker peptide.

[0014] The recombinant hemoglobin provided by the present invention further has the following characteristics: the three linker peptides sequentially connect the amino acid sequences of the four polypeptide chains from the N-terminus to the C-terminus in any order.

[0015] The recombinant hemoglobin provided by the present invention further has the following characteristics: the number of amino acids in the linker peptide is 1 to 90, 1 to 30, 1 to 35, or 1 to 40.

[0016] The recombinant hemoglobin provided by the present invention further has the following characteristics: the linker peptide connecting two α chains is a first linker peptide, and the number of amino acids in the first linker peptide is 1 to 5, preferably 1, 2, or 3; the linker peptide connecting 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 5 to 90, 5 to 85, 5 to 30, 5 to 35, 5 to 40, 10 to 30, 10 to 35, or 10 to 40.

[0017] The recombinant hemoglobin provided by the present invention further has the following characteristics: the first linker peptide has one amino acid, glycine.

[0018] The recombinant hemoglobin provided by the present invention is further characterized in that the second linker peptide contains major amino acid residues selected from any one or more of G, S, T, and A, and the number of such 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 include one or more of G and S, and the total amount accounts for at least 40%, or more than 45%, or more than 60%, or more than 70%, or more than 80% of the total number of amino acids in the second linker peptide.

[0019] The recombinant hemoglobin provided by the present invention further has the following characteristics: the second linker peptide comprises at least a flexible unit.

[0020] The recombinant hemoglobin provided by the present invention further has the following characteristic: the second linker peptide comprises an affinity unit in which hydrophilic amino acids and hydrophobic amino acids are arranged alternately, and the order of amino acids in the second linker peptide from the N-terminus to the C-terminus is flexible unit-affinity unit in which hydrophilic amino acids and hydrophobic amino acids are arranged alternately-flexible unit.

[0021] The recombinant hemoglobin provided by the present invention further has the following characteristics: the structure of the flexible unit is (GGGGS)a, preferably, a is 1 to 4; More preferably, the flexible unit is (1)GGGGS, (2)GGGGSGGGGS, (3)GGGGSGGGGSGGGGSGGGGS is selected from.

[0022] The recombinant hemoglobin provided by the present invention further has the following characteristic: the affinity unit structure is one or more of (RADA)b, (KLDL)c, (LK)d, and (LD)f, and preferably, b is 1 to 5, c is 1 to 5, d is 1 to 10, and f is 1 to 10; More preferably, the affinity unit is (1) RADARADA RADARADA, (2) KLDLKLDLKLDL, (3) LKLKLKLKLKLK, (4)LDLDLDLDLDLD is selected from.

[0023] The recombinant hemoglobin provided by the present invention further has the following characteristics: the amino acid sequence of the second linker peptide is any of SEQ ID NOs: 1 to 6.

[0024] The recombinant hemoglobin provided by the present invention further has the following characteristic: the amino acid sequences of the four polypeptide chains are linked, from the N-terminus to the C-terminus, in any one 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.

[0025] The recombinant hemoglobin provided by the present invention further has the following characteristics: the α chain is derived from one or more of human, porcine or bovine origin, and the β chain is derived from one or more of human, porcine or bovine origin; and / or the amino acid sequence of the alpha chain is any one or more of SEQ ID NOs: 7, 13 and 14, or has a percentage of sequence identity of at least 80%, 85%, 90%, 95%, 97% or 99% with any one or more of SEQ ID NOs: 7, 13 and 14, respectively; The amino acid sequence of the beta chain is any one or more of SEQ ID NOs: 8, 15 and 16, or has a percentage of sequence identity of at least 80%, 85%, 90%, 95%, 97% or 99% with any one or more of SEQ ID NOs: 8, 15 and 16, respectively.

[0026] The recombinant hemoglobin provided by the present invention is further characterized in that it further comprises hemin bound to each of the polypeptide chains.

[0027] The present invention further provides a nucleic acid comprising nucleotides encoding the aforementioned recombinant hemoglobin.

[0028] The present invention further provides a vector comprising the aforementioned nucleic acid.

[0029] The present invention further provides a host cell, characterized in that it contains the above-mentioned nucleic acid and / or the above-mentioned vector, Preferably, the host cell is derived from a prokaryotic cell or a eukaryotic cell, and further, the host cell is derived from a bacterium, a mammalian cell, a human cell, a plant cell, a yeast cell, or an insect cell, and is preferably selected from Escherichia coli or a yeast cell, and further, the yeast cell is selected from one or more combinations of Saccharomyces cerevisiae, Kluyveromyces, and in another preferred embodiment, the Kluyveromyces is selected from one or more combinations of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanski.

[0030] Use of the aforementioned nucleic acid, vector or host cell of the invention in the production of the aforementioned recombinant hemoglobin.

[0031] The present invention further provides an in vitro cell-free protein synthesis system, characterized by comprising a cell extract and an mRNA or DNA template encoding the recombinant hemoglobin described above, wherein the cell extract is preferably an Escherichia coli extract or a yeast cell extract, and further, the cell extract is derived from any combination of one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces, and preferably the yeast cell extract is derived from Kluyveromyces, more preferably from any combination of one or more of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii, Additionally, the following ingredients: The enzyme comprises one or more of an amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent.

[0032] The present invention further provides a method for in vitro cell-free synthesis of the aforementioned recombinant hemoglobin, the method comprising: To provide an in vitro cell-free protein synthesis system, The method includes adding an mRNA or a DNA template encoding the recombinant hemoglobin to a cell-free protein synthesis system and carrying out an in vitro synthesis reaction to obtain the recombinant hemoglobin, and the volume ratio of the DNA template to the in vitro cell-free protein synthesis system is preferably 1:10 to 1:50, more preferably 1:20 to 1:40, most preferably 1:25 to 1:35, and particularly preferably 1:30.

[0033] The in vitro cell-free protein synthesis method provided by the present invention further comprises adding hemin to the cell-free protein synthesis system to produce recombinant hemoglobin bound to the hemin. The order of addition of hemin is not limited, and the hemin can be added before or after in vitro synthesis using the cell-free protein synthesis system.

[0034] Preferably, the added hemin contains ferric iron.

[0035] The in vitro cell-free synthesis method provided by the present invention further has the following characteristics: the cell-free protein synthesis system includes a cell extract; Furthermore, the extracellular protein synthesis system comprises any combination of one or more of an amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent; and / or The cell extract is an Escherichia coli extract, a yeast cell extract, and further derived from any combination of one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces, preferably the yeast cell extract is derived from Kluyveromyces, more preferably derived from any combination of one or more of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii, or Yeast extracts account for 50-80% of all in vitro cell-free protein synthesis systems.

[0036] The present invention further provides the aforementioned recombinant hemoglobin for use as a blood substitute and in the treatment of cancer, stroke, hemorrhagic shock, acute mountain sickness (AMS), peripheral arterial disease (PAD) and Parkinson's disease (PD).

[0037] The recombinant hemoglobin, the nucleic acid encoding it, and the method for producing it provided by the present invention have at least the following advantages: (1) Recombinant hemoglobin has four polypeptide chains linked in tandem in the primary structure via a linker peptide. Therefore, during the synthesis and expression of recombinant hemoglobin in vivo or in vitro, the tandem α and β chains are likely to interact to form a monomeric structure with similar functions to tetrameric hemoglobin. The linkage in the primary structure makes such a structure more stable and less likely to dissociate, allowing for the formation of a stable tetramer. Furthermore, the tandem α and β chain ratio is 1:1, which avoids the formation of other aggregate structures due to insufficient matching between the separated α and β chains. This results in a higher yield and more convenient production. No additional chemical cross-linking is required, which avoids the side effects of chemical modification, simplifies the process, and reduces costs. (2) The present invention enables direct production of recombinant hemoglobin by in vitro cell-free synthesis using a nucleic acid encoding the recombinant hemoglobin, which is obtained by linking four polypeptide chains in tandem in the primary structure, as a template, resulting in rapid production speed and low cost. [Brief explanation of the drawings]

[0038] [Figure 1] The results of activity tests of EGFP-fused hemoglobin (scHemoglobin-EGFP) obtained by six types of in vitro synthesis reactions are shown in terms of fluorescence values. [Figure 2] This is a graph showing the test results of the hemoglobin purification effect of six types of scHemoglobin-EGFP, where a higher fluorescence value indicates a larger amount of protein obtained. [Figure 3] The figure shows the results of SDS-PAGE electrophoresis of protein bands in the eluate after purification of six types of scHemoglobin-EGFP. [Figure 4] The figure shows the results of SDS-PAGE electrophoresis of protein bands of hemoglobin (scHemoglobin) not containing EGFP fusion protein obtained by six types of IVTT reactions and purification. [Figure 5] 5 and 6 are graphs showing different experimental results regarding the effect of adding hemin to a reaction solution for synthesizing EGFP protein (PC) in an in vitro synthesis reaction. [Figure 6] 5 and 6 are graphs showing different experimental results regarding the effect of adding hemin to a reaction solution for synthesizing EGFP protein (PC) in an in vitro synthesis reaction. [Figure 7] The photographs show the reaction mixture of 15 types of scHemoglobin synthesized in an in vitro synthesis reaction, to which hemin was added at a final concentration of 20 μM, and after expression and purification, each solution was replaced with PBS. [Figure 8] This shows the results of SDS-PAGE electrophoresis after standard expression and purification in an in vitro synthesis reaction system containing hemin but no DNA template. [Figure 9] 1 shows the spectral characteristics of hemoglobin in the oxygen-carrying and anoxic states as disclosed in Reference 1. [Figure 10] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 11] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 12] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 13] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 14] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 15] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 16]In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 17] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 18] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 19] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 20] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 21] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 22] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 23] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. [Figure 24] In Example 4, 15 types of scHemoglobin were bound to hemin, and then the detected spectral characteristics were shown with reference to Reference 1. DETAILED DESCRIPTION OF THE INVENTION

[0039] Specific embodiments of the present disclosure are described below in conjunction with the accompanying drawings. Regarding the specific methods or materials used in the examples, those skilled in the art can make conventional alternative selections based on the technical concept of the present invention and existing techniques, and are not limited to the specific descriptions of the examples of the present disclosure.

[0040] The methods used in the examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0041] The definitions of terms used herein are intended to incorporate generally accepted prior art definitions for each term in the biotechnology field. Examples are provided where appropriate. These definitions apply to terms as they are used throughout this specification, unless otherwise limited in specific instances, either alone or as part of a larger group.

[0042] As used herein, "recombinant hemoglobin" refers to a hemoglobin molecule and / or variants thereof having a molecular size of at least about 65 kDa and which has not been isolated or purified from an animal or human source but has been synthesized by standard molecular biology techniques.

[0043] As used herein, "variant" refers to a polypeptide or polynucleotide sequence that differs from a reference polypeptide or polynucleotide sequence, but retains essential properties thereof. Typically, variants are generally closely similar, and in many regions identical, to the reference polypeptide or polynucleotide sequence.

[0044] For example, a variant may comprise the amino acid sequence of a parent polypeptide sequence with at least one conservative amino acid substitution, or a variant may comprise the amino acid sequence of a parent polypeptide sequence with at least one non-conservative amino acid substitution, where the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant, and the non-conservative amino acid substitution may enhance the biological activity of the variant, increasing the biological activity of the variant compared to the parent polypeptide.

[0045] The term "percentage of sequence identity" when used with respect to polypeptide or polynucleotide sequences refers to a comparison between a polynucleotide and a polypeptide, and is determined by comparing two optimally 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 (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues occur, calculating the number of matching positions, dividing this 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 calculate the percentage of sequence identity. Homology can be evaluated using any of a variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW. In certain embodiments, the Basic Local Alignment Search Tool ("BLAST"), known in the art, is used to assess protein and nucleic acid sequence homology (see, e.g., 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 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402).

[0046] As used herein, "heme" refers to ferrous heme, an iron porphyrin compound that is a cofactor for hemoglobin, as well as a cofactor for myoglobin, cytochrome, peroxidase, catalase, and the like.

[0047] As used herein, the term "active structure" refers to the fact that the produced recombinant hemoglobin has a function such as the ability to transport oxygen and can be produced or used as a blood substitute; for example, the protein before binding to hemin has a function such as the ability to transport oxygen after binding to hemin and can be produced as a blood substitute, and the protein before binding to hemin is considered to have an active structure.

[0048] 1. Recombinant hemoglobin The recombinant hemoglobin provided by the present invention comprises four polypeptide chains, each of which is composed of two α chains and two β chains, and the amino acid sequences of the four polypeptide chains are connected by linker peptides. The order and position of the linker peptides connecting the amino acid sequences of the four polypeptide chains are not particularly specified and may be in any order or position. That is, the four polypeptide chains are connected to each other in the primary structure by one or more linker peptides.

[0049] In a preferred example, the three linker peptides sequentially link the amino acid sequences of the four polypeptide chains in any order from the N-terminus to the C-terminus, i.e., the four polypeptide chains are linked in tandem, 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, where "-" represents a linker peptide, and the amino acid sequences of the three linker peptides may be the same, or only two may be the same, or all may be different.

[0050] The number of amino acids in the linker peptide is intended to ensure that the recombinant hemoglobin forms a functional active structure and maintains a certain level of stability, and preferably, the number of amino acids in the linker peptide is 1 to 90, 1 to 30, 1 to 35, or 1 to 40.

[0051] In one example, the linker peptide comprises two types: One is used to link the two α chains and is called the first linker peptide. Preferably, the number of amino acids in the first linker peptide is 1 to 5, more preferably 1, 2, or 3; The other is used to link two β chains or one α chain and one β chain, and is called a second linker peptide. Preferably, the number of amino acids in the second linker peptide ranges from 5 to 90, 5 to 85, 5 to 30, 5 to 35, 5 to 40, 10 to 30, 10 to 35, and 10 to 40.

[0052] In one example, the first linker peptide has one amino acid that is glycine.

[0053] In one example, the second linker peptide contains one or more major amino acid residues selected from G, S, T, and A, and the number of these 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 include one or more G and S amino acids, and the total amount accounts for at least 40%, 45%, 60%, 70%, or 80% of the total number of amino acids in the second linker peptide. The amino acid sequence of the second linker peptide is, for example, SEQ ID NOs: 10 to 12.

[0054] In one example, the second linker peptide comprises at least a flexible unit.

[0055] In one example, the flexible unit has a structure of (GGGGS)a, where a is preferably 1 to 4. More preferably, the flexible unit has a structure of: (1)GGGGS, (2)GGGGSGGGGS, (3)GGGGSGGGGSGGGGSGGGGS is selected from.

[0056] In one example, the second linker peptide comprises an affinity unit in which hydrophilic amino acids and hydrophobic amino acids are arranged alternately, and the order of amino acids in the second linker peptide from the N-terminus to the C-terminus is flexible unit-affinity unit in which hydrophilic amino acids and hydrophobic amino acids are arranged alternately-flexible unit.

[0057] As used herein, alternating hydrophilic and hydrophobic amino acids refers to the following different situations: (1) One hydrophilic amino acid and one hydrophobic amino acid alternate, (2) One or more consecutive hydrophilic amino acids form a hydrophilic group, and one or more consecutive hydrophobic amino acids form a hydrophobic group, wherein the amino acids in each group may be the same or different; for example, in each hydrophilic group, the hydrophilic amino acids may be the same or different, and the amino acid composition between different hydrophilic groups or different hydrophobic groups may be the same or different, for example.

[0058] In one example, the structure of the affinity unit is one or more of (RADA)b, (KLDL)c, (LK)d, and (LD)f, and preferably, b is 1 to 5, c is 1 to 5, d is 1 to 10, and f is 1 to 10.

[0059] More preferably, the affinity unit is (1) RADARADA RADARADA, (2) KLDLKLDLKLDL, (3) LKLKLKLKLKLK, (4)LDLDLDLDLDLD is selected from.

[0060] In one example, the amino acid sequence of the second linker peptide is SEQ ID NO: 1 to SEQ ID NO: 6.

[0061] In one example, the amino acid sequence of the alpha chain has SEQ ID NO: 7 or a percentage of sequence identity with SEQ ID NO: 7 of at least 80%, 85%, 90%, 95% or 99%, and the amino acid sequence of the beta chain has SEQ ID NO: 8 or a percentage of sequence identity with SEQ ID NO: 8 of at least 80%, 85%, 90%, 95% or 99%.

[0062] 2. The present invention further provides a nucleic acid comprising a nucleotide sequence encoding the aforementioned recombinant hemoglobin.

[0063] The present invention further provides a vector comprising the above nucleic acid.

[0064] The present invention further provides a host cell comprising the nucleic acid and / or the vector, wherein the host cell is of prokaryotic or eukaryotic origin, and further wherein the cell is selected from one or more combinations of E. coli cells, human cells, Chinese hamster cells, ovary cells, insect cells, wheat germ cells, rabbit reticulocytes, and yeast cells.

[0065] Further, the host cell is selected from a yeast cell, and further, the yeast cell is selected from one or more combinations of Saccharomyces cerevisiae, Kluyveromyces, and in another preferred embodiment, the Kluyveromyces is selected from one or more combinations of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii.

[0066] The nucleic acids, vectors or host cells provided by the present invention can be used to produce recombinant hemoglobin.

[0067] The present invention further provides an in vitro cell-free protein synthesis system comprising a cell extract and an mRNA or DNA template encoding the aforementioned recombinant hemoglobin.

[0068] In one example, the cell extract is an Escherichia coli extract, a yeast cell extract, or further derived from one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces, preferably the yeast cell extract is derived from Kluyveromyces, more preferably derived from any combination of one or more of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii.

[0069] In one example, the in vitro cell-free synthesis system further comprises one or more of an amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent.

[0070] 3. A method for in vitro cell-free synthesis of recombinant hemoglobin, comprising: To provide an in vitro cell-free protein synthesis system, The method comprises adding the mRNA or DNA template encoding the recombinant hemoglobin to a cell-free protein synthesis system and carrying out an in vitro synthesis reaction of a single-chain polypeptide.

[0071] The volume ratio of the DNA template to the in vitro cell-free protein synthesis system is 1:10 to 1:50, preferably 1:20 to 1:40, most preferably 1:25 to 1:35, and particularly preferably 1:30.

[0072] In one example, the in vitro cell-free protein synthesis system comprises a cell extract.

[0073] In one example, the in vitro cell-free protein synthesis system includes any combination of one or more of an amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent.

[0074] In one example, the cell extract is an Escherichia coli extract, a yeast cell extract, and further derived from one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces, preferably the yeast cell extract is derived from Kluyveromyces, more preferably derived from any combination of one or more of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii.

[0075] In one example, the yeast extract accounts for 50 to 80% of the entire in vitro cell-free protein synthesis system.

[0076] An in vitro protein synthesis reaction refers to a protein synthesis reaction in an in vitro cell-free synthesis system that includes at least a translation process. Examples include, but are not limited to, an IVT reaction (in vitro translation reaction), an IVTT reaction (in vitro transcription / translation reaction), and an IVDTT reaction (in vitro replication / transcription / translation reaction). In the present invention, an IVTT reaction is preferred. The IVTT reaction, which corresponds to the IVTT system, is a process of transcribing and translating DNA into protein outside the body. Therefore, this type of in vitro protein synthesis system is also called a D2P system, a D-to-P system, a D_to_P system, or a DNA-to-protein system, and the corresponding in vitro protein synthesis method is also called a D2P method, a D-to-P method, a D_to_P method, or a DNA-to-protein method.

[0077] In a preferred embodiment, in the in vitro cell-free synthesis method of the present invention, the technical elements of the in vitro protein synthesis system of the present invention, the template, the plasmid, the target protein, the in vitro protein synthesis reaction (culture reaction), various production methods, various detection methods, etc. can be further independently selected from the following documents as appropriate embodiments or implementation methods: CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109 These include, but are not limited to, CN423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, and CN110964736A. Unless inconsistent with the objectives of the present invention, these documents and the references therein are incorporated by reference in their entirety for all purposes.

[0078] The present invention can synthesize the above-mentioned recombinant hemoglobin by the above-mentioned in vitro cell-free synthesis method, which has a short cycle, simple operation, low cost, and can provide stable hemoglobin having the above-mentioned active structure.

[0079] Furthermore, the in vitro cell-free synthesis method The method further comprises adding hemin to the cell-free protein synthesis system simultaneously or after the in vitro synthesis reaction to produce recombinant hemoglobin bound to the hemin, and preferably the added hemin contains ferric iron. [Example]

[0080] The present invention will be further described below with reference to specific examples.

[0081] In the following examples: (1) The IVTT reaction system consisted of 4-hydroxyethylpiperazineethanesulfonic acid at a final concentration of 22 mM, pH 7.4, 30 to 150 mM potassium acetate, 1.0 to 5.0 mM magnesium acetate, 1.5 to 4 mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, uridine triphosphate), 0.08 to 0.24 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine), 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphate kinase, and 0.027 to 0.054 mg / mL T7. Add RNA polymerase, 1%–4% polyethylene glycol, 0.5%–2% sucrose, and finally 50–80% of the volume of yeast cell extract. The yeast cell extract was derived from Kluyveromyces.

[0082] (2) Transformation of each target plasmid was carried out as follows: 1 μl of target plasmid was added to 20 μl of DH5α, left on ice for 30 minutes, heat shocked at 42°C for 45 seconds, left on ice for 2 minutes, 500 μl of medium was added, and cultured with shaking (200 rpm) at 37°C for 1 hour. 100 μl was pipetted onto an LB (antibody-containing) plate and cultured inverted at 37°C for 12–16 hours, then removed and stored in a 4°C refrigerator.

[0083] (4) Extraction and amplification of transformation plasmid: Amplification system: Final concentrations of 20-30 μM random primers, 0.05-0.15 μg / mL plasmid template, 0.5-1 mM dNTPs, 2x BSA, 1x phi29 reaction buffer (components: 50 mM Tris-HCl, 10 mM MgCl, 10 mM (NH)SO, 4 mM DTT, pH 7.5). For example, for a 10 mL amplification system: 10 ml of amplification system, 4 ng / μl of plasmid DNA at a final concentration, and 5 μl of 0.05-0.1 mg / mL phi29 DNA polymerase were mixed and incubated at 37°C and 30 rpm for 2 hours. The resulting product was then electrophoresed on a 1% agarose gel and identified. The resulting product was used as a DNA template.

[0084] In the following examples, all structures constructed are from N- to C-terminus unless otherwise noted.

[0085] Example 1. Expression and purification of EGFP-fused hemoglobin Different linkers were designed to link the four hemoglobin subunits. The four polypeptide chains were linked in tandem to form a single-chain scHemoglobin. EGFP protein was fused to the C-terminus of scHemoglobin to detect protein expression. The sequences are numbered hemo006, hemo007, hemo008, hemo009, hemo010, and hemo011, respectively. The corresponding tandem sequences for each subunit are listed in Table 1. In this example, 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.

[0086] [Table 1]

[0087] Single-chain hemoglobin genes with different linker peptides were synthesized by gene synthesis and directly inserted into our optimized plasmid (D2P1.08t) using BamHI and HindIII as insertion sites. A total of six plasmids containing the target gene fragments hemo006 to hemo011 were obtained, and DNA templates encoding the structures shown in Table 1 were obtained by transformation and amplification.

[0088] 15 ng / μL of DNA template was added to each IVTT reaction system. After uniform mixing, the mixture was placed in an environment of 25-30°C and reacted for 3 hours. After the reaction was completed, the reaction solution was collected and the RFU fluorescence value of the EGFP fusion protein was measured using a microplate reader.

[0089] The products of the six proteins synthesized and expressed by IVTT were detected by fluorescence, and it was confirmed that hemoglobins of these six fusion methods could be successfully expressed, as shown in Figure 1.

[0090] After confirming the successful expression of these scHemoglobin-EGFP fusion proteins, we purified the post-IVTT reaction mixture and tested the purification efficiency. Nickel magnetic beads were added to 1 mL of the post-IVTT reaction mixture, and the fluorescence values ​​of the flow-through and eluate were recorded to obtain the purification efficiency, which is shown in Figure 2. These results demonstrate that the target protein can be purified using these six scHemoglobin-EGFP design schemes. In Figure 2, the fluorescence values ​​for each protein are, from left to right, the post-IVTT reaction mixture (whole, i.e., before adding magnetic beads), the flow-through after magnetic bead binding (FT), and the final eluate (Elution).

[0091] The eluate was subjected to SDS-PAGE electrophoresis to detect the protein bands. The results are shown in Figure 3, and the protein size was found to be consistent with the theoretical molecular weight.

[0092] Example 2. Expression and purification of scHemoglobin Only scHemoglobin was constructed without adding EGFP fusion protein. Six types of scHemoglobin were constructed based on Example 1, and the construction methods are shown in Table 2.

[0093] [Table 2]

[0094] Following the same method as in Example 1, except that EGFP was not fused, a DNA template was obtained, and then reaction and purification were carried out using an IVTT reaction system without adding hemin. The resulting protein was verified by SDS-PAGE electrophoresis as shown in Figure 4, which demonstrated that scHemoglobin could be correctly expressed.

[0095] Example 3. Co-expression and purification of scHemoglobin and hemin Because natural hemoglobin must bind to heme to exert its oxygen-carrying capacity, the artificial hemoglobin we produce must also bind to heme to function properly. However, heme binds to ferrous iron ions, which are highly unstable and easily oxidized to ferric iron ions in the air. The ferrous iron ions in heme can only remain stable once bound 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 out of stock, has long delivery times, and is expensive.

[0096] We have attempted to use hemin, a type of hemoglobin containing iron(III), as a cofactor for scHemoglobin. First, we examined the effect of hemin concentration added to the IVTT reaction system. We added different concentrations of hemin to the IVTT reaction solution for EGFP protein expression synthesis and tested the expression level of EGFP protein to determine whether the addition of hemin affected the expression level of PC. As shown in Figure 5, hemin at a final concentration of 3.3 μM did not affect the IVTT expression level of PC. We then continued the test with increasing concentrations of hemin, as shown in Figure 6. Hemin at a final concentration of 99 μM did not affect the IVTT expression of PC. That is, the amount of hemin added does not affect the expression of the IVTT reaction, and there is no particular limit to the amount added. The amount added was higher than the expected hemoglobin yield, primarily to ensure that all the expressed hemoglobin was in a hemin-bound state. Next, scHemoglobin was expressed and synthesized using the constructs shown in Tables 2 and 3. The resulting IVTT reaction mixture was added with 20 μM hemin for expression and purification. Gel electrophoresis of the results for each construct confirmed the molecular weight, indicating the target protein was obtained. Furthermore, after PBS immersion, the purified proteins corresponding to the various constructs exhibited a blood-red color. (As shown in Figure 7, the first row, from left to right, shows the structures constructed by binding HM001, HM002, HM003, HM004, HM005, and HEM006 with hemin. The second row, from left to right, shows HM007, HM008, HM009, HEM010, HM013, HM014, and HEM015. The third row, from left to right, shows HEM023 and HEM024.)

[0097] [Table 3-1]

[0098] [Table 3-2]

[0099] Hemoglobin itself is colorless, so the blood-red color is due to hemin. This was done to eliminate the possibility of free hemin remaining in the final product after purification. We performed the following control experiment (HEM-NC). During the IVTT reaction, we performed a standard expression purification procedure with hemin added but without adding DNA template. The resulting protein eluate was also buffer-exchanged to a final PBS buffer solution. SDS-PAGE electrophoresis and color observation revealed no sc hemoglobin band (see Figure 8). After the PBS solution was exchanged, the solution was colorless and transparent, and no blood-red color was observed. This indicates that the blood-red color obtained previously only appears after the protein binds to hemin.

[0100] Example 4. Oxygen transport experiment The proteins corresponding to the structures in Tables 2 and 3, which were obtained by purification according to Example 3, were subjected to oxygen transport experiments as follows. The spectral characteristics of hemoglobin in oxygen-carrying and oxygen-free states 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. 12 1599-1607): As shown in Figure 9, in the oxygen-carrying state, hemoglobin shows peaks at 415, 541, and 577 nm, and Figure 9 significantly shows that the absorbance of hemoglobin in the oxygen-carrying state at 415 nm is higher than the absorbance of hemoglobin in the anaerobic state at 430 nm. In the anaerobic state, hemoglobin shows peaks at 430 nm and 555 nm, and Figure 9 significantly shows that the absorbance of hemoglobin in the anaerobic state at 430 nm is higher than the absorbance of hemoglobin in the oxygen-carrying state at 430 nm. The conversion of hemoglobin from an oxygen-carrying state to an oxygen-free state is catalyzed by sodium dithionite. When sodium dithionite is added to oxygen-carrying hemoglobin, the hemoglobin is reduced and simultaneously loses oxygen to become deoxygenated, resulting in a change in spectral characteristics. Based on this, the spectral detection results of hemin-binding proteins corresponding to the compositions in Tables 2 and 3 obtained in Example 3 are shown in Figures 10 to 24.

[0101] As can be seen from Figures 10 to 24, the spectral characteristics of the 15 hemin-binding proteins obtained in Example 3 were consistent with the schematic diagram in Figure 9. In other words, it was confirmed that the present invention enables functional hemoglobin to be obtained by linking the amino acid sequences of four polypeptide chains via a linker peptide.

[0102] Example 5. Stability Test Protein stability was determined by Tm values, which were measured using the Unchained Uncle Protein Stability Analyzer. The intrinsic fluorescence of proteins comes from amino acids containing a benzene ring (tryptophan, tyrosine, and phenylalanine), which emit absorbed photons (fluorescence) when excited by ultraviolet wavelengths. When the environment of these aromatic amino acids in a protein changes, the intensity of the emitted fluorescence also changes. Therefore, the change in intrinsic fluorescence can be used as an indicator of conformational changes in the tertiary structure of a protein, and the conformational stability of a protein can be analyzed. By detecting the change in the fluorescent signal during the heating process, a thermal denaturation curve was obtained that describes one or more phase transitions that occur as the protein unfolds. The midpoint of this transition (Tm) can be used to describe the stability of the protein. The higher the Tm value, the more stable the protein. When the Tm values ​​of the same hemoglobin used in the oxygen transport experiment were tested as in Example 4, the average Tm values ​​were not significantly different from the Tm values ​​of the control bovine hemoglobin, indicating that the stability of the modified hemoglobin was maintained at the level of the original protein. The sequences referred to herein are summarized in Table 4.

[0103] [Table 4-1]

[0104] [Table 4-2]

Claims

1. four polypeptide chains, each of which is two α chains and two β chains; Here, the recombinant hemoglobin is characterized in that the amino acid sequences of the four polypeptide chains are linked by a linker peptide.

2. The recombinant hemoglobin of claim 1, wherein the three linker peptides sequentially link the amino acid sequences of the four polypeptide chains from the N-terminus to the C-terminus in any order.

3. The recombinant hemoglobin according to claim 1 or 2, characterized in that the number of amino acids in the linker peptide is 1 to 90, 1 to 30, 1 to 35, or 1 to 40.

4. the linker peptide used to link the two α chains is a first linker peptide, and the number of amino acids in the first linker peptide is 1 to 5, preferably 1, 2, or 3; The recombinant hemoglobin according to claim 3, wherein the 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 of the second linker peptide is 5 to 90, 5 to 85, 5 to 30, 5 to 35, 5 to 40, 10 to 30, 10 to 35, or 10 to 40.

5. The recombinant hemoglobin of claim 4, wherein the first linker peptide has one amino acid, glycine.

6. 6. The recombinant hemoglobin of claim 4 or 5, wherein the second linker peptide comprises major amino acid residues selected from any one or more of G, S, T and A, and the number of said major amino acid residues accounts for at least 50%, 60%, 70%, 80% or more than 90% of the total number of amino acid residues of the second linker peptide, more preferably, the major amino acid residues comprise one or more of G and S amino acids, and the total amount accounts for at least more than 40%, or more than 45%, or more than 60%, or more than 70%, or more than 80% of the total number of amino acids of the second linker peptide.

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

8. The recombinant hemoglobin of claim 7, wherein the second linker peptide comprises an affinity unit in which hydrophilic amino acids and hydrophobic amino acids are arranged alternately, and wherein the order of amino acids in the second linker peptide from the N-terminus to the C-terminus is flexible unit - affinity unit in which hydrophilic amino acids and hydrophobic amino acids are arranged alternately - flexible unit.

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

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

11. The recombinant hemoglobin according to any one of claims 4, 5 and 7-1, characterized in that the amino acid sequence of the second linker peptide is SEQ ID NO: 1 to 6.

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

13. the α chain is of human, porcine or bovine origin, and the β chain is of human, porcine or bovine origin, and / or the amino acid sequence of the alpha chain is any one or more of SEQ ID NOs: 7, 13 and 14, or has a percentage of sequence identity of at least 80%, 85%, 90%, 95%, 97% or 99% with any one or more of SEQ ID NOs: 7, 13 and 14, respectively; 13. The recombinant hemoglobin of any one of claims 1 to 12, wherein the amino acid sequence of the beta chain is any one or more of SEQ ID NOs: 8, 15 and 16, or has a percentage of sequence identity of at least 80%, 85%, 90%, 95%, 97% or 99% with any one or more of SEQ ID NOs: 8, 15 and 16, respectively.

14. 12. The recombinant hemoglobin of claim 1, further comprising hemin bound to each of the polypeptide chains.

15. A nucleic acid comprising nucleotides encoding the recombinant hemoglobin according to any one of claims 1 to 14.

16. A vector comprising the nucleic acid of claim 15.

17. A host cell, characterized in that it contains a nucleic acid according to claim 15 and / or a vector according to claim 16, said host cell preferably being of prokaryotic or eukaryotic origin, Further, the host cell is derived from a bacterium, a mammalian cell, a human cell, a plant cell, a yeast cell, or an insect cell, and is preferably selected from Escherichia coli or a yeast cell, and further, the yeast cell is selected from one or more combinations of Saccharomyces cerevisiae, Kluyveromyces, and in another preferred embodiment, the Kluyveromyces is selected from one or more combinations of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii.

18. Use of the nucleic acid according to claim 15, the vector according to claim 16 or the host cell according to claim 17 in the production of a recombinant hemoglobin according to any one of claims 1 to 14.

19. cell extracts, and The recombinant hemoglobin according to any one of claims 1 to 14, comprising an mRNA or DNA template encoding the hemoglobin, wherein the cell extract is preferably an Escherichia coli extract, a yeast cell extract, and further derived from any combination of one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces, and preferably the yeast cell extract is derived from Kluyveromyces, more preferably derived from any combination of one or more of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii; The in vitro cell-free protein synthesis system further comprises one or more of an amino acid mixture, dNTP, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent.

20. To provide an in vitro cell-free protein synthesis system, 15. The method for in vitro cell-free synthesis of recombinant hemoglobin according to any one of claims 1 to 14, comprising the step of adding an mRNA or a DNA template encoding the recombinant hemoglobin according to any one of claims 1 to 14 to the cell-free protein synthesis system and carrying out an in vitro synthesis reaction to obtain the recombinant hemoglobin, wherein the volume ratio of the DNA template to the in vitro cell-free protein synthesis system is preferably 1:10 to 1:50, preferably 1:20 to 1:40, most preferably 1:25 to 1:35, and particularly preferably 1:

30.

21. The in vitro cell-free synthesis method according to claim 20, further comprising adding hemin to the cell-free protein synthesis system to produce recombinant hemoglobin bound to the hemin, and preferably the added hemin contains trivalent iron.

22. The in vitro cell-free protein synthesis system comprises a cell extract, Furthermore, the in vitro cell-free protein synthesis system comprises any combination of one or more of an amino acid mixture, dNTP, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent; and / or The cell extract is an Escherichia coli extract, a yeast cell extract, and further derived from any combination of one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces, preferably the yeast cell extract is derived from Kluyveromyces, more preferably derived from any combination of one or more of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii, or 22. The in vitro cell-free protein synthesis method according to claim 20 or 21, wherein the yeast extract accounts for 50 to 80% of the entire in vitro cell-free protein synthesis system.

23. Use of a recombinant hemoglobin according to any one of claims 1 to 14 as a blood substitute and for use in cancer, stroke, hemorrhagic shock, acute mountain sickness (AMS), peripheral arterial disease (PAD) and Parkinson's disease (PD).

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