Cell-free method for synthesizing hemoprotein or peptide chain

Abstract

A cell-free method for synthesizing a hemoprotein or peptide chain. The method comprises: adding a DNA or RNA template encoding a heme-binding protein or peptide chain to a cell-free in-vitro synthesis system so as to synthesize the heme-free form of the heme-binding protein in vitro; and incubating the heme with a binding system containing the heme-binding protein or peptide chain for a predetermined period of time so as to produce a hemoprotein or peptide chain bound to the heme. The method does not require modifications to a strain and fermentation broth, involves simple production, and has universal applicability.

Description

A method for cell-free synthesis of heme proteins or peptide chains Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for cell-free synthesis of heme protein or peptide chains. Background Technology

[0002] Currently, heme-binding proteins, such as hemoglobin, which function by binding heme, are generally expressed by cells. During the expression process, in order to bind heme, a certain amount of heme needs to be added to the fermentation broth, and heme transport proteins and heme synthases need to be added to the gene of the heme-binding protein to achieve the binding of heme by the heme-binding protein.

[0003] This method often requires cell modification and the addition of heme to the fermentation broth, resulting in a complicated and cumbersome process. Furthermore, this means that the cells used in this production process are only suitable for expressing heme-binding proteins and cannot be used for expressing other proteins, thus lacking versatility. Summary of the Invention

[0004] This invention provides a cell-free method for synthesizing heme proteins or peptide chains, which simplifies the production of heme proteins or peptide chains and allows for the use of common cells or fermentation broths, facilitating continuous recycling and replacement of the target protein to be expressed.

[0005] Therefore, the present invention provides the following technical solution:

[0006] This invention provides a method for cell-free synthesis of heme protein or peptide chain, characterized by comprising: adding a DNA or RNA template encoding a heme-binding protein or peptide chain to a cell-free in vitro synthesis system for in vitro synthesis of heme-binding protein without heme; and incubating the heme with a binding system containing the heme-binding protein or peptide chain for a predetermined time to generate heme protein or peptide chain bound to the heme.

[0007] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention is further characterized by the following: the heme is added before, at the start of, during, or after the in vitro synthesis; and / or

[0008] The binding system consists of the cell-free in vitro synthesis system or the elution buffer after purifying the heme-binding protein that has not bound to heme.

[0009] The cell-free method for synthesizing heme proteins or peptide chains provided by the present invention is further characterized in that the heme-binding protein is selected from any one or more of the following: hemoglobin, peroxidase, cytochrome, bacterial ferritin, hydroxylamine oxidoreductase, nitroprotein, cyclooxygenase, catalase, cytochrome, chloroperoxidase, PAS domain heme sensor, H-NOX heme sensor, and nitric oxide synthase.

[0010] The heme-binding peptide chain refers to any single subunit of the heme-binding protein mentioned above.

[0011] Preferably, the heme-binding protein is selected from hemoglobin or peroxidase, and the heme-binding peptide chain is selected from any subunit of hemoglobin; more preferably, the amino acid sequence of the peroxidase is SEQ ID NO: 23 or has a sequence homology percentage of at least 85%, 90%, 95%, 97%, 98% or 99% with SEQ ID NO: 23.

[0012] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention also has the following feature: wherein each polymer is composed of two or more subunits connected in series from the N-terminus to the C-terminus in any order.

[0013] The cell-free method for synthesizing heme proteins or peptide chains provided by the present invention is further characterized in that each of the subunits originates from any subunit of a heme-binding protein of any species.

[0014] The cell-free method for synthesizing heme protein or peptide chains provided by the present invention also has the following features: wherein the heme includes a unit heme, the unit heme includes at least two polypeptide chains, the polypeptide chains being any one or more combinations of α chains and β chains, wherein the amino acid sequences of at least two polypeptide chains are linked by a linking peptide;

[0015] Preferably, it comprises four polypeptide chains, namely two α chains and two β chains, and the amino acid sequences of the four polypeptide chains are linked by a linker peptide.

[0016] The cell-free method for synthesizing heme protein or peptide chains provided by this invention also has the following feature: wherein the heme protein comprises two or more of the aforementioned unit heme proteins.

[0017] Two or more hemoglobin units are linked together by inter-unit linking peptides.

[0018] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention also has the following characteristics: wherein 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.

[0019] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention also has the following feature: wherein the number of amino acids of 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.

[0020] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention further has the following feature: the intra-unit linker peptide used to connect the two α chains is a first linker peptide, and the number of amino acids in the first linker peptide is 1-5, preferably 1, 2 or 3.

[0021] 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.

[0022] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention also has the following feature: the amino acid of the first linker peptide is one glycine.

[0023] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention further 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 them 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.

[0024] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention also has the following feature: the second linker peptide contains at least a flexible unit.

[0025] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention also has the following feature: 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.

[0026] The cell-free method for synthesizing heme protein or peptide chains provided by this invention also has the following feature: the structure of the flexible unit is (GGGGS)a, preferably, a is 1-4.

[0027] More preferably, the flexible unit is selected from one or more of the following group:

[0028] (1) GGGGS;

[0029] (2) GGGGSGGGGS;

[0030] (3)GGGGSGGGGSGGGGSGGGGS.

[0031] The cell-free method for synthesizing heme proteins or peptide chains provided by this invention also has the following characteristic: the structure of the affinity / reluctance unit is any one or more of (RADA)b, (KLDL)c, (LK)d, and (LD)f.

[0032] Preferably: b is 1-5, c is 1-5, d is 1-10, f is 1-10.

[0033] More preferably, the affinity unit is selected from one or more of the following group:

[0034] (1) RADARADARADARADA;

[0035] (2) KLDLKLDLKLDL;

[0036] (3)LKLKLKLKLKLK;

[0037] (4)LDLDLDLDLDLD.

[0038] The cell-free method for synthesizing heme protein or peptide chains provided by the present invention further comprises the following feature: wherein the amino acid sequence of the second linker peptide is any one of SEQ ID NO: 1-6, 10-12, 21, and 22, or has a sequence homology percentage of at least 85%, 90%, 95%, 97%, 98%, or 99% with any one of SEQ ID NO: 1-6, 10-12, 21, and 22.

[0039] Preferably, the amino acid sequences of all the second linker peptides are all the same, partially the same, or all different.

[0040] The method for cell-free synthesis of heme protein or peptide chains 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 linked in any of the following order:

[0041] α chain - α chain - β chain - β chain, β chain - β chain - α chain - α chain, β chain - α chain - α chain

[0042] β chain, α chain-β chain-α chain-β chain and β chain-α chain-β chain-α chain.

[0043] The cell-free method for synthesizing heme protein or peptide chains provided by this invention further comprises the following features: wherein 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

[0044] The amino acid sequence of the α chain is as follows: SEQ ID NO: 7, 13, and 14.

[0045] Any one or more of them, or any one or more of them in SEQ ID NO: 7, 13 and 14, have a sequence homology percentage of at least 80%, 85%, 90%, 95%, 97 or 99%, respectively;

[0046] 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.

[0047] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention further has the following characteristics: wherein 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 linking peptide contains a combination of any one or more amino acid residues of G, S, A, and T, and / or the component linking peptide contains a combination of any one or two amino acid residues of E and D; preferably, the percentage of any one or more amino acid residues of G, S, T, and A in the inter-unit linking peptide relative to the total number of amino acid residues in the inter-unit linking peptide is at least greater than 45%, 50%, 60%, 70%, 80%, or 90%; furthermore, the amino acid sequence of the inter-unit 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.

[0048] and / or

[0049] The number of units is 2, 3, or 4.

[0050] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention also has the following characteristics: the volume ratio of the DNA template to the in vitro 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.

[0051] The method for cell-free synthesis of heme protein or peptide chains provided by this invention also has the following feature: wherein the in vitro synthesis system includes at least: yeast cell extract,

[0052] The composition comprises any one or more of the following: an amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, an energy supply system, polyethylene glycol, and an aqueous solvent.

[0053] Furthermore: the yeast cell extract is derived from any one or more of the following group: Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces kuhlii;

[0054] Preferably, the yeast cell extract is derived from Kluyveromyces kluyveromyces, more preferably from Kluyveromyces lactis; or the yeast extract accounts for 20-80% of the entire cell-free in vitro protein synthesis system, more preferably 50-80%.

[0055] The method for cell-free synthesis of heme protein or peptide chains provided by the present invention also has the following feature: the predetermined time is any time between 5 minutes and 3 hours, starting from 5 minutes and increasing by 5 minutes at intervals of 5 minutes, for example, greater than or equal to 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes... and greater than or equal to 180 minutes.

[0056] The cell-free method for synthesizing heme protein or peptide chains provided by the present invention also has the following characteristics: the final concentration of heme added to the reaction is 0-160 μM, preferably 0-80 μM, 10-80 μM, or 25-80 μM.

[0057] The present invention also provides a cell-free in vitro synthesis system for synthesizing the aforementioned heme protein or peptide chain, characterized in that it comprises:

[0058] Cell extracts;

[0059] The mRNA or DNA template encoding a heme-binding protein or peptide chain, preferably, the cell extract is the yeast cell extract, further, derived from any one or more of the following group: Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces var. kusnezoffii; more preferably, the yeast cell extract is derived from Kluyveromyces var. kusnezoffii, more preferably Kluyveromyces lactis.

[0060] And heme;

[0061] Furthermore, it also includes one or more of the following components:

[0062] Amino acid mixture, dNTPs, RNA polymerase, DNA polymerase, energy supply system, polyethylene glycol, and aqueous solvent;

[0063] Furthermore, the final concentration of heme added to the reaction is 0–160 μM, preferably 0–80 μM, 10–80 μM, or 25–80 μM.

[0064] Invention Function and Effect

[0065] The method provided by this invention has at least the following advantages:

[0066] (1) Only any target protein (heme-binding protein) template that can bind to heme needs to be provided, added to a cell-free system, and incubated with heme to obtain heme-bound heme protein. No changes to the strain or fermentation broth are required. The production is simple and universally applicable. The target protein can be continuously produced and modified, saving production steps.

[0067] (2) In the synthesis system and synthesis method of the present invention, when the concentration of added heme is 0 to 80 μM, the expression of heme protein has a high yield. Only when the concentration of heme is higher than 80 μM will the expression of heme protein be inhibited. Compared with the prior art, the concentration gradient range of added heme in the present invention is larger, the control reaction is simpler, and the large concentration range can meet the binding needs as much as possible. It can avoid the process of separating excess heme due to the inability to saturate the binding of heme, and is suitable for higher yield at one time and large-scale production. Attached Figure Description

[0068] Figure 1 shows the results of fluorescence quantification when Hemin is added to the purified EGFP sample.

[0069] Figure 2 shows the gel electrophoresis results of Hemin added to the purified EGFP sample;

[0070] Figure 3 shows the gel electrophoresis results of the heme concentration gradient test during the hemoglobin synthesis process.

[0071] Figure 4 shows the gel electrophoresis results of the heme concentration gradient test during the synthesis of catalase.

[0072] Figure 5 shows the gel electrophoresis results of the heme concentration gradient test during EGFP synthesis.

[0073] Figure 6 shows the appearance and gel image of hemoglobin β subunit after binding with heme;

[0074] Figure 7 shows the spectroscopic properties of hemoglobin β subunit bound to heme under oxygen-carrying and anaerobic conditions.

[0075] Figure 8 shows the appearance and gel image of the hemoglobin β-α fusion protein after binding with erythrin;

[0076] Figure 9 shows the spectroscopic properties of the hemoglobin β-α fusion protein after binding with erythrin under oxygen-carrying and anaerobic conditions.

[0077] Figure 10 shows the spectroscopic properties of hemoglobin under oxygen-carrying and anaerobic conditions as described in the references.

[0078] Figure 11 shows the gel electrophoresis results of EGFP after expression and purification following the addition of buffers containing different concentrations of hemin at the initial stage of the IVTT reaction of EGFP.

[0079] Figure 12 shows the fluorescence spectrum obtained after adding different concentrations of hemin-dissolving buffer in the initial stage of the EGFP IVTT reaction. Detailed Implementation

[0080] 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.

[0081] Unless otherwise specified, the methods used in the embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0082] 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.

[0083] In this article, heme-binding proteins or peptide chains refer to a class of proteins or peptide chains that can bind to heme to form corresponding functional proteins. The combination of these proteins with heme is referred to as heme protein or peptide chain. In other words, heme protein or peptide chain refers to a protein or peptide chain containing heme as a prosthetic group, and is a general term for the combination of heme and heme-binding proteins or peptide chains.

[0084] Heme-binding proteins, such as hemoglobin, peroxidase, cytochrome, bacterial ferritin, hydroxylamine oxidoreductase, nitroprotein, cyclooxygenase, catalase, cytochrome, chloroperoxidase, PAS domain heme sensor, H-NOX heme sensor, and nitric oxide synthase, refer to aggregates or tandem aggregates of any number of subunits that make them up, for example, hemoglobin refers to aggregates of two, three, or four subunits, or aggregates or tandem aggregates of two or more aggregates; in this context, aggregates refer to more than two subunits tandemly or self-aggregated together.

[0085] Heme-binding peptide chain refers to any subunit that makes up the heme-binding protein mentioned above, such as a single α chain or β chain of hemoglobin.

[0086] The subunits of hemoglobin involved in the reaction described herein for binding heme can be any subunit of hemoglobin from any species, such as any one or more from humans, cattle, and pigs.

[0087] In this article, “monohemaglobin” refers to hemoglobin molecules and / or their variants with a molecular size of at least about 65 kDa, and synthesized by any standard molecular biology technique, rather than isolated or purified from any animal or human source.

[0088] 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.

[0089] 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.

[0090] 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 generate 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 embodiments, basic localization known in the art is used.

[0091] Comparison search tools (“BLAST”) are 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 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402).

[0092] 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.

[0093] In this article, "active structure" refers to the ability of prepared hemoglobin to carry oxygen, and to be prepared as or used as a blood substitute. For example, the protein before it is bound to heme. As long as it can bind to heme and have the function of carrying oxygen, it can be prepared as a blood substitute. The protein before it is bound to heme is considered to have an active structure.

[0094] The heme added in this article can be at the following time points:

[0095] Heme is added before the in vitro synthesis is performed: the incubation time is from the start of the IVTT reaction to the end of the reaction.

[0096] Heme is added at the start of the in vitro synthesis: the incubation time is from the start of the IVTT reaction to the end of the reaction.

[0097] Heme may be added at any point during the in vitro reaction synthesis process: the incubation time shall be at least the time from the time of heme addition to the end of the reaction.

[0098] The heme was added after the in vitro synthesis was completed;

[0099] The incubation time for each of the above, i.e. the predetermined time, is any time between 5 minutes and 3 hours, starting from 5 minutes and increasing by 5 minutes every 5 minutes. That is, starting from 5 minutes, greater than or equal to 5 minutes, or greater than or equal to 5+5=10 minutes, or 10+5=15 minutes, and so on. For example, any time within the range of greater than or equal to 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, and 180 minutes.

[0100] The binding system is the in vitro synthesis system and the eluent after purifying the heme.

[0101] This invention further categorizes hemoglobin into several cases:

[0102] 1. Monohemoglobin

[0103] The monohemoglobin 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 monohemoglobin. 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.

[0104] 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 sequences: 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.

[0105] The number of amino acids in the linker peptide within the unit is designed to ensure that 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.

[0106] In one example, the intra-unit linking peptides include two types:

[0107] 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.

[0108] 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.

[0109] In one example, the first linker peptide has one glycine amino acid.

[0110] 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. The amino acid sequence of the second linker peptide is, for example, SEQ ID NO: 10-12.

[0111] In one example, the second linker peptide contains at least a flexible unit.

[0112] 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 one or more of the following group:

[0113] (1) GGGGS;

[0114] (2) GGGGSGGGGS;

[0115] (3)GGGGSGGGGSGGGGSGGGGS.

[0116] 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.

[0117] The alternation of hydrophilic and hydrophobic amino acids in this article refers to the following different situations:

[0118] (1) Alternating between a hydrophilic amino acid and a hydrophobic amino acid;

[0119] (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.

[0120] 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.

[0121] More preferably, the affinity unit is selected from one or more of the following group:

[0122] (1) RADARADARADARADA;

[0123] (2) KLDLKLDLKLDL;

[0124] (3)LKLKLKLKLKLK;

[0125] (4)LDLDLDLDLDLD.

[0126] 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.

[0127] Preferably, the amino acid sequences of all the second linker peptides are all the same, partially the same, or all different.

[0128] 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.

[0129] 2. Hemoglobin composed of multiple unit hemoglobins linked together

[0130] It refers to a hemoglobin comprising two or more of the aforementioned units, and the units are linked together by inter-unit linking peptides.

[0131] In one example, the inter-unit linking peptide connects the units sequentially from the N-terminus to the C-terminus.

[0132] 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.

[0133] In one example, the interunit linker peptide contains a combination of any one or more amino acid residues from G, S, A, and T.

[0134] In one example, the component linker peptide contains a combination of any one or more amino acid residues from E and D.

[0135] 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.

[0136] 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.

[0137] There is no particular limitation on the number of units; it can be 2, 3, or 4, or even more.

[0138] 3. The present invention also provides a nucleic acid comprising: a nucleotide sequence encoding the aforementioned heme-binding protein or peptide chain.

[0139] The present invention also provides a carrier comprising the above-described nucleic acid.

[0140] 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.

[0141] 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.

[0142] The nucleic acid, vector, or host cell provided by this invention can be used in the preparation of hemoglobin.

[0143] The present invention also provides an in vitro cell-free protein synthesis system, comprising: cell extract and mRNA or DNA template encoding the aforementioned heme-binding protein.

[0144] In one example, the cell extract is a yeast cell extract, further, derived from any one or more of the following group: Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces var. kusnezoffii; preferably, the yeast cell extract is derived from Kluyveromyces kusnezoffii, more preferably Kluyveromyces lactis.

[0145] 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.

[0146] 4. In vitro cell-free synthesis methods for heme-binding proteins, including:

[0147] Provides a cell-free in vitro synthesis system (also known as a cell-free in vitro protein synthesis system, in vitro protein synthesis system, in vitro protein synthesis system, in vitro synthesis system, etc.);

[0148] The heme-binding protein was synthesized in vitro by adding an mRNA or DNA template encoding the aforementioned heme-binding protein to a cell-free in vitro synthesis system.

[0149] 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, particularly preferably 1:30, or 8-20 ng / μL.

[0150] 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.

[0151] 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.

[0152] In one example, yeast extract constitutes 20-80%, 20%-80%, or more preferably 50-80% of the entire cell-free in vitro protein synthesis system.

[0153] .

[0154] 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.

[0155] 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, CN108949801A, CN108642076A, CN109022478A, and CN1094234. References include CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, and CN110964736A. Unless otherwise stated, these references and their cited documents are incorporated herein by reference in their entirety and for all purposes.

[0156] The present invention synthesizes the aforementioned heme protein using the aforementioned in vitro cell-free synthesis method, which has a short cycle, is easy to operate, has low cost, and can obtain a stable heme-binding protein with the aforementioned active structure.

[0157] Furthermore, in vitro cell-free synthesis methods also include:

[0158] Simultaneously, heme is added to the cell-free protein synthesis system or after the in vitro synthesis reaction to generate heme protein bound with the heme. Preferably, the added heme contains ferric iron.

[0159] The following specific experimental examples further illustrate the content of this invention.

[0160] Example 1 IVTT reaction and purification

[0161] I. IVTT reaction:

[0162] (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.

[0163] The yeast cell extract was obtained from Kluyveromyces oryzae, specifically, in this embodiment, Kluyveromyces lactis was used.

[0164] (2) The transformation of each target plasmid is carried out as follows:

[0165] 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 antibiotics), incubate upside down at 37℃ for 12–16 h, and then store at 4℃.

[0166] (4) The transformed plasmid was extracted and amplified:

[0167] 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).

[0168] Taking a 10ml amplification system as an example:

[0169] 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.

[0170] (5) IVTT reaction

[0171] 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.

[0172] II. Purification:

[0173] Add nickel magnetic beads to the collected reaction solution (sample) and incubate at 4 degrees Celsius for 10 minutes;

[0174] Wash three times with imidazole-free PBS solution;

[0175] Wash three times with PBS solution containing 20 mM imidazole;

[0176] The target protein was eluted with PBS containing 250 mM imidazole.

[0177] Example 2: Construction of Hemoglobin

[0178] An intra-unit linker was designed to tandemly link the four subunits of hemoglobin, i.e., the four polypeptide chains, into a single chain, scHemoglobin (monohemoglobin), resulting in hemoglobin with the designation HEM006. The tandem linking methods of each subunit are shown in Table 1. 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.

[0179] A hemoglobin gene containing a linker peptide was synthesized using gene synthesis methods. This gene was then directly inserted into our optimized plasmid (D2P1.08t, specifically described in Figure 1 of patents 2022111062129 or 2023104761829), with insertion sites at BamHI and HindIII. The resulting plasmid containing the target gene fragment, HEM006, was transformed and amplified to obtain a DNA template encoding the structure shown in Table 1.

[0180] 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.

[0181] We attempted to use Hemin (heme chloride) as the cofactor of scHemoglobin. Hemin is heme with ferric iron.

[0182] First, the potential impact of Hemin addition on IVTT quantitation was tested:

[0183] (1) Following the method in Example 1, EGFP template was added to IVTT, and the EGFP protein was obtained after purification following the reaction.

[0184] (2) Centrifuge the obtained EGFP protein and add 0.5 mg / ml of the supernatant to 4 ml;

[0185] (3) Set up 10 groups of EGFP protein, prepare 10 2ml centrifuge tubes, add 300ul of protein to each tube, add different final concentrations of Hemin as follows, vortex to mix, and let stand for 15min:

[0186] 0, 5, 10, 20, 30, 40, 50, 80, 100 and 160uM;

[0187] (4) The RFU value of the result of (3) is measured and the result is shown in Figure 1; the same volume of gel is run for the result of (3) and the result is shown in Figure 2.

[0188] As shown in Figure 1, when free heme is not removed, the concentration of EGFP protein at the same concentration decreases with increasing heme concentration, according to the RFU results. However, the gel electrophoresis results in Figure 2 show that the protein concentration remains consistent regardless of the heme concentration added. This indicates that the addition of heme affects the quantitative fluorescence method.

[0189] Secondly, it was verified that the purification process had eliminated the influence of heme:

[0190] The constructed structure shown in Table 1 was expressed and synthesized. Hemin was added to the synthesized IVTT reaction solution at a final concentration of 20 μM. The IVTT reaction and purification were performed according to the method in Example 1. The results obtained from the construction were analyzed by gel electrophoresis. The gel electrophoresis results were consistent with the molecular weight, indicating that the target protein was obtained. To rule out the possible influence of heme in the purified protein, the following control experiment was performed: Hemin was added to the IVTT reaction without adding a DNA template, and the same expression and purification were performed. SDS-PAGE electrophoresis and color observation showed no scHemoglobi bands, and after replacement with PBS solution, the solution was colorless and transparent, without any blood-red color. This indicates that the protein we obtained was hemoglobin that successfully bound heme.

[0191] In subsequent tests, unless otherwise specified, the purified target protein (with free heme removed) was used for gel electrophoresis for quantification.

[0192] Example 3: Time gradient of heme addition during hemoglobin IVTT synthesis

[0193] Perform according to the method of Example 1:

[0194] 1. Add the template of HEM006 to IVTT for reaction. After thorough mixing, divide the reaction solution into 8 equal portions, each 50 mL.

[0195] 2. Each sample was given a final concentration of 20 μM heme at different times during the IVTT reaction: 0, 1 h, 2 h, 2 h 10 min, 2 h 40 min, 2 h 50 min, 2 h 55 min, and 3 h, for a total of 8 time points.

[0196] 3. Samples are collected 3 hours after the IVTT reaction. For samples at the 3-hour mark, heme is added after 3 hours, and all 8 IVTT samples are collected.

[0197] 4. Add an equal amount of nickel magnetic beads to each sample and purify them according to the method in Example 1.

[0198] The results showed that the incubation time, which in this embodiment is the time from the addition of heme to IVTT until the reaction ends, should be at least 5 minutes and preferably more than 1 hour.

[0199] Example 4: Time gradient of heme addition after hemoglobin IVTT synthesis.

[0200] Perform according to the method of Example 1:

[0201] 1. The template of HEM006 was added to IVTT for reaction. After 3 hours, the reaction solution was divided into 8 equal portions, each 50 mL, and stored at 4 degrees Celsius.

[0202] 2. Under 4-degree conditions, each sample was incubated with 20 μM of heme at different times: 0, 1 h, 2 h, 2 h 10 min, 2 h 40 min, 2 h 50 min, 2 h 55 min, and 3 h, for a total of 8 time points.

[0203] 3. After adding heme to the samples at 3 hours, collect all 8 IVTT samples.

[0204] 4. Add an equal amount of nickel magnetic beads to each sample and purify them according to the method in Example 1.

[0205] The results showed that the incubation time should be at least 5 minutes. In this example, the time for adding heme after the IVTT reaction should also be at least 5 minutes and preferably more than 1 hour.

[0206] Example 5 Stability Test

[0207] Protein stability is determined by the Tm value. Tm is measured using the Unchained Uncle Protein Stability Analyzer.

[0208] 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.

[0209] 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.

[0210] The Tm value of HM006 hemoglobin bound to heme obtained according to Example 1 was tested. The average Tm value was not much different from the Tm value of bovine hemoglobin in the control group, indicating that the stability of hemoglobin after binding to heme maintained the original protein level.

[0211] Example 6 P50 Test

[0212] The heme-bound HM006 hemoglobin obtained according to the method of Example 1 was subjected to a P50 test.

[0213] P50 Experimental Principle:

[0214] 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.

[0215] 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.

[0216] This 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.

[0217] When the oxygen on hemoglobin is replaced by an inert gas, such as nitrogen, hemoglobin undergoes the following reaction:

[0218] Specific testing process:

[0219] (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.

[0220] (2) Install the oxygen permeable membrane. Rinse the surface of the oxygen electrode with distilled water and shake off excess water to ensure the surface 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 adheres tightly to the front end of the electrode. Minimize the amount of air bubbles generated on the oxygen permeable membrane and avoid air bubbles between the membrane and the electrode.

[0221] (3) Slightly moisten the sample cell with distilled water, slowly push in the electrode, keep the O-ring fixed in the electrode groove and do not move, so as to achieve a sealing effect.

[0222] (4) Ensure that the oxygen-permeable membrane is installed correctly, the sample cell is equipped with a magnetic stir bar, the air and nitrogen compression bottles are open and the outlet pressure is within the range of 0.1-0.2MPa, and press the power switch on the back of the main unit.

[0223] (5) Activate the oxygen-permeable membrane. Inhale 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. The PO2 value will increase after air is introduced and decrease after nitrogen is introduced. 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.

[0224] (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, and 2.9 ml of buffer. Add an appropriate amount of NaOH to adjust the pH to neutral, then add 4 μL of Tween 20 and mix well. It is recommended that the added sample contain 3-6 mg of hemoglobin.

[0225] (7) After the oxygen-permeable membrane is activated, drain the distilled water from the sample cell and draw the sample into the sample cell. Click the Ready button on the touch screen to start the test.

[0226] Test results showed that the P50 of HM006 hemoglobin was 13.53 mmHg. This proves that a functional hemoglobin bound to heme was obtained.

[0227] Example 7: Effect of Heme Concentration on Hemoglobin Expression

[0228] Following the method in Example 1, the obtained HEM006 DNA template was transferred to IVTT, and at the initial stage of the reaction, different final concentrations of heme (0–160 μM, respectively) were added, followed by IVTT reaction for 3 hours. Hemoglobin was then purified using the method in Example 1. Electrophoretic analysis of the eluted protein revealed that the hemoglobin yield was similar at heme concentrations ranging from 0–40 μM, but the hemoglobin concentration decreased significantly when the added heme concentration reached 80 μM (see Figure 3). This indicates that the amount of high-concentration heme added affects the hemoglobin yield.

[0229] Example 8: Effect of Heme Concentration on Catalase Expression

[0230] Similarly, following the method in Example 1, the obtained catalase (CAT) DNA template was added to IVTT, and at the initial stage of the reaction, different final concentrations of heme (0–160 μM, respectively) were added, and the IVTT reaction was carried out for 3 hours. The catalase was then purified using the method in Example 1. Electrophoretic analysis of the eluted protein revealed that when the added heme concentration reached 80 μM, the catalase concentration significantly decreased (see Figure 4). This indicates that the amount of high-concentration heme added affects the catalase yield.

[0231] Example 9: Effect of Heme Concentration on EGFP Expression

[0232] Similarly, following the method in Example 1, the obtained EGFP DNA template was added to IVTT, and at the initial stage of the reaction, different final concentrations of heme (0–160 μM, respectively) were added, and the IVTT reaction was carried out for 3 hours. EGFP was then purified using the method in Example 1. Electrophoretic analysis of the eluted protein revealed that when the added heme concentration reached 160 μM, the EGFP concentration significantly decreased (see Figure 5). This further demonstrates that the amount of high-concentration heme added does indeed affect the protein yield of the IVTT reaction system.

[0233] Example 10 Expression of hemoglobin subunits

[0234] Following the method in Example 1, the obtained individual hemoglobin β subunit DNA template was added to IVTT. At the initial stage of the reaction, 20 μM of heme was added to a final concentration. After 3 hours of reaction, the sample was collected for purification to obtain the β subunit protein. The electrophoresis gel image is shown in Figure 6. As can be seen from Figure 6, the hemoglobin β subunit successfully bound to heme.

[0235] Example 11 Expression of hemoglobin subunit fusion

[0236] The β-α subunit and α subunit of hemoglobin were fused to obtain a β-α fusion protein plasmid. Similarly, following the method in Example 1, the obtained fusion plasmid DNA template was added to IVTT. At the initial stage of the reaction, 20 μM of heme was added to a final concentration. After 3 hours of reaction, the sample was collected for nickel affinity purification to obtain the β-α subunit fusion protein. The electrophoresis gel image is shown in Figure 8. Figure 8 shows that the hemoglobin β-α subunit fusion protein successfully bound to heme.

[0237] Example 12 Oxygen-carrying experiment

[0238] The spectroscopic properties of hemoglobin under oxygen-carrying and anaerobic conditions follow the pattern shown in Figure 10 (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):

[0239] As shown in Figure 10, under oxygen-carrying conditions, hemoglobin exhibits peaks at 415, 541, and 577 nm. Figure 10 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 10 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.

[0240] 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, losing oxygen and becoming deoxygenated, resulting in a transformation of its spectroscopic properties. Based on this, redox peak analysis was performed on the proteins expressed in Examples 8 and 9 to obtain the peaks in the oxygen-carrying and deoxygenated states, as shown in Figures 7 and 9. Figures 7 and 9 show that the method of this invention successfully synthesized functional hemoglobin that successfully binds heme.

[0241] In addition, according to the method of Example 6, the proteins obtained in Examples 10 and 11 were tested for P50. The result for Example 10 was 7.92 mmHg and the result for Example 11 was 13.51 mmHg, which further confirmed that the two examples successfully obtained functional hemoglobin that binds to heme.

[0242] Example 13: Eliminating the effect of the Hemin-dissolving buffer on IVTT

[0243] According to the Hemin concentration in Example 9, the corresponding buffer was added to the IVTT at the initial stage of the EGFP IVTT reaction, without adding Hmein.

[0244] Electrophoresis and fluorescence detection after purification according to the method in Example 1 show (see Figures 11-12) that within the tested concentration range, the buffer corresponding to Hemin does not have a significant effect on the IVTT reaction activity, which means that high concentrations of Hemin will affect the IVTT reaction activity.

[0245] The sequences mentioned above in this invention are summarized in Table 2.

[0246] Based on the above-described preferred embodiments according to this application, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical concept of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for cell-free synthesis of a heme protein or peptide chain, characterized in that, The method comprises: adding a DNA or RNA template encoding a heme-binding protein or a peptide chain to a cell-free in vitro synthesis system for synthesizing a heme-binding protein without heme in vitro; incubating the heme with a binding system containing the heme-binding protein or the peptide chain for a predetermined time to generate a heme protein or a peptide chain with the heme bound thereto.

2. The method of claim 1, wherein: wherein the heme is added before the in vitro synthesis, at the beginning of the in vitro synthesis, in the reaction of the in vitro synthesis, or after the completion of the in vitro synthesis; and / or the binding system comprises the cell-free in vitro synthesis system or an eluate after purifying the heme-binding protein without heme.

3. The method of claim 1 or 2, wherein: wherein the heme-binding protein is selected from any one or more of the following: hemoglobin, peroxidase, cytochrome, bacterial ferritin, hydroxylamine oxidoreductase, nitroprotein, epoxidase, catalase, cytochrome, chloroperoxidase, PAS domain heme sensor, H-NOX heme sensor, and nitric oxide synthase; the heme-binding peptide chain refers to any single subunit of the above heme-binding protein; preferably, the heme-binding protein is selected from hemoglobin or peroxidase, and the heme-binding peptide chain is selected from any subunit of hemoglobin; further preferably, the amino acid sequence of the peroxidase is SEQ ID NO: 23 or has a sequence homology percentage of at least 85%, 90%, 95%, 97%, 98%, or 99% or more with SEQ ID NO:

23.

4. The method of claim 3, wherein: wherein, each of the aggregates is formed by two or more subunits connected in any order from N-terminus to C-terminus.

5. The method of any one of claims 1-4, wherein: wherein each of the subunits is derived from any subunit of a heme-binding protein of any species.

6. The method of any one of claims 3-5, wherein: wherein the hemoglobin comprises a unit hemoglobin, and the unit hemoglobin comprises at least two polypeptide chains, which are any one or more of an alpha chain and a beta chain, wherein the amino acid sequences of the at least two polypeptide chains are connected by a connecting peptide; preferably, the hemoglobin comprises four polypeptide chains, which are two alpha chains and two beta chains, and the amino acid sequences of the four polypeptide chains are connected by a connecting peptide.

7. The method of claim 6, wherein: wherein the hemoglobin comprises two or more of the unit hemoglobin, the two or more unit hemoglobins are connected by an inter-unit connecting peptide.

8. The method of claim 6 or 7, wherein: wherein, the intra-unit connecting peptide connects the amino acid sequences of the four polypeptide chains in any order from N-terminus to C-terminus, and the inter-unit connecting peptide connects the units in order from N-terminus to C-terminus.

9. The method of any one of claims 6-8, wherein: wherein, the number of amino acids of the intracellular linker is greater than or equal to 1, 1-140, 1-120, 1-90, 1-30, 1-35, or 1-40.

10. The method of any one of claims 6-9, wherein: the intracellular linker for connecting between two alpha chains is a first linker, and the number of amino acids of the first linker is 1-5, preferably 1, 2, or 3, the intracellular linker for connecting between two beta chains or between one alpha chain and one beta chain is a second linker, and the number of amino acids of the second linker 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.

11. The method of any one of claims 6-10, wherein: the amino acid of the first linker is 1 glycine.

12. The method of any one of claims 6-11, wherein: the second linker contains any one or more of G, S, T, and A as the main amino acid residues, and the number of the main amino acid residues is at least greater than 45%, 50%, 60%, 70%, 80%, or 90% of the total number of amino acid residues of the second linker, more preferably, among the main amino acid residues, any one or more of G and S is contained, and the total amount contained is at least greater than 40% or greater than 45% or greater than 60% or greater than 70% or greater than 80% of the total number of amino acid residues of the second linker.

13. The method of any one of claims 6-12, wherein: the second linker contains at least a flexible unit.

14. The method of claim 13, wherein: the second linker further contains a hydrophilic-hydrophobic unit composed of alternating hydrophilic and hydrophobic amino acids, and in this case, the amino acid sequence of the second linker from N-terminus to C-terminus is: flexible unit-hydrophilic-hydrophobic unit composed of alternating hydrophilic and hydrophobic amino acids-flexible unit.

15. The method of claim 12 or 13, wherein: the structure of the flexible unit is (GGGGS)a, preferably, a is 1-4, more preferably, the flexible unit is selected from one or more of the following group: (1) GGGGS; (2) GGGGSGGGGS; (3) GGGGSGGGGSGGGGSGGGGS.

16. The method of claim 14 or 19, wherein: the structure of the hydrophilic-hydrophobic 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 hydrophilic-hydrophobic unit is selected from one or more of the following group: (1) RADARADARADARADA; (2) KLDLKLDLKLDL; (3) LKLKLKLKLKLK; (4) LDLDLDLDLDLD.

17. The method of any one of claims 6-16, wherein: wherein, the amino acid sequence of the second connecting peptide is any one of SEQ ID NOs: 1-6, 10-12, 21, and 22, or has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence homology to any one of SEQ ID NOs: 1-6, 10-12, 21, and 22, preferably, the amino acid sequences of all the second connecting peptides are all the same, partially the same, or each different.

18. The method of any one of claims 6-17, wherein: the amino acid sequences of the four polypeptide chains are in any one of the following orders from N-terminus to C-terminus: a chain-a chain- b chain- b chain, b chain- b chain- a chain- a chain, b chain- a chain- a chain- b chain, a chain- b chain- a chain- b chain, and b chain- a chain- b chain- a chain.

19. The method of any one of claims 6-18, wherein: wherein, the a chain is from any one or more of human, pig, or cow, and the b chain is from any one or more of human, pig, or cow; and / or the amino acid sequence of the a chain is any one or more of SEQ ID NOs: 7, 13, and 14, or has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence homology to any one or more of SEQ ID NOs: 7, 13, and 14, respectively; the amino acid sequence of the b chain is any one or more of SEQ ID NOs: 8, 15, and 16, or has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence homology to any one or more of SEQ ID NOs: 8, 15, and 16, respectively.

20. The method of any one of claims 6-19, wherein: wherein, the inter-unit connecting peptide has a number of amino acids that is greater than or equal to 0, 0-100, or 0-90, or 0-85, and / or the inter-unit connecting peptide contains a combination of any one or more of G, S, A, and T amino acid residues, and / or the assembly connecting peptide contains a combination of any one or both of E and D amino acid residues: preferably, the number of any one or more of G, S, T, and A amino acid residues contained in the inter-unit connecting peptide is at least greater than 45%, 50%, 60%, 70%, 80%, or 90% of the total number of amino acid residues of the inter-unit connecting peptide, further, the amino acid sequence of the inter-unit connecting peptide is any one of SEQ ID NOs: 17-20, or has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence homology to any one or more of SEQ ID NOs: 17-20, respectively; and / or the units are 2, 3, or 4.

21. The method of any one of claims 1-20, wherein: the volume ratio of the DNA template to the in vitro synthesis system is 1:10-1:50, preferably 1:20-1:40; most preferably 1:25-1:35, and particularly preferably 1:

30.

22. The method of claim 21, wherein: wherein the in vitro synthesis system comprises at least any one or more of the following components: yeast cell extract, amino acid mixture, dNTP, RNA polymerase, DNA polymerase, energy supply system, polyethylene glycol, and aqueous solvent; further: the yeast cell extract is from any one or more of the following group: S. cerevisiae, S. pombe, and S. kluyveri; preferably: the yeast cell extract is from S. kluyveri, more preferably from S. kluyveri lactis; or the yeast extract is 20-80%, 20%-80%, preferably 50-80% of the entire cell-free in vitro protein synthesis system.

23. The method of any one of claims 1-22, wherein: the predetermined time is any one of the following: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, 200 minutes, 205 minutes, 210 minutes, 215 minutes, 220 minutes, 225 minutes, 230 minutes, 235 minutes, 240 minutes, 245 minutes, 250 minutes, 255 minutes, 260 minutes, 265 minutes, 270 minutes, 275 minutes, 280 minutes, 285 minutes, 290 minutes, 295 minutes, 300 minutes, 305 minutes, 310 minutes, 315 minutes, 320 minutes, 325 minutes, 330 minutes, 335 minutes, 340 minutes, 345 minutes, 350 minutes, 355 minutes, 360 minutes, 365 minutes, 370 minutes, 375 minutes, 380 minutes, 385 minutes, 390 minutes, 395 minutes, 400 minutes, 405 minutes, 410 minutes, 415 minutes, 420 minutes, 425 minutes, 430 minutes, 435 minutes, 440 minutes, 445 minutes, 450 minutes, 455 minutes, 460 minutes, 465 minutes, 470 minutes, 475 minutes, 480 minutes, 485 minutes, 490 minutes, 495 minutes, 500 minutes, 505 minutes, 510 minutes, 515 minutes, 520 minutes, 525 minutes, 530 minutes, 535 minutes, 540 minutes, 545 minutes, 550 minutes, 555 minutes, 560 minutes, 565 minutes, 570 minutes, 575 minutes, 580 minutes, 585 minutes, 590 minutes, 595 minutes, 600 minutes, 605 minutes, 610 minutes, 615 minutes, 620 minutes, 625 minutes, 630 minutes, 635 minutes, 640 minutes, 645 minutes, 650 minutes, 655 minutes, 660 minutes, 665 minutes, 670 minutes, 675 minutes, 680 minutes, 685 minutes, 690 minutes, 695 minutes, 700 minutes, 705 minutes, 710 minutes, 715 minutes, 720 minutes, 725 minutes, 730 minutes, 735 minutes, 740 minutes, 745 minutes, 750 minutes, 755 minutes, 760 minutes, 765 minutes, 770 minutes, 775 minutes, 780 minutes, 785 minutes, 790 minutes, 795 minutes, 800 minutes, 805 minutes, 810 minutes, 815 minutes, 820 minutes, 825 minutes, 830 minutes, 835 minutes, 840 minutes, 845 minutes, 850 minutes, 855 minutes, 860 minutes, 865 minutes, 870 minutes, 875 minutes, 880 minutes, 885 minutes, 890 minutes, 895 minutes, 900 minutes, 905 minutes, 910 minutes, 915 minutes, 920 minutes, 925 minutes, 930 minutes, 935 minutes, 940 minutes, 945 minutes, 950 minutes, 955 minutes, 960 minutes, 965 minutes, 970 minutes, 975 minutes, 980 minutes, 985 minutes, 990 minutes, 995 minutes, 1000 minutes, 1005 minutes, 1010 minutes, 1015 minutes, 1020 minutes, 1025 minutes, 1030 minutes, 1035 minutes, 1040 minutes, 1045 minutes, 1050 minutes, 1055 minutes, 1060 minutes, 1065 minutes, 1070 minutes, 1075 minutes, 1080 minutes, 1085 minutes, 1090 minutes, 1095 minutes, 1100 minutes, 1105 minutes, 1110 minutes, 1115 minutes, 1120 minutes, 1125 minutes, 1130 minutes, 1135 minutes, 1140 minutes, 1145 minutes, 1150 minutes, 1155 minutes, 1160 minutes, 1165 minutes, 1170 minutes, 1175 minutes, 1180 minutes, 1185 minutes, 1190 minutes, 1195 minutes, 1200 minutes, 1205 minutes, 1210 minutes, 1215 minutes, 1220 minutes, 1225 minutes, 1230 minutes, 1235 minutes, 1240 minutes, 1245 minutes, 1250 minutes, 1255 minutes, 1260 minutes, 1265 minutes, 1270 minutes, 1275 minutes, 1280 minutes, 1285 minutes, 1290 minutes, 1295 minutes, 1300 minutes, 1305 minutes, 1310 minutes, 1315 minutes, 1320 minutes, 1325 minutes, 1330 minutes, 1335 minutes, 1340 minutes, 1345 minutes, 1350 minutes, 1355 minutes, 1360 minutes, 1365 minutes, 1370 minutes, 1375 minutes, 1380 minutes, 1385 minutes, 1390 minutes, 1395 minutes, 1400 minutes, 1405 minutes, 1410 minutes, 1415 minutes, 1420 minutes, 1425 minutes, 1430 minutes, 1435 minutes, 1440 minutes, 1445 minutes, 1450 minutes, 1455 minutes, 1460 minutes, 1465 minutes, 1470 minutes, 1475 minutes, 1480 minutes, 1485 minutes, 1490 minutes, 1495 minutes, 1500 minutes, 1505 minutes, 1510 minutes, 1515 minutes, 1520 minutes, 1525 minutes, 1530 minutes, 1535 minutes, 1540 minutes, 1545 minutes, 1550 minutes, 1555 minutes, 1560 minutes, 1565 minutes, 1570 minutes, 1575 minutes, 1580 minutes, 1585 minutes, 1590 minutes, 1595 minutes, 1600 minutes, 1605 minutes, 1610 minutes, 1615 minutes, 1620 minutes, 1625 minutes, 1630 minutes, 1635 minutes, 1640 minutes, 1645 minutes, 1650 minutes, 1655 minutes, 1660 minutes, 1665 minutes, 1670 minutes, 1675 minutes, 1680 minutes, 1685 minutes, 1690 minutes, 1695 minutes, 1700 minutes, 1705 minutes, 1710 minutes, 1715 minutes, 1720 minutes, 1725 minutes, 1730 minutes, 1735 minutes, 1740 minutes, 1745 minutes, 1750 minutes, 1755 minutes, 1760 minutes, 1765 minutes, 1770 minutes, 1775 minutes, 1780 minutes, 1785 minutes, 1790 minutes, 1795 minutes, 1800 minutes, 1805 minutes, 1810 minutes, 1815 minutes, 1820 minutes, 1825 minutes, 1830 minutes, 1835 minutes, 1840 minutes, 1845 minutes, 1850 minutes, 1855 minutes, 1860 minutes, 1865 minutes, 1870 minutes, 1875 minutes, 1880 minutes, 1885 minutes, 1890 minutes, 1895 minutes, 1900 minutes, 1905 minutes, 1910 minutes, 1915 minutes, 1920 minutes, 1925 minutes, 1930 minutes, 1935 minutes, 1940 minutes, 1945 minutes, 1950 minutes, 1955 minutes, 1960 minutes, 1965 minutes, 1970 minutes, 1975 minutes, 1980 minutes, 1985 minutes, 1990 minutes, 1995 minutes, 2000 minutes, 2005 minutes, 24. The method of any one of claims 1-23, wherein: ​ 25. A cell-free in vitro synthesis system for synthesizing the heme protein or peptide chain according to any one of claims 1 to 24, characterized in that ​ ​ ​ ​ ​ ​ ​

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