Hemoglobin mutant with improved stability and application thereof

Abstract

The invention discloses a hemoglobin mutant with improved stability and application thereof, and belongs to the technical field of gene engineering. According to the invention, a high-throughput screening method for structural stability of human hemoglobin and heme binding reinforcement mutants is designed, so that a foundation is laid for obtaining ideal stable human hemoglobin mutants. Hot amino acid sites for structural stability and thermal stability modification of human hemoglobin are determined through semi-rational design, iterative saturated mutation is further performed to obtain an ideal mutant rHb9th, and the mutant rHb9th has the advantages of structural stability, thermal stability, autoxidation resistance, heme binding rate and the like. Compared with the rHbwild-type, the anti-heme loss rate, the yield of human hemoglobin and the like are remarkably improved.

Description

Technical Field

[0001] The invention relates to a hemoglobin mutant with improved stability and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Human hemoglobin is a tetrameric protein consisting of two identical alpha globins (141 residues) and two identical beta globins (146 residues). Each globin binds to one molecule of hemoglobin, which further carries one molecule of oxygen. Hemoglobin plays a vital role in transporting oxygen, with an efficiency more than 2 times higher than that of red blood cells. Currently, heterologous synthesis of recombinant human hemoglobin (rHb) in Escherichia coli or Saccharomyces cerevisiae has many advantages over the traditional method of extracting hemoglobin from human blood, including reduced risk of disease transmission and cost savings. However, the synthesis of rHb lacks the antioxidant system (superoxidase and catalase) present in red blood cells, which makes rHb unstable, further affecting its application as an artificial oxygen carrier.

[0003] The instability of rHb mainly includes unstable protein structure and rapid loss of cofactor heme due to rapid self-oxidation of rHb. Unstable protein structure can easily decompose human hemoglobin into dimers and monomers, causing rHb degradation and causing serious damage to the kidneys. The rapid loss of heme will cause rHb to lose the ability to transport oxygen and accelerate the degradation of rHb. At present, the methods to enhance the stability of rHb mainly include chemical modification and genetic engineering mutagenesis. However, chemical modification not only increases the cost of rHb synthesis, but also is not conducive to its subsequent large-scale application. The current genetic engineering mutagenesis strategy for rHb stability modification only relies on strategies such as rational design of a few sites, and the efficiency of the modification is limited.

[0004] Therefore, in order to obtain more stable rHb mutants, random mutagenesis, directed evolution or rational modification of neglected key residues can be used to generate a large number of potential mutants. Then, high-throughput screening based on improved fluorescent biosensors can be performed to quickly obtain ideal rHb mutants. Summary of the invention

[0005] The present invention first designs a high-throughput screening strategy for human hemoglobin structural stability and heme binding enhanced mutants, and simultaneously uses a semi-rational design strategy to determine the corresponding hotspot amino acid sites for modification, ultimately achieving the gradual enhancement of the structural stability and heme binding ability of human hemoglobin.

[0006] The present invention provides a hemoglobin mutant having a mutation shown in (a) or (b) based on the starting sequence:

[0007] (a) The glycine at position 25 of α-globin was mutated to cysteine, the glycine at position 57 was mutated to lysine, and the proline at position 114 was mutated to isoleucine; the histidine at position 2 of β-globin was mutated to glutamic acid, the histidine at position 97 was mutated to tryptophan, and the asparagine at position 102 was mutated to threonine; the obtained mutants were named α-G25C / G57K / P114I and β-H2E / H97W / N102T;

[0008] (b) The glycine at position 25 of α-globin was mutated to cysteine, the tyrosine at position 42 was mutated to serine, the glycine at position 57 was mutated to lysine, and the proline at position 114 was mutated to isoleucine; the histidine at position 2 of β-globin was mutated to glutamic acid, the serine at position 44 was mutated to aspartic acid, the glycine at position 57 was mutated to lysine, the histidine at position 97 was mutated to tryptophan, and the asparagine at position 102 was mutated to threonine; the obtained mutants were named α-G25C / Y42S / G57K / P114I, β-H2E / S44D / P51N / H97W / N102T.

[0009] In one embodiment, the starting sequence of α-globin is shown as SEQ ID NO.1; and the starting sequence of β-globin is shown as SEQ ID NO.2.

[0010] In one embodiment, the amino acid sequences of the mutants α-G25C / G57K / P114I, β-H2E / H97W / N102T are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0011] In one embodiment, the amino acid sequences of the mutants α-G25C / Y42S / G57K / P114I, β-H2E / S44D / P51N / H97W / N102T are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0012] The present invention also provides a gene encoding the hemoglobin mutant.

[0013] The invention also provides a recombinant plasmid carrying the gene.

[0014] The present invention also provides a recombinant microorganism expressing the hemoglobin mutant.

[0015] The present invention also provides a biosensor that can be used to screen structurally stable hemoglobin, wherein the biosensor has a structure such as "α-globin-(SSG)2-CysG A –(G4S)3-β-globin”; CysG AIt is the siroheme synthetase encoded by the sequence shown in SEQ ID NO.8, (SSG)2 is the connecting peptide SSGSSG, and (G4S)3 is the connecting peptide GGGGSGGGGSGGGGS.

[0016] In one embodiment, the biosensor is constructed on a plasmid, including but not limited to pETDuet-1.

[0017] In one embodiment, the starting strain of Escherichia coli required for the structural stability modification of rHb includes but is not limited to Escherichia coli BL21 (DE3).

[0018] The present invention also provides the use of the recombinant microorganism in preparing the hemoglobin.

[0019] In one embodiment, the use is to culture the recombinant microorganism in a culture medium, and add heme during the culture process.

[0020] In one embodiment, the concentration of hemoglobin in the culture environment is 0-20 mg / L.

[0021] The present invention provides a high-throughput screening method for human hemoglobin heme binding enhanced mutants, which is to introduce a double plasmid screening system into the starting strain cells of Escherichia coli; the double plasmid includes a heme detection element HS1 M7A The recombinant plasmid pTf16-HS1 M7A and the recombinant plasmid pET-rHb carrying the hemoglobin encoding gene.

[0022] In one embodiment, the human hemoglobin rHb wild-type The amino acid sequence is shown in PDB: 2DN2, the PDB ID of the red fluorescent protein mKATE2 is 3PJ5, and the PDB ID of the green fluorescent protein eGFP is 2GX2.

[0023] In one embodiment, the hemoglobin detection element HS1 M7A The sequence is shown in SEQ ID NO.7.

[0024] In one embodiment, the Escherichia coli starting strain includes but is not limited to Escherichia coli R11-HEME, which has been disclosed in the paper "Whole-cell P450 biocatalysis using engineered with fine-tuned hemebiosynthesis".

[0025] In one embodiment, the dual plasmid screening system determined by the high-throughput screening strategy for rHb heme binding enhancement is rHb-HS1 M7A(RSF ori). Among them, the pETDuet-1 plasmid screened positive clones by ampicillin resistance, the replicon was pBR322 ori, and the rHb encoding gene was fused downstream of the T7 promoter, and the expression of rHb was induced by isopropyl-β-D-thiogalactoside (IPTG). The pTf16 (RSF ori) plasmid screened positive clones by chloramphenicol resistance, the replicon was RSF ori, and HS1 was fused downstream of the araB promoter. M7A Encoding gene, HS1 induced by arabinose M7A expression.

[0026] The present invention provides a method for improving the structural stability of human hemoglobin, which is to mutate one or more of the following amino acid sites of human hemoglobin: α-G15, α-K16, α-G25, α-G57, α-V93, α-D94, α-P114, β-H2, β-G16, β-G69, β-S72, β-H97 and β-N102.

[0027] In one embodiment, the method is to mutate the glycine at position 25 of α-globin to cysteine, the glycine at position 57 to lysine, and the proline at position 114 to isoleucine; mutate the histidine at position 2 of β-globin to glutamic acid, the histidine at position 97 to tryptophan, and the asparagine at position 102 to threonine; and obtain mutants α-G25C / G57K / P114I, β-H2E / H97W / N102T.

[0028] The present invention also provides the use of the hemoglobin mutant and the recombinant microorganism in preparing hemoglobin or related products.

[0029] Beneficial effects:

[0030] (1) The present invention designs high-throughput screening strategies for mutants with enhanced structural stability and heme binding ability of multi-subunit protein human hemoglobin, which is conducive to the efficient acquisition of ideal mutants.

[0031] (2) The present invention uses a semi-rational design strategy to determine the key amino acid sites for the structural stability of human hemoglobin and the heme binding enhancement iterative saturation library, and obtains mutant rHb 9th Compared with rHb, the thermal stability (TM value increased by 4.2°C), structural stability (increased by 26.7%), self-oxidation ability (reduced by 15.4 times), heme binding rate (increased by 37.4%), heme loss rate (reduced by 15.5 times and 2.3 times in α and β globin, respectively) and protein yield (increased by 1.63 times) of α-globin and β-globin were significantly improved. wild-type All have been significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 High-throughput screening strategy for structural stability modification of human hemoglobin. a is a high-throughput screening strategy for determining the appropriate one through linker optimization. b is rHb-CysG with different linkers. A CysG fusion protein A Red fluorescence intensity. c is rHb-CysG A Correlation analysis between the fluorescence intensity of the fusion protein and the mutation energy of the mutant. The red line represents the junction between the C-terminus of β-globin and CysG A The C-terminal (G4S)3 linker, the green line represents the link between the β-globin C-terminus and CysG A The C-terminus of the (G4S)2 linker.

[0033] Figure 2 A high-throughput screening strategy for the heme binding enhancement modification of human hemoglobin. Among them, a is the construction of a high-throughput screening strategy for human hemoglobin variants (PDB ID of human hemoglobin: 2DN2, PDB ID of red fluorescent protein mKATE2: 3PJ5, PDB ID of green fluorescent protein eGFP: 2GX2). b is rHb-HS1 under different replicon (RSF ori and p15Aori) or induction conditions. M7A eGFP / mKATE2 fluorescence of the system. 1 and 2 represent the addition of the inducer IPTG first, followed by the addition of arabinose. 3 represents the simultaneous addition of two inducers (IPTG and arabinose). c is the verification of the heme loss rate in human hemoglobin by adding different heme dissociation reagents. d is rHb-HS1 M7A Correlation analysis between the fluorescence ratio and the mutation energy of rHb mutants.

[0034] Figure 3 Semi-rational design and modification of human hemoglobin to improve its structural stability. Among them, a is the mutation site determined by semi-rational design to improve rHb stability. Green amino acids represent candidate sites after virtual saturation mutation, and red amino acids represent sites ultimately used for iterative saturation mutation. b is the process of iterative saturation mutation. cg are the properties of the ideal mutant determined by each round of iterative saturation mutation: c is the fluorescence intensity, d is the biomass and human hemoglobin yield, e is the secondary structure of the mutant determined by circular dichroism spectroscopy, f is the thermal stability of the mutant determined by differential scanning calorimetry, and g is the self-oxidation rate of human hemoglobin mutants.

[0035] Figure 4Semi-rational design and modification of human hemoglobin to improve its heme binding ability. Among them, a is the selection of appropriate mutation sites to enhance the heme binding strength of hemoglobin. Black amino acids represent the sites that are ultimately used for iterative saturation mutation. b is the process of iterative saturation mutation. cg are the properties of the ideal mutants determined by each round of iterative saturation mutation: c is the eGFP / mKATE2 fluorescence ratio, d is the CysG A Fluorescence intensity, e is the biomass and human hemoglobin production, f is the heme loss rate, and g is the heme binding rate. DETAILED DESCRIPTION

[0036] Purification and quantification method of human hemoglobin: The synthetic rHb was suspended in 20.0 mM phosphate buffer (PBS, including 5.0 mM NaH2PO4, 48.0 mM Na2HPO4, 0.5 M NaCl, pH 7.4), and phenylmethylsulfonyl fluoride was added at a final concentration of 1 mM as a protease inhibitor to prevent rHb degradation. The cells were disrupted at 4°C using a high-pressure homogenizer (Union Biotech). The supernatant was collected by centrifugation at 4°C for 10 minutes (7000 rpm) and added to a His-tagged affinity gravity column. 10 column volumes of PBS buffer A (pH 7.4, final concentration of 20.0 mM imidazole) were added to wash the gravity column, and then 10 column volumes of PBS buffer B (pH 7.4, final concentration of 80.0 mM imidazole) were used to wash away other proteins, and rHb was eluted with PBS buffer C (pH7.4, 0.5 M imidazole). Use The imidazole concentration of the purified rHb was reduced to below 50.0 mM using an Ultra-0.5 centrifuge (Merck KGaA), and the protein concentration was determined using a Bradford protein assay kit (Beyotime Biotech). The quality of the purified rHb was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0037] Determination of oxygen saturation: Sodium dithionite was used as a deoxygenating agent and reducing agent for methemoglobin. rHb and sodium dithionite solution were mixed in a 96-well black plate at a ratio of 1:13, and then rHb was converted into deoxyhemoglobin. Then, the mixed solution was incubated at 37°C and A was measured by a microplate reader (BioTek synergy H1). 556 and A 523 The absorbance value of each round of measurement was measured. The 96-well black plate was shaken at 300 rpm / min for 1 minute. The oxygen saturation of rHb was determined according to the previous method:

[0038]

[0039] K Hb K is the absorbance of rHb when it is completely hypoxic; HbO2 is the absorbance when rHb is completely oxidized, K x It is the absorbance when oxygenation reaches a specific time (t).

[0040] Oxygen balance curve: The oxygen balance curve of rHb was determined using an oxygen binding / release instrument (BL00DOX-2018), and ferrous hemoglobin was prepared using a 0.1M sodium dithionite solution. During the measurement, the partial pressures of air and nitrogen were set to 0.1MPa, and the reaction temperature was maintained at 37°C. A mixture of 3.0mg rHb and 4.0mL buffer (including 50.0mM HEPES, 0.1MNaCl, 0.1mM EDTA, and 0.1mM bovine serum albumin) was injected into the sample cell of the oxygen binding / release instrument. Open the air valve and let in air at a constant rate (13.0mL / min) until P O2 Then, nitrogen was introduced at a constant rate (13.0 mL / min) to measure the oxygen dissociation rate until P O2 The value stabilizes again, and the P of rHb is calculated 50 Value and Hill coefficient.

[0041] Circular dichroism spectroscopy to detect the structural stability of rHb: Circular dichroism measurements were performed using a Chirascan spectropolarimeter. 6.0 M guanidine hydrochloride (GdnHCl) was prepared in 0.2 M potassium phosphate buffer (containing 68.0 mM KH2PO4 and 131.0 mM K2HPO4, pH 7.0), and the pH of the potassium phosphate buffer was adjusted to 7.0 after the addition of the denaturant GdnHCl. 12.0 μM rHb and different concentrations of GdnHCl (0.5, 1.0, 1.5, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, and 4.0 M) were dissolved in 0.2 M potassium phosphate buffer (pH 7.0) to prepare a mixture. The rHb-GdnHCl mixture was incubated in a 10 °C water bath for 1 hour before spectral measurements. The circular dichroism spectra of each sample were recorded in the range of 190 to 250 nm using a JASCO 1700CD spectropolarimeter, and the negative ellipticity changes at 222 nm were monitored to characterize the secondary structure changes of rHb.

[0042] Differential Scanning Calorimetry to Determine the Thermal Stability of rHb: The thermal denaturation process of rHb was studied by nano-DSC in the temperature range of 40-90°C. 0.3 mL of rHb (15.5 μM) was loaded into the sample cell, and the differential scanning curve of the degassed sample (equilibrated for 600 seconds) was recorded at a constant external pressure of 3 atm and a scanning rate of 1°C per minute. wild-typeas a control, and the data were analyzed using Nano Analyzer.

[0043] Determination of the auto-oxidation rate of rHb: 10.0 μM rHb solution was diluted in 0.2 M potassium phosphate (pH 7.0) buffer. Deoxyhemoglobin was prepared by a concentration ratio of 1:13 (rHb solution: sodium dithionite solution). The complete spectrum was recorded in the wavelength range of 400-700 nm at 37°C for 64 hours using a microplate reader (BioTek synergy H1). The values ​​of oxyhemoglobin and methemoglobin were calculated by multicomponent analysis, and the auto-oxidation rate in the percentage of oxygenated hemoglobin was fitted to a single exponential expression by nonlinear least squares curve fitting.

[0044] Determination of heme binding rate: 0.13 μM rHb was added to a solution containing 3.5 mL pyridine and 1.5 mL 1 M NaOH, and then mixed with a 0.2 M sodium dithionite solution. The characteristic absorption peak at 410 nm was immediately detected, and the molar ratio of heme to rHb (i.e., the molar percentage of heme bound per mole of rHb) was determined based on the extinction coefficient to characterize the heme binding rate:

[0045] ε 410 =342500M -1 cm -1

[0046] Determination of heme loss rate: 0.45 M sucrose was added to 0.1 M PBS buffer (pH 7.4), and 6.5 μM methemoglobin was mixed with 40.0 μM sperm whale apomyoglobin mutant apoMb H64Y / V68F Mixed. Among them, apoMb H64Y / V68F As a high affinity heme scavenger, high concentration of sucrose was used as an osmotic agent to prevent apohemoglobin precipitation. The absorbance in the wavelength range of 400-650 nm was recorded for 4 hours at 37°C. 410 The specific absorbance was used to calculate the heme loss rate using a biexponential expression fitted by nonlinear least squares curve fitting.

[0047] Example 1 Construction of a high-throughput detection strategy for human hemoglobin structural stability mutants

[0048] The structural stability of human hemoglobin is mainly reflected in the fact that structurally stable hemoglobin mutants are not easily cleaved during intracellular synthesis, which is conducive to the synthesis of higher yields of target proteins. A ) can convert uroporphyrin III into proporphyrin-2, which emits bright red fluorescence in cells. A G364 The target protein is inserted into the site. If the target protein structure is stable, the fusion can continue to emit fluorescence, while if the target protein is unstable, it will cleave, resulting in the CysG in the fusion being divided into two segments. A The fluorescent signal is lost because the protein cannot bind normally. Based on this principle, the purpose of high-throughput screening of stable protein mutants can be achieved. CysG was amplified from the genome of Escherichia coli BL21 (DE3) strain A The rHb gene (nucleotide sequence is shown in SEQ ID NO.8) was cloned into the downstream of the T7 promoter of the pETDuet-1 vector. The rHb gene (nucleotide sequence of α globin is shown in SEQ ID NO.9, and the nucleotide sequence of β globin is shown in SEQ ID NO.10) was inserted into the CysG A The 364th amino acid (G 364 site), and linkers of different lengths (5-54 bp) and glycine percentages (33.0-80.0%) were selected to achieve CysG A The C-terminus of α-globin (nucleotide sequence as shown in SEQ ID NO.8) and β-globin (nucleotide sequence as shown in SEQ ID NO.10) were effectively connected to construct multiple "α-globin-linker 1-CysG A -linker 2-β-globin" rHb-CysG A The fusion protein, wherein linker1 is (SSG)2, and linker2 is selected from the connecting peptides shown in Table 1.

[0049] Table 1 for CysG A The C-terminus of β-globin was fused to linkers of varying lengths and glycine ratios

[0050]

[0051]

[0052] The constructed rHb-CysG A The fusion proteins were expressed in BL21 (DE3) strains using pETDuet-1 as a vector. The constructed recombinant Escherichia coli was cultured in Luria-Bertani medium (0.5% yeast extract, 1.0% peptone, 1.0% sodium chloride, 100 mg / L ampicillin) at 37°C (220 rpm) until OD 600When the value reached 0.6, the strain was induced with 10.0 μM IPTG, and 20.0 mg / L heme was supplemented at the same time to promote the synthesis of rHb. The strain was cultured at 30°C for 10 h (220 rpm), and the cells were washed twice with 20.0 mM PBS buffer (pH 7.4). CysG was measured at a specific wavelength (excitation wavelength 357 nm, emission wavelength 620 nm) using a microplate reader (BIO-RAD). A Select CysG A The fusion protein rHb-CysG with the lowest fluorescence intensity A (G4S)3 (Linker is (G4S)3) as a candidate for rHb stability modification

[0053] The Gibson assembly method was used to construct a mutant with the structure of "α-globin mutant-(SSG)2-CysG A -(G4S)3-β-globin mutant" is a fusion protein with a structure shown in the figure, wherein the rHb mutant is selected from 6 rHb mutants disclosed in the prior art, including three mutants with better stability (α-G15A, α-G15A / β-G16A and α-G15A / β-G16A / β-H116I) and three mutants with weaker stability (α-P119S, α-L29W and β-L28W). The stability mutation energies of the six mutants were calculated based on the Calculate Mutation Energy (Stability) module of the molecular simulation tool Discovery Studio 2019 (α-P119S>α-L29W>β-L28W>wild-type>α-G15A>α-G15A / β-G16A>α-G15A / β-G16A / β-H116I). In addition, according to the above rHb-CysG A CysG fusion protein A The fluorescence value detection method was used to determine the six rHb mutants-CysG A (G4S)3 The fluorescence intensity of the fusion protein was determined based on the expression of these rHb mutants-CysG A The correlation between the fluorescence intensity of the fusion protein and the stable mutation energy of the rHb mutant verifies the rHb-CysG A The reliability of the folded sensor. A (G4S)3 The correlation was significantly higher than that of the control rHb-CysG (R2=0.95, P<0.05). A (G4S)2 (R2=0.72,P>0.05, Figure 1 ), indicating that the folding sensor rHb-CysGA (G4S)3 It can be applied to the screening of hemoglobin structural stability mutants.

[0054] Example 2 Construction of a high-throughput detection strategy for human hemoglobin heme binding enhanced mutants

[0055] Due to the trace amount of heme binding and the rapid loss of heme during rHb synthesis, the current methods for enhancing heme binding capacity cannot meet the needs of high-throughput screening. Therefore, this embodiment designs a unique dual-plasmid screening system (rHb-HS1 M7A ), the first recombinant plasmid is used for the heme detection element HS1 M7A The expression of heme in hemoglobin is used to detect the heme escape rate. pTf16 is used as the plasmid backbone, the screening marker is chloramphenicol resistance, the replicon is RSFori / p15Aori, and the heme detection element HS1 M7A It is constructed downstream of the araB promoter and expressed by arabinose induction. Heme detection element HS1 M7A (The sequence is shown in SEQ ID NO.7) It consists of a fluorescent protein eGFP that is sensitive to heme and a fluorescent protein mKATE 2 that is insensitive to heme, wherein the cytochrome b in eGFP 562 The eGFP fluorescence intensity decreases when the heme binding domain of the recombinant plasmid binds heme. The second recombinant plasmid is used for the expression of hemoglobin. Its plasmid backbone is pETDuet-1, the selection marker is ampicillin resistance, the replicon is pBR322 ori, and the hemoglobin encoding gene is constructed downstream of the T7 promoter and expressed by IPTG induction. M7A It was co-transformed with the recombinant plasmid pET-rHb carrying the hemoglobin encoding gene into the Escherichia coli HEME-R11 strain with enhanced heme supply disclosed in the prior art (disclosed in the paper "Whole-cell P450 biocatalysis using engineered with fine-tuned heme biosynthesis"), thereby obtaining a strain with a corresponding dual-plasmid expression system.

[0056] According to the above strategy, we constructed wild-type The recombinant plasmid pET-rHb (the coding gene sequence is shown in SEQ ID NO.9 and SEQ ID NO.10) wild-type rHb wild-type and containing rHb β-K82D (amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.11)β-K82D rHb β-K82D The strain rHb wild-type and strain rHb β-K82D The cells were cultured in TB medium (50 mg / L ampicillin and 17 mg / L chloramphenicol) at 37°C and 220 rpm until OD 600 The value reached 0.6, and induced by 40mM IPTG and 10mM arabinose at the final concentration. The strain was further cultured at 30°C for 5 hours, and then the cells were washed twice with 20.0mM PBS buffer (pH 7.4). The fluorescence ratio of eGFP / mKATE2 in these cells was measured respectively (eGFP: excitation wavelength 488nm, emission wavelength 523nm; mKATE2: excitation wavelength 588nm, emission wavelength 620nm). rHb-HS1 M7A The eGFP / mKATE2 fluorescence ratio of (RSF ori) reached 4.52 ( Figure 2 ), indicating that the response range of the system to heme is significantly enhanced.

[0057] In addition, in order to accelerate the dissociation rate of rHb-bound heme, rHb-HS1 was expressed in M7A Different heme dissociation reagents were added to 2 mL of fermentation broth of (RSF ori), including cuprous chloride (1.0, 10.0, 100.0, 1000.0 μM), hexadecyltrimethylammonium bromide (2.0, 20.0, 200.0, 2000.0 μM), sodium dodecyl sulfate (50.0, 500.0, 2000.0 μM) and acidified ethanol (0.05%, 0.25%, 5% v / v). After culturing at 30°C for 8 hours (rotation speed 220 rpm), the fluorescence ratio of eGFP / mKATE2 in these cells was detected. The results showed that when 50.0 μM sodium dodecyl sulfate was added, rHb wild-type and the heme binding enhanced mutant rHb disclosed in the prior art β-K82D The eGFP / mKATE2 fluorescence ratios decreased by 39.8±2.5% and 41.9±4.0%, respectively, indicating that this concentration of sodium dodecyl sulfate can accelerate the dissociation rate of heme in rHb.

[0058] Finally, to verify rHb-HS1 M7AThe screening efficiency was high, and seven reported rHb mutants were selected (the starting sequences of rHb mutants are shown in SEQ ID NO.1 and SEQ ID NO.2), including five mutants with enhanced heme binding ability (β-V67T, β-F41Y, β-T84Y, β-K82D and β-S44H) and two mutants with reduced heme binding ability (β-H92Q and α-H58L). The correlation between the fluorescence ratio of eGFP / mKATE2 and the binding mutation energy of these mutants was studied using the molecular simulation tool Discovery Studio 2019. The results showed that rHb-HS1 M7A The correlation between rHb-HS1 and RSF ori was 0.89 (P<0.05), indicating that rHb-HS1 M7A (RSF ori) can be effectively used to screen rHb mutants with enhanced heme-binding ability.

[0059] Example 3 Screening of structurally stable human hemoglobin mutants using a semi-rational design strategy

[0060] Based on the structural characteristics of rHb (the coding gene sequences are shown in SEQ ID NO.9 and SEQ ID NO.10), appropriate mutation sites were determined by a semi-rational design strategy. The rHb mutation sites were selected mainly from four aspects: (1) subunit contacts at the α1β1 interface (34 amino acid residues) and the α1β2 interface (19 amino acid residues); (2) surface amino acid sites of rHb (33 amino acid residues in α-globin and 30 amino acid residues in β-globin); (3) amino acid residues with B factor values ​​higher than 40 (potential unstable sites, 8 sites); (4) potential amino acid sites for improving rHb stability screened by the virtual alanine scanning module of Discovery Studio 2019 (Stability Mutation Energy <-0.40 kcal / mol, 15 residues). Among these residues, first, sites with specific functions were removed, including Bohr effect (α-H45, β-L141 and β-H146) and allosteric function (α-K99, α-R141), etc. Secondly, 31 key residues that may improve stability were identified by virtual saturation mutagenesis (Stability Mutation Energy <-2.00 kcal / mol). In order to further improve the screening efficiency, a saturation mutation library was constructed using the NNK degenerate bases for the 31 key residues of rHb. A CysG fusion protein A The experimental process of fluorescence signal detection was to detect the CysG AThe fluorescence was verified. wild-type ) A The ratio of fluorescence intensity (>1.2) identified 13 hotspot residues associated with rHb stability, namely α-G15, α-K16, α-G25, α-G57, α-V93, α-D94, α-P114, β-H2, β-G16, β-G69, β-S72, β-H97 and β-N102. These residues can then be used for iterative saturation mutagenesis. In addition, α-P114I (rHb 1st ) A The highest fluorescence intensity is rHb wild-type 1.75 times ( Figure 3 ), indicating that α-P114I can be used as the starting mutant for subsequent iterative saturation mutagenesis.

[0061] In order to effectively screen ideal mutants, firstly, negative mutants with reduced stability, including rHb (α-G25D) and (β-H97P) mutants, were removed using the HbVar database (http: / / globin.bx.psu.edu / hbvar / ). The 13 hotspot residues mentioned above were subjected to saturation mutagenesis using NNK degenerate bases to construct plasmid pETDuet-1 carrying rHb mutants, in which the starting sequence of hemoglobin mutants (nucleotide sequence of α-globin as shown in SEQ ID NO.9, nucleotide sequence of β-globin as shown in SEQ ID NO.10) was constructed downstream of the T7 promoter. The plasmid carrying rHb mutants was transformed into the BL21 (DE3) strain, and 100 clones were screened for each amino acid residue site to achieve 95% coverage in the library. Each single colony was further transferred to a 96-well plate containing 0.2 mL of Luria-Bertani medium (100 mg / L ampicillin) and cultured at 37 °C (220 rpm) until OD 600 The value reached 0.6, and the strain was induced with 10.0 μM IPTG, and 20.0 mg / L heme was supplemented to promote the synthesis of rHb. The strain was cultured at 30°C for 10 hours (220 rpm), and the cells were washed twice with 20.0 mM PBS buffer (pH 7.4). The CysG A The fluorescence value was calculated and the mutant with the highest fluorescence value in each round of iterative saturation mutagenesis was selected for the next round of saturation mutagenesis. 6th (α-G25C / G57K / P114I, β-H2E / H97W / N102T) CysG A Fluorescence intensity ratio rHb wild-type The result was 7.87 times higher than that of the ideal mutant rHb obtained by the previous round of iterative saturation mutation.5th (α-G25C / G57K / P114I, β-H2E / N102T) CysG A The fluorescence values ​​were similar, indicating that the ideal stable rHb mutant rHb had been obtained. 6th Meanwhile, the oxygen affinity of all ideal mutants in each iteration was checked to ensure their future applications in the field of artificial oxygen carriers.

[0062] To verify rHb 6th The stability of rHb was investigated by measuring its secondary structure, thermal stability and self-oxidation rate. The secondary structure of rHb was verified by circular dichroism signal value at 222 nm, and guanidine hydrochloride (GdnHCl) was used as the denaturant of rHb. 6th [GdnHCl] midpoint (GdnHCl concentration at which 50% of the secondary structure is dissociated) increased to 1.90 M, which is higher than rHb wild-type (1.50M) increased by 26.7%. In addition, rHb 6th The melting temperature (TM) of rHb increased to 69.74±0.05℃, which was higher than that of rHb wild-type (64.04±0.13℃) ​​increased by 8.9%. In addition, rHb 6th The self-oxidation rate (k autox =0.011±0.005 / h) than rHb wild-type (k autox =0.167±0.034 / h) decreased by 14.83 times, indicating that rHb 6th It has good resistance to self-oxidation. In addition, due to the improved stability, rHb 6th The yield (83.23±1.51mg / L) is rHb wild-type Therefore, compared with rHb wild-type In comparison, rHb 6th It has significant advantages in commercial production and application, including higher stability, oxidation resistance and yield.

[0063] Example 4 Screening of human hemoglobin mutants with enhanced heme binding using a semi-rational design strategy

[0064] rHb-HS1 constructed in Example 2 M7A The (RSF ori) system can be used to screen rHb mutants with enhanced heme binding ability. In order to determine the appropriate modification site through semi-rational design, rHb 6th Around the heme pocket in α and β globin Virtual saturation mutations were performed on residues within the range of rHb, as well as residues of the CE helix that affect heme binding in β-globin. Then, 26 sites were selected as candidate sites based on the mutations that significantly enhanced heme affinity (Binding Mutation Energy <-0.60 kcal / mol) in the virtual saturation mutations. Subsequently, in order to narrow the screening range, saturation mutations were performed on these 26 candidate residues using NNK degenerate base pairs, and the plasmid carrying the rHb mutant was transformed into the Escherichia coli R11-HEME strain. At least 100 clones were selected for each key site to verify the effect of the mutation. And based on the comparison between the mutant and the control (rHb 6th ) between the two groups (>1.50-fold), we further identified nine sites for subsequent iterative saturation mutagenesis, namely: α-F33, α-Y42, α-F43, β-W15, β-S44, β-P51, β-K66, β-K82 and β-D99.

[0065] In order to efficiently obtain the ideal mutant, the above 9 sites were saturatedly mutated using NNK degenerate bases to construct a plasmid pETDuet-1 carrying the rHb mutant, in which the coding gene (nucleotide sequence shown in SEQ ID NO.9 and SEQ ID NO.10) of the hemoglobin mutant (amino acid sequence shown in SEQ ID NO.1 and SEQ ID NO.2) was constructed downstream of the T7 promoter and expressed by IPTG induction. M7A The pTf16 plasmid was co-transformed into the E. coli HEME-R11 strain, and 100 clones were screened for each amino acid residue site to achieve 95% coverage in the library. Each single colony was further transferred to a 96-well plate containing 0.2 mL TB medium (50 mg / L ampicillin, 17 mg / L chloramphenicol) and cultured at 37°C and 220 rpm until OD 600 The value reached 0.6. The strain was induced with 40mM IPTG and 10mM arabinose and cultured at 30°C and 220rpm for 5 hours. Subsequently, 0.05mM SDS was added to the fermentation broth to accelerate the rate of heme loss in rHb. The strain was cultured for another 8 hours at 30°C and 220rpm, the bacterial cells were collected by centrifugation, and the cells were washed twice with 20.0mM PBS buffer (pH 7.4). The eGFP / mKATE2 fluorescence ratio of these cells was measured, and the mutant with the highest fluorescence ratio in each round of iterative saturation mutagenesis was selected for the next round of saturation mutagenesis. After three rounds of iterative saturation mutagenesis, rHb 9th(α-G25C / Y42S / G57K / P114I, β-H2E / S44D / P51N / H97W / N102T) eGFP / mKATE2 fluorescence ratio compared to rHb 6th Increased by 2.64 times ( Figure 4 ). In addition, rHb 9th CysG A Fluorescence intensity ratio rHb 6th 3.6% higher, indicating that subsequent mutations did not affect rHb 6th stability, so rHb 9th It is an ideal mutant with significantly enhanced heme binding ability.

[0066] Finally, due to rHb 9th The heme pocket region in the cytoplasm has changed, resulting in a significant decrease in the rate of heme loss (k -H β=1.32±0.10 / h,k -H α=0.02±0.004 / h), and rHb 6th In comparison, rHb 9th The heme dissociation rate of β-globin was reduced by 1.40 times, and that of α-globin was reduced by 6.00 times. 9th The enhanced heme binding capacity also helped to increase rHb production (84.94±2.70mg / L) and heme binding rate (55.7±1.8% mol heme / mol rHb), and reduce the autoxidation rate (k autox =0.008±0.003 / h), while rHb 9th With rHb 6th There were no significant differences in other properties.

[0067] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A hemoglobin mutant, characterized in that Based on the starting sequence, one or more of the following sites were mutated: G15, K16, G25, G57, V93, D94, P114 of α-globin, and H2, G16, G69, S72, H97 and N102 of β-globin.

2. The hemoglobin mutant according to claim 1, characterized in that Having the mutation shown in (a) or (b): (a) The glycine at position 25 of α-globin is mutated to cysteine, the glycine at position 57 is mutated to lysine, and the proline at position 114 is mutated to isoleucine; the histidine at position 2 of β-globin is mutated to glutamic acid, the histidine at position 97 is mutated to tryptophan, and the asparagine at position 102 is mutated to threonine; (b) The glycine at position 25 of α-globin was mutated to cysteine, the tyrosine at position 42 was mutated to serine, the glycine at position 57 was mutated to lysine, and the proline at position 114 was mutated to isoleucine; the histidine at position 2 of β-globin was mutated to glutamic acid, the serine at position 44 was mutated to aspartic acid, the glycine at position 57 was mutated to lysine, the histidine at position 97 was mutated to tryptophan, and the asparagine at position 102 was mutated to threonine.

3. The hemoglobin mutant according to claim 1 or 2, characterized in that: The starting sequence of α-globin is shown in SEQ ID NO.1; the starting sequence of β-globin is shown in SEQ ID NO.

2.

4. A gene encoding the hemoglobin mutant according to any one of claims 1 to 3.

5. A recombinant plasmid carrying the gene according to claim 4.

6. A recombinant microorganism expressing the hemoglobin mutant according to any one of claims 1 to 3.

7. A recombinant Escherichia coli, characterized in that pETDuet-1 is used as an expression vector to express the hemoglobin mutant according to any one of claims 1 to 3.

8. A method for preparing hemoglobin, characterized in that: The recombinant microorganism according to claim 6 or the recombinant Escherichia coli according to claim 7 is cultured in a culture medium, and the heme concentration in the culture environment is controlled to be 0-20 mg / L.

9. A biosensor for screening structurally stable hemoglobin, characterized in that: With "α-globin-(SSG)2-CysG A -(G4S)3-β-globin"; wherein CysG A It is the siroheme synthetase encoded by the sequence shown in SEQ ID NO.8, (SSG)2 is the connecting peptide SSGSSG, and (G4S)3 is the connecting peptide GGGGSGGGGSGGGGS.

10. A recombinant microorganism containing a dual plasmid selection system, characterized in that: The dual plasmid system consists of a heme detection element HS1 M7A The recombinant plasmid pTf16-HS1 M7A and the recombinant plasmid pET-rHb carrying the hemoglobin encoding gene.

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