Heat-resistant fibronectin and its preparation method, uses, nucleic acid, expression vector and strain

Abstract

The present invention discloses a heat-resistant fibronectin and its preparation method, uses, nucleic acid, expression vector and strain. Among them, the amino acid sequence of the heat-resistant fibronectin is the amino acid sequence as set forth in SEQ ID NO.6. The technical solution of the present invention obtains a heat-resistant fibronectin by means of genetic engineering and directed evolution. The heat-resistant fibronectin has high thermal stability, does not change the biological activity of the heat-resistant fibronectin, and significantly improves the application prospect of the heat-resistant fibronectin in the fields such as cosmetic raw materials.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to heat-resistant fibronectin, its preparation method and uses, nucleic acid, expression vector and strain. Background Art

[0002] Fibronectin (Fn) is a major non-collagenous glycoprotein present in the extracellular matrix and basement membrane, which participates in regulating cell polarity, differentiation and growth and plays a key role in cell adhesion. Fibronectin is involved in many key life activities of the human body: 1) It participates in cell migration, adhesion, proliferation, hemostasis and tissue repair, as well as embryonic development, and can promote the orderly proliferation of cells and collagen; 2) It promotes fibroblasts to secrete proteases to decompose protein impurities; 3) It induces the reconstruction of the subepidermal basement membrane and the normal keratinization process, and accelerates the comprehensive repair of cells and tissues in terms of structure and function. Therefore, fibronectin has been widely used in fields such as cosmetic skin care ingredients, medical wound dressings, and in vivo treatments, and has extremely high biological and medical application values.

[0003] Natural fibronectin is a dimeric glycoprotein with a molecular weight of 230-250 kDa for each monomer, and has variable molecular conformations and splicing variants. Fibronectin contains different functional regions and can bind to integrin, heparin, collagen, DNA, etc. to play important physiological functions. Although fibronectin has multiple biological activities such as promoting wound repair and cell adhesion, its low thermal stability greatly limits its application in fields such as cosmetics and in vitro medical devices. Wild-type fibronectin is extremely prone to protein denaturation during the emulsification process of cosmetics, thus losing its biological activity and application value. Summary of the Invention

[0004] The main object of the present invention is to provide a heat-resistant fibronectin, its preparation method and uses, nucleic acid, expression vector and strain, aiming to improve the thermal stability of fibronectin.

[0005] To achieve the above object of the invention, the following technical solutions are adopted.

[0006] A heat-resistant fibronectin, comprising at least one of the following:

[0007] A heat-resistant fibronectin with the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.2:

[0008] PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDVPST;

[0009] Heat-resistant fibronectin with the amino acid sequence shown in SEQ ID NO.4, SEQ ID NO.4:

[0010] PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVRGD;

[0011] Heat-resistant fibronectin with the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.6:

[0012] PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGTEYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLKVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATPTTITISWRTKTEPITGFQVDAVPANGQTPIQRTIPPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSPPLIGRKKTDELPQLVRGD;

[0013] Heat-resistant fibronectin with the amino acid sequence shown in SEQ ID NO.8, SEQ ID NO.8:

[0014] SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDEL;

[0015] Heat-resistant fibronectin with the amino acid sequence shown in SEQ ID NO.10, SEQ ID NO.10:

[0016] SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEPTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLPSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVPEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDEL。

[0017] The above-mentioned heat-resistant fibronectin has good thermal stability, with Tm values all exceeding 50 °C, and the highest Tm value even exceeding 80 °C. This temperature is much higher than the minimum emulsification temperature of 75 °C for cosmetics. Therefore, the heat-resistant fibronectin of the present invention can be used in the preparation of cosmetics. Similarly, due to the excellent thermal stability of the heat-resistant fibronectin of the present invention, it is also suitable for fields that require heat resistance of fibronectin, such as the preparation of drugs, as pharmaceutical excipients, and the preparation of culture media.

[0018] The present invention also discloses a nucleic acid that encodes the above-mentioned heat-resistant fibronectin.

[0019] Preferably, the above nucleic acid is DNA.

[0020] More preferably, the sequence of the above DNA is:

[0021] The sequence encoding the heat-resistant fibronectin with the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO. 1:

[0022]

[0023] The sequence of a heat-resistant fibronectin having an encoded amino acid sequence as shown in SEQ ID NO. 4, SEQ ID NO. 3:

[0024]

[0025] The sequence of a heat-resistant fibronectin having an encoded amino acid sequence as shown in SEQ ID NO. 6, SEQ ID NO. 5:

[0026]

[0027] The sequence of a heat-resistant fibronectin having an encoded amino acid sequence as shown in SEQ ID NO. 8, SEQ ID NO. 7:

[0028]

[0029] The sequence of heat-resistant fibronectin with the encoded amino acid sequence shown in SEQ ID NO. 10, SEQ ID NO. 9:

[0030]

[0031] The present invention also discloses a preparation method of the above-mentioned heat-resistant fibronectin, comprising the following steps:

[0032] Design a recombinant fibronectin;

[0033] Perform site-directed mutagenesis on the amino acid residues of the recombinant fibronectin;

[0034] Express the protein after site-directed mutagenesis to obtain the heat-resistant fibronectin.

[0035] More preferably, the specific operation of the step of designing the recombinant fibronectin is as follows:

[0036] Based on sequence and active domain screening, design and obtain a recombinant fibronectin.

[0037] More preferably, the specific operation of the step of expressing the recombinant fibronectin is as follows:

[0038] Insert the codon-optimized and artificially synthesized base sequence of the recombinant fibronectin into a plasmid vector to obtain an expression vector. Transfer the expression vector into Escherichia coli, culture the recombinant fibronectin by shake flask culture, and then purify the recombinant fibronectin.

[0039] Even more preferably, the above plasmid vector is a pET28a vector, and the inserted restriction enzyme sites are NdeI / XhoI. The vector is not limited to pET28a, and other common plasmids in Escherichia coli such as pET22a, pET22b, pET22c, pET14, pET21, pET30, and pET42 can all achieve the expression of recombinant fibronectin. The strain is not limited to Escherichia coli BL21(DE3), and other types of Escherichia coli, or yeast, or Bacillus subtilis can also be selected.

[0040] Even more preferably, in the site-directed mutagenesis of amino acid residues, it includes at least one of the following amino acid residues:

[0041] G39, K140, T179, G251, A269, T335, N357, V170, E213, T226, K248, E369, V72, D73, I94, M151, M169, A206, V232, R245, and K375. Among them, the letters G, K, T, A, N, V, E, D, I, M, R are the single-letter abbreviations of amino acids, and the numbers following them represent the positions of the amino acids.

[0042] Even more preferably, in the site-directed mutagenesis of amino acid residues, it includes at least one of the following mutation methods:

[0043] G39P, K140P, T179P, G251P, A269P, T335P, N357P, V170P, E213P, T226P, K248P, E369P, V72T, D73E, I94L, M151V, M169K, A206L, V232I, R245V, and K375V. Among them, the letters G, K, T, A, N, V, E, D, I, M, R before the numbers are single-letter abbreviations of amino acids, the numbers represent the positions of the amino acids, and the letters P, T, E, L, V, L, I, V after the numbers represent the amino acids at that position after mutation.

[0044] More preferably, the mutation primers for G39 include G39P-F and G39P-R, and their sequences are SEQ ID NO.27 and SEQ ID NO.28 respectively; and / or,

[0045] The sequences of the mutation primers for K140 are SEQ ID NO.29 and SEQ ID NO.30 respectively; and / or,

[0046] The sequences of the mutation primers for T179 are SEQ ID NO.31 and SEQ ID NO.32 respectively; and / or,

[0047] The sequences of the mutation primers for G251 are SEQ ID NO.33 and SEQ ID NO.34 respectively; and / or,

[0048] The sequences of the mutation primers for A269 are SEQ ID NO.35 and SEQ ID NO.36 respectively; and / or,

[0049] The sequences of the mutation primers for T335 are SEQ ID NO.37 and SEQ ID NO.38 respectively; and / or,

[0050] The sequences of the mutation primers for N357 are SEQ ID NO.39 and SEQ ID NO.40 respectively and / or,

[0051] The sequences of the mutation primers for V170 are SEQ ID NO.41 and SEQ ID NO.42 respectively and / or,

[0052] The sequences of the mutation primers for E213 are SEQ ID NO.43 and SEQ ID NO.44 respectively and / or,

[0053] The sequences of the mutation primers for T226 are SEQ ID NO.45 and SEQ ID NO.46 respectively and / or,

[0054] The sequences of the mutant primers for K248 are SEQ ID NO.47, SEQ ID NO.48 and / or,

[0055] The sequences of the mutant primers for E369 are SEQ ID NO.49, SEQ ID NO.50 and / or,

[0056] The sequences of the mutant primers for V72 are SEQ ID NO.51, SEQ ID NO.52 and / or,

[0057] The sequences of the mutant primers for D73 are SEQ ID NO.51, SEQ ID NO.52 and / or,

[0058] The sequences of the mutant primers for I94 are SEQ ID NO.53, SEQ ID NO.54 and / or,

[0059] The sequences of the mutant primers for M151 are SEQ ID NO.55, SEQ ID NO.56 and / or,

[0060] The sequences of the mutant primers for M169 are SEQ ID NO.57, SEQ ID NO.58 and / or,

[0061] The sequences of the mutant primers for A206 are SEQ ID NO.59, SEQ ID NO.60 and / or,

[0062] The sequences of the mutant primers for V232 are SEQ ID NO.61, SEQ ID NO.62 and / or,

[0063] The sequences of the mutant primers for R245 are SEQ ID NO.63, SEQ ID NO.64 and / or,

[0064] The sequences of the mutant primers for K375 are SEQ ID NO.65, SEQ ID NO.66.

[0065] More preferably, the specific operation of the site-directed mutagenesis step of amino acid residues of recombinant fibronectin includes:

[0066] Using the environment of the Alphafold 2.2.0 structure prediction algorithm, inputting the protein sequence for tertiary structure prediction, and selecting the mutation sites according to the prediction results for mutation.

[0067] More preferably, the preparation method of the above heat-resistant fibronectin includes the following steps:

[0068] Designing recombinant fibronectin;

[0069] Recombine the domains in recombinant fibronectin;

[0070] Perform site-directed mutagenesis of amino acid residues on the recombinant fibronectin with recombined domains;

[0071] Express the protein after site-directed mutagenesis to obtain heat-resistant fibronectin.

[0072] More preferably, the method for preparing the above heat-resistant fibronectin includes the following steps:

[0073] Design recombinant fibronectin;

[0074] Express recombinant fibronectin;

[0075] Recombine the domains in recombinant fibronectin;

[0076] Express the fibronectin with recombined domains;

[0077] Perform site-directed mutagenesis of amino acid residues on the recombinant fibronectin with recombined domains;

[0078] Express the protein after site-directed mutagenesis to obtain heat-resistant fibronectin.

[0079] More preferably, the specific operation of the step of recombining the domains in recombinant fibronectin is as follows:

[0080] Express and purify the domains contained in recombinant fibronectin respectively, and detect their Tm values; combine different domains to obtain a recombinant fibronectin with higher heat stability and improved heat resistance.

[0081] The fibronectin type I region contains 12 highly similar repeat domain units, the type II region contains 2 repeat domain units, and the type III region contains 15 repeat domain units (abbreviated as Fn1-Fn15). Among them, the fibronectin type III region is the most functionally important, containing the central binding domain Fn1-12 (its Fn10 domain contains the arginine-glycine-aspartic acid sequence (Arg-Gly-Asp, RGD), which recognizes and binds to cell surface integrins, thereby achieving cell adhesion, migration, etc.), and the heparin-binding domain Fn12-14. According to the structural characteristics of fibronectin, the fibronectin type III region is studied to design a miniaturized recombinant fibronectin with high biological activity and high stability.

[0082] More preferably, the recombined domains include at least one of the following:

[0083] Fn8, Fn9, Fn10, Fn12, Fn13, Fn14.

[0084] More preferably, the specific operation of the site-directed mutagenesis step of amino acid residues on the recombined fibronectin with domain recombination is as follows:

[0085] Use Alphafold 2.2.0 to perform structural calculations and three-dimensional modeling on the recombined fibronectin with domain recombination. Subsequently, perform molecular dynamics simulations and rational design on the recombined fibronectin with domain recombination, select potential unstable amino acid residues, and mutate them. Construct, purify, screen the mutated sites with improved thermal stability and combine them to obtain the final highly heat-resistant recombinant fibronectin variant.

[0086] The heat-resistant fibronectin prepared by the above steps has good thermal stability, and at the same time has extremely high biological activity while having good thermal stability. Through testing, it is found that rFN2 and HythermFN have extremely high activities in promoting the production of type I collagen in human fibroblasts, promoting cell adhesion, and promoting cell migration, and have good application prospects.

[0087] The present invention also discloses an expression vector for preparing heat-resistant fibronectin, and the expression vector includes nucleic acids encoding the proteins shown in SEQ ID NO.2, 4, 6, 8, 10.

[0088] Preferably, the expression vector includes nucleic acids shown in at least one of the sequences of SEQ ID NO.1, 3, 5, 7, 9.

[0089] More preferably, the expression vector further includes at least one pair of the above mutation primer sequences.

[0090] More preferably, the expression vector further includes at least one pair of primers for domain screening, and the primers for domain screening include SEQ ID NO.11 - SEQ ID NO.26.

[0091] The present invention also discloses an expression strain for preparing heat-resistant fibronectin, including the above expression vector for preparing heat-resistant fibronectin.

[0092] Preferably, the strain is at least one of Escherichia coli, yeast, and Bacillus subtilis.

[0093] More preferably, the strain is Escherichia coli BL21(DE3).

[0094] The present invention also discloses the uses of the above heat-resistant fibronectin, including at least one of the following:

[0095] Use in the preparation of cosmetics;

[0096] Use in the preparation of drugs and / or drug delivery;

[0097] Use in the preparation of cell culture media.

[0098] Preferably, the heat-resistant fibronectin includes the proteins shown in SEQ ID NO.2, 4, 6, 8, 10.

[0099] More preferably, the heat-resistant fibronectin includes the proteins shown in SEQ ID NO. 4, 6, 10.

[0100] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0101] By means of genetic engineering and directed evolution, the technical solution of the present invention obtains a heat-resistant fibronectin. This heat-resistant fibronectin has high thermal stability, does not change the biological activity of the heat-resistant fibronectin, and significantly improves the application prospects of fibronectin in the fields of cosmetic raw materials and the like. The heat-resistant fibronectin of the present invention has good thermal stability, and the Tm values all exceed 50 °C, and the highest Tm value even exceeds 80 °C. It has good thermal stability and extremely high biological activity while having good thermal stability. Through tests, it is found that rFN2 and HythermFN have extremely high activities in promoting the production of type I collagen in human fibroblasts, promoting cell adhesion, and promoting cell migration, and have good application prospects. Description of the Drawings

[0102] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the structures shown in these drawings.

[0103] Figure 1 It is the SDS-PAGE result diagram of rFN1 protein purification;

[0104] Figure 2 It is the predicted model of the rFN1 protein structure;

[0105] Figure 3 It is the predicted model of the rFN2 protein structure;

[0106] Figure 4 It is the ∆∆G calculation flow chart of the rFN2 protein;

[0107] Figure 5 It is the sequence alignment result of the type III fibronectin domain;

[0108] Figure 6 It is the structural diagram of the R245V and K375V mutant amino acid residues;

[0109] Figure 7 It is the experimental result of HythermFN promoting human fibroblast adhesion;

[0110] Figure 8 Comparison results of the ability to promote human fibroblast adhesion before and after HythermFN heating;

[0111] Figure 9 Comparison results of the ability to promote human fibroblast collagen production between HythermFN and wild-type fibronectin;

[0112] Figure 10 Purification diagram of rFn protein expression;

[0113] Figure 11 Predicted tertiary structure diagram of rFn protein.

[0114] The realization, functional characteristics and advantages of the object of the present invention will be further described in conjunction with the embodiments with reference to the accompanying drawings. Specific embodiments

[0115] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0116] Example 1

[0117] Preparation of recombinant fibronectin rFN1

[0118] 1. Construction of rFN1 expression vector

[0119] The Fn8-10 domain (cell-binding domain, containing the RGD sequence) of type III fibronectin and the Fn12-14 domain (heparin-binding domain) were linked by Linker (amino acid sequence: KPSA) to form a new recombinant fibronectin (abbreviation: rFN1). Subsequently, after codon optimization, the optimized rFN1 gene sequence was cloned between the NdeI / XhoI restriction enzyme sites of the pET28a vector (entrusted Nanjing Genscript Biotech Co., Ltd. for vector construction) to obtain the pET28a-rFN1 vector. The base sequence corresponding to the rFN1 protein is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2.

[0120] SEQ ID NO. 1:

[0121]

[0122] SEQ ID NO.2:

[0123] PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDVPST*。

[0124] 2. Construction of rFN1 expression strain: pET28a-rFN1 was transformed into BL21(DE3) strain to obtain the recombinant strain BL21 / pET28a-rFN1.

[0125] 3. Expression of rFN1 protein: The above recombinant strain was streaked on a LB solid medium plate with Km resistance and cultured overnight at 37 °C. The next day, a single colony was picked and inoculated into 10 mL of LB liquid medium (Km resistance) and cultured at 37 °C and 230 rpm on a shaker for about 6 - 8 h. Then all 10 mL of the bacterial solution was added to 1 L of large flask LB liquid medium (Km resistance) and cultured at 37 °C and 230 rpm on a shaker for about 3 h, and the OD600 was measured to be 0.5 - 0.6. IPTG solution was added to the large flask medium to a final concentration of 500 µM, and cultured overnight at 18 °C and 230 rpm on a shaker.

[0126] 4. Purification of rFN1 protein: (i) Centrifuge the overnight cultured BL21(DE3) bacterial solution expressing the protein at 4°C and 6000 g for 10 min, and discard the supernatant; (ii) Add 40 mL of PBS buffer, vortex to resuspend the bacterial solution until there are no obvious bacterial clumps; (iii) Use a high-pressure cell disruptor to lyse Escherichia coli to release the protein, and lyse at 80 MPa for 8 min. The high-pressure cell disruptor has been pre-equilibrated with a protein purification bacterial lysis buffer and pre-cooled to 4°C; (iv) Centrifuge the lysate from the previous step at 4°C and 9500 g at high speed for 30 min; (v) Pour the supernatant into a new sterile centrifuge tube and filter it through a 0.45 µm filter membrane to further remove impurities; (vi) Load all the filtrate onto a Ni column pre-equilibrated with PBS buffer; (vii) Wash the Ni column with 40 mL of PBS buffer containing 50 mM imidazole to remove impurity proteins; (viii) Elute the target protein from the Ni column with 30 mL of 250 mM imidazole PBS buffer; (ix) Transfer the protein solution to a protein concentration ultrafiltration tube, centrifuge at 5500 g until the volume reaches 2 mL, then add PBS buffer without imidazole to 10 mL and mix well; (x) Repeat step (ix) once, and at this time the imidazole concentration in the protein solution is diluted to about 10 mM; (xi) Load all 10 mL of the protein solution from the previous step onto a heparin column for further purification to further improve the protein purity. The gel filtration column has been pre-equilibrated with PBS buffer; (xii) Mix the conventional PBS buffer and the PBS buffer containing 1 M sodium chloride according to a program (the PBS buffer containing 1 M sodium chloride increases linearly from 0% to 100% in content), and collect the protein according to the UV absorption peak; (xiii) Dialyze the protein solution to replace the protein buffer with a conventional PBS buffer, then dilute the protein to 1 mg / mL and store it in a -80°C refrigerator for a long time. Load the rFn protein onto a denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and the result shows that the protein purification effect is good. The results are as Figure 1 shown.

[0127] 5. Detection of the thermal stability of rFN1 protein: The Protein Thermal Shift™ kit (purchased from ThermoFisher SCIENTIFIC) was used to detect the thermal stability of rFn1 protein. The thermal stability reaction system was 20 µL (12.5 µL protein solution (concentration 1 mg / mL), 2.5 µL 8× fluorescent dye, 5 µL reaction buffer). After mixing, the Tm value of the protein was detected using a fluorescence PCR instrument, and the sample addition process was carried out on ice throughout. The heating program of the fluorescence PCR instrument was as follows: incubate at 25 °C for 2 min, then uniformly increase the temperature to 99 °C at a rate of 0.5 °C / s (monitor the fluorescence value in real time throughout the heating process), incubate at 99 °C for 1 min, and after the reaction ended, the fluorescence data was analyzed according to the internal program of the fluorescence PCR instrument to obtain the Tm value. The thermal stability experiment was repeated three times. The measurement results showed that the Tm value of rFn1 protein was 55.5 °C (Table 2), which was lower than the minimum emulsification temperature of 75 °C for cosmetics.

[0128] Example 2

[0129] Preparation of recombinant fibronectin rFN2 No. 2

[0130] 1. Prediction of the rFn1 protein structure: The environment of the local Alphafold 2.2.0 structure prediction algorithm was set up, and the rFn1 protein sequence was input to calculate its tertiary structure. The prediction results are as Figure 2 shown.

[0131] 2. Expression and purification of domains: According to the prediction results, six single domains, namely Fn8, Fn9, Fn10, Fn12, Fn13, and Fn14, and two triple domains, namely Fn8-10 and Fn12-14, were expressed and purified separately from a structural perspective. The expression plasmids for single domains, the Fn8-10 triple domain, and the Fn12-14 triple domain were constructed by homologous recombination: using the pET28a-rFN1 plasmid as a template, the pET28a plasmid backbone and the target gene fragment were amplified by PCR respectively (the primers are shown in Table 1).

[0132] Table 1. Primers for domain screening

[0133]

[0134] The amplified product was detected for the target band by agarose gel electrophoresis, and finally the target gene fragment was cloned between the NdeI / XhoI restriction sites of the pET28a vector by homologous recombination. The above ligation product was transformed into Escherichia coli DH5α strain, and finally the correctly constructed expression vector was verified by Sanger sequencing. The correctly constructed expression vector was transformed into BL21(DE3) strain to obtain an expression strain, and the expression strain was used for expression; after expression, purification was carried out, and the protein expression and purification steps were the same as those of the rFN1 purification step in Example 1.

[0135] 3. Thermal stability test of the domain: The experimental procedure was the same as that of the rFN1 protein. The results of the thermal stability experiment are shown in Table 2.

[0136] Table 2. Thermal stability of the domain

[0137] Domain (combination) Tm (°C) rFN1 55.5 Fn8 63.7 Fn9 49.4 Fn10 82.5 Fn12 77.2 Fn13 66.3 Fn14 70.9 Fn8-10 54.0 Fn12-14 72.6 rFN2 73.0

[0138] From the results of the thermal stability test, it can be seen that the thermal stabilities of the Fn8 and Fn9 domains are relatively low, 63.7 °C and 49.4 °C respectively, which are lower than those of other domains. In addition, compared with the thermal stability of the Fn12-14 triple domain (Tm value is 54 °C), it is also much higher than that of the Fn8-10 triple domain protein (Tm value is 72.6 °C). Finally, the Fn12-14 triple domain protein with good thermal stability was fused with the Fn10 domain (containing the RGD sequence) to obtain rFN2 protein, whose corresponding base sequence is SEQ ID NO.3 and amino acid sequence is SEQ ID NO.4;

[0139] SEQ ID NO.3:

[0140]

[0141] SEQ ID NO.4:

[0142] PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVRGD。

[0143] 4. Construction, protein expression and purification of rFN2 protein expression vector: The experimental steps are the same as those of the above single-domain protein, and the primers are shown in Table 1.

[0144] 5. Thermal stability detection of rFN2 protein: The experimental steps are the same as those of rFN1 protein. The results show that the Tm value of rFN2 protein reaches 73 °C (Table 2), which is significantly better than that of rFN1 protein.

[0145] Example 3

[0146] Directed evolution to improve the thermal stability of rFN2

[0147] Although the Tm value of rFN2 protein reaches 73 °C, it is still lower than the minimum emulsification temperature of 75 °C for cosmetics. Therefore, based on the rFN2 variant, directed evolution is carried out to further improve the thermal stability of rFn2 protein to meet the usage requirements.

[0148] 1. Predict the structure of rFn2 protein: Use the local Alphafold 2.2.0 structure prediction algorithm to predict the structure of rFN2 protein, and the results are as Figure 3 shown.

[0149] 2. Determine the mutation sites:

[0150] The 800-ns all-atom molecular dynamics simulation of the rFN2 protein sequence was carried out using the Gromacs toolkit. By calculating the root mean square fluctuation (rmsf) of the α-carbon atoms of the protein backbone, the vulnerable residues potentially affecting the protein backbone stability at 368 K (73 °C) were determined.

[0151] For the rFN2 protein, a high-throughput calculation process of cartesian ∆∆G in the Rosetta-MPI toolkit was established ( Figure 4 ), and the ∆∆G energy calculation was performed on the vulnerable residues obtained from the above molecular dynamics simulation analysis to quantitatively predict the impact of mutating specific residues at specific sites on the structural backbone stability of rFN2.

[0152] Through the calculation of ∆∆G energy, the amino acid residues V170, E213, T226, K248, and E369 were respectively mutated to proline (P) to improve the protein stability.

[0153] Sequence alignment was performed between different domains of type III fibronectin ( Figure 5 ), and the sequence conservation was analyzed. The residues with relatively low conservation, V72, D73, I94, M151, M169, A206, and V232, were respectively mutated to threonine (T), glutamate (E), leucine (L), valine (V), lysine (K), leucine (L), and isoleucine (I) with higher conservation at the corresponding sites.

[0154] Through the analysis of the rFN2 protein structure, it was found that the side chains of the amino acid residues R245 and K375 were deflected outward, with relatively large steric hindrance, which was not conducive to the stability of the domain ( Figure 6 ). Therefore, the amino acid residues R245 and K375 were respectively mutated to valine (V) with relatively small steric hindrance of the side chain.

[0155] 3. Preparation of mutant proteins

[0156] According to the mutant sites screened above, the 14 mutations of V170P, E213P, T226P, K248P, E369P, V72T, D73E, I94L, M151V, M169K, A206L, V232I, R245V, and K375V were introduced into the rFN2 protein in the form of single mutations or combined mutations; the primers required for mutation and construction of the expression vector are shown in Table 3. The expression vector was constructed by homologous recombination using the pET28a-rFN2 vector as the PCR amplification template, and finally, the correctly constructed expression vector was verified by sanger sequencing. The correctly constructed expression vector was transformed into the BL21(DE3) strain to obtain the expression strain, and the protein expression and purification steps were the same as those of the rFN1 purification step in Example 1.

[0157] Table 3 Primers required for amino acid point mutations

[0158]

[0159] 4. Thermal stability detection

[0160] The mutants containing single mutation sites and combinations of mutation sites were subjected to thermal stability experiments, and the results are shown in Table 4. In Table 4, ∆Tm = Tm(Mutant) - Tm(rFN2).

[0161] Table 4 Thermal stability of mutants

[0162]

[0163] The thermal stability experiments showed that the HythermFN mutant containing 7 mutations (V72T / D73E / M169K / E213P / T226P / K248P / E369P) increased the Tm value of the rFN2 protein from 73 °C to 78.2 °C, significantly improving the thermal stability of fibronectin and broadening the application prospects of fibronectin.

[0164] Example 4

[0165] Preparation of recombinant fibronectin rFN

[0166] 1. Construction of rFN expression vector

[0167] Based on structural analysis, the FNIII subunit Fn10 domain containing the RGD sequence was linked to the Fn12 - 14 domain through a Linker (amino acid sequence: KPSA) to form a new recombinant fibronectin (abbreviation: rFn). After codon optimization, the optimized rFn sequence was cloned between the NdeI / XhoI restriction sites of the pET28a vector (commissioned Nanjing Genscript Biotech Co., Ltd. to construct the expression vector) to obtain the pET28a - rFn vector. The nucleotide sequence corresponding to the rFn protein is SEQ ID NO.7, and the amino acid sequence is SEQ ID NO.8;

[0168] SEQ ID NO. 7:

[0169]

[0170] SEQ ID NO.8:

[0171] SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDEL。

[0172] 2. Construction of rFN expression strain: pET28a-rFn was transformed into BL21(DE3) strain to obtain the recombinant strain BL21 / pET28a-rFn.

[0173] 3. rFN protein expression: The above recombinant strain was streaked on an LB solid medium plate with Km resistance and cultured overnight at 37 °C. The next day, a single colony was selected and inoculated into 10 mL of LB liquid medium (with Km resistance), and cultured at 37 °C and 230 rpm in a shaker for about 6 - 8 h. Then, the 10 mL of bacterial solution was added to 1 L of large flask LB liquid medium (with Km resistance), and cultured at 37 °C and 230 rpm in a shaker for about 3 h, and the OD600 was measured to be 0.5 - 0.6. IPTG solution was added to the large flask medium to a final concentration of 500 μM, and cultured overnight at 18 °C and 230 rpm in a shaker.

[0174] 4. rFN Protein Purification: (i) Centrifuge the overnight-cultured BL21(DE3) bacterial solution expressing the protein at 4 °C and 6000 g for 10 min, and discard the supernatant; (ii) Add 40 mL of PBS buffer, vortex to resuspend the bacterial solution until there are no obvious bacterial clumps; (iii) Use a high-pressure cell disruptor to lyse Escherichia coli to release the protein, and lyse it at 80 MPa for 8 min. The high-pressure cell disruptor is pre-equilibrated with a protein purification bacterial lysis buffer and pre-cooled to 4 °C; (iv) Centrifuge the lysate from the previous step at 4 °C and 9500 g at high speed for 30 min; (v) Pour the supernatant into a new sterile centrifuge tube and filter it through a 0.45 µm filter membrane to further remove impurities; (vi) Load all the filtrate onto a Ni column pre-equilibrated with PBS buffer; (vii) Wash the Ni column with 40 mL of PBS buffer containing 50 mM imidazole to remove contaminant proteins; (viii) Elute the target protein from the Ni column with 30 mL of 250 mM imidazole PBS buffer; (ix) Transfer the protein solution to a protein concentration ultrafiltration tube, centrifuge at 5500 g until the volume reaches 2 mL, and then add PBS buffer without imidazole to 10 mL and mix well; (x) Repeat step (ix) once, and at this time, the imidazole concentration in the protein solution is diluted to about 10 mM; (xi) Load all 10 mL of the protein solution from the previous step onto a heparin column for further purification to further improve the protein purity. The gel chromatography column has been pre-equilibrated with PBS buffer; (xii) Collect the protein according to the UV absorption peak, transfer the protein solution to a protein concentration ultrafiltration tube, concentrate the protein to 1 mg / mL, and store it in a -80 °C refrigerator for a long time. Loading the rFn protein onto a denaturing polyacrylamide gel electrophoresis (SDS-PAGE) shows that the protein purification effect is good, and the results are as Figure 10 shown.

[0175] Example 5

[0176] 1. Prediction of the rFn2 Protein Structure: Set up the environment of the local Alphafold 2.2.0 structure prediction algorithm, input the rFn protein sequence for tertiary structure prediction, and the prediction results are as Figure 11 shown.

[0177] 2. Determination of Mutation Sites: According to the prediction results, select 7 amino acid residues, namely G39, K140, T179, G251, A269, T335, and N357, to mutate into proline (P).

[0178] 3. Preparation of Mutant Proteins

[0179] The mutants were all amplified using the pET28a-rFn plasmid as a template. The successfully constructed template was transformed into the BL21(DE3) strain to express the protein. The expression and purification methods of the mutants were the same as those of the rFn in Example 4. The mutant primers are shown in Table 5.

[0180] Table 5 Primers required for amino acid point mutations

[0181]

[0182] 4. Thermal stability detection

[0183] The detection kit was Protein Thermal Shift™ kit (ThermoFisher SCIENTIFIC). The thermal stability reaction system was 20 μL (12.5 μL protein solution (concentration 1 mg / mL), 2.5 μL 8× fluorescent dye, 5 μL reaction buffer). Mix well and use a fluorescence PCR instrument to detect the Tm value of the protein. The whole process of adding samples was carried out on ice. The heating program of the fluorescence PCR instrument was: incubate at 25 °C for 2 min, then increase the temperature uniformly to 99 °C at a rate of 0.5 °C / s (monitor the fluorescence value in real time during the heating process), incubate at 99 °C for 1 min, and the reaction ended. Analyze the fluorescence data according to the internal program of the fluorescence PCR instrument to obtain the Tm value. The thermal stability experiment of each mutant was repeated three times, and the experimental results are shown in Table 6.

[0184] Table 6 Thermal stability of mutants

[0185]

[0186] The thermal stability experiment results showed that compared with the melting temperature of 72.2 °C of the rFn protein, the single amino acid residue mutations of K140P, N357P and T335P increased its Tm to 74.5 °C, 76.0 °C and 75.2 °C respectively. Finally, three mutations of K140P, N357P and T335P were introduced simultaneously, namely highly heat-resistant ThrFn. The thermal stability experiment showed that its Tm value reached 81.0 °C, meeting the heat resistance requirements during the emulsification process of cosmetics.

[0187] Test Example 1

[0188] Detection of heat resistance and biological activity of HythermFN mutants

[0189] The biological activity of fibronectin is manifested in many aspects. Among them, promoting cell adhesion and type I collagen production are important activity indicators. Therefore, the biological activity of fibronectin was evaluated by detecting its effects on the adhesion of human fibroblasts and the secretion of type I collagen.

[0190] Sample treatment: The 1 mg / ml fibronectin solution was filtered and sterilized through a 0.2 μm filter membrane; an appropriate amount of the solution was placed in a water bath at 70, 72, and 75 °C for 30 min respectively.

[0191] Cell adhesion detection: The fibronectin solution was diluted to 100 μg / ml, and an appropriate amount of the solution was added to a 48-well plate to cover the surface. The plate was placed at 4 °C overnight, and the excess solution was aspirated to form a fibronectin coating layer on the surface. Human dermal fibroblasts (HDF) P6 were cultured in complete medium (DMEM + 1% Pen / Strep + 10% FBS). When the cell density reached 70%-80%, the cells were collected and seeded in the fibronectin-coated plate. After 6 h, the cells were photographed under a microscope to observe the cell adhesion state, and the cell adhesion rate was calculated using ImageJ.

[0192] Collagen content detection: Human dermal fibroblasts (HDF) P6 were cultured in complete medium (DMEM + 1% Pen / Strep + 10% FBS). When the cell density reached 70%-80%, the cells were collected and seeded in a 96-well plate. After 24 h, the medium was replaced with fresh medium and fibronectin samples were added according to the detection concentrations. The cells were continued to be cultured in an incubator at 37 °C. After 24 h, the cell supernatant was collected. The collagen content in the cell supernatant was detected using a human type I collagen (Col-1) ELISA Kit. The operation was carried out according to the kit instructions, and the fluorescence absorption intensity was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0193] Figure 7 It shows that HythermFN after heating still exhibits a significant effect of improving cell adhesion. Figure 8 It shows that compared with the blank control, HythermFN can significantly promote the production of type I collagen after heating at room temperature and 75 °C. Figure 9 It shows that there is no significant difference in the effect of promoting the production of type I collagen between HythermFN and wild-type WT fibronectin. The above results prove that the HythermFN mutant exhibits extremely high heat resistance while retaining the biological activity of the protein.

[0194] The above are only optional embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. All equivalent structural transformations made under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A heat-resistant fibronectin, characterized in that The amino acid sequence of the heat-resistant fibronectin is as follows: SEQ ID NO.6: PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGTEYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLKVATKYEVSVYALKDTLTSRPAQGVV TTLENVSPPRRARVTDATPTTITISWRTKTEPITGFQVDAVPANGQTPIQRTIPPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSPPLIGRKKTDELPQLVRGD.

2. A nucleic acid, characterized in that The nucleic acid encodes the heat-resistant fibronectin according to claim 1.

3. An expression vector for preparing heat-resistant fibronectin, characterized in that: The expression vector comprises the nucleic acid according to claim 2.

4. An expression strain for preparing heat-resistant fibronectin, characterized in that: The strain comprises the expression vector according to claim 3.

5. The use of the heat-resistant fibronectin according to claim 1, characterized in that: Include at least one of the following: Use in the preparation of cosmetics; Use in preparing cell culture medium.

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