CN116396397A - Collagen short peptide mutant fusion protein and application thereof in preparation of lactoferrin peptide - Google Patents
Collagen short peptide mutant fusion protein and application thereof in preparation of lactoferrin peptide Download PDFInfo
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- CN116396397A CN116396397A CN202211737882.0A CN202211737882A CN116396397A CN 116396397 A CN116396397 A CN 116396397A CN 202211737882 A CN202211737882 A CN 202211737882A CN 116396397 A CN116396397 A CN 116396397A
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- 239000012138 yeast extract Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
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Abstract
The invention discloses a collagen short peptide mutant fusion protein and application thereof in preparation of lactoferrin peptide, wherein the amino acid sequence of the collagen short peptide mutant fusion protein is shown as SEQ ID NO. 3. The invention can better guide the expression of the bovine lactoferrin peptide by modifying the collagen short peptide as carrier protein, increase the expression quantity of the bovine lactoferrin peptide, improve the expression quantity by 10-15 times, and can be degraded into amino acid by reasonably designed collagen short peptide and connecting acidolysis peptide in the subsequent acidolysis process to release the lactoferrin peptide, and the lactoferrin peptide can be used in the fields of medicines, foods, feeds and the like.
Description
Field of the art
The invention relates to a collagen short peptide mutant fusion protein and application thereof in preparation of lactoferrin peptide.
(II) background art
Lactoferrin (LF) was first isolated from cow's milk in 1960 and was initially referred to as "hemoglobin" because it binds iron to form a red complex. Lactoferrin is a glycoprotein found in multiple glands in mammals, such as milk, tears, saliva and other exocrine or neutrophil cells, plasma, and has been shown to be the highest in colostrum.
The bovine lactoferrin is decomposed into smaller polypeptides with 25 amino acid residues in sequence through the alimentary canal, the polypeptides are amino acid residues from 17 th to 41 th in the bovine lactoferrin, the isoelectric point PI of the active small peptides is more than 12, the active small peptides have extremely strong cationic property, and cysteines at 19 th and 36 th positions in the structure form intramolecular disulfide bonds, so that the natural lactoferrin peptide presents an alpha helical structure. These properties provide the bovine lactoferrin with antibacterial, bactericidal and cell-infecting effects.
For the current report, the secretion yield of lactoferrin peptide is lower, isui and Jose et al successfully clone cDNA genes of bovine lactoferrin peptide (LfcinB), form fusion proteins with thioredoxin, construct pET32-bLf recombinant expression vector, transfer into escherichia coli for expression, and after nickel column separation and purification, the yield is 15.3mg/L; designing and synthesizing an LfcinB derived peptide multimeric sequence by using the Zigang Tian et al, inserting an expression vector pET32a, and performing IPTG induced expression, wherein the expression quantity is up to 10mg/L; liuying and the like, in which bovine lactoferrin peptide and Cry60Ba crystal protein are fused in bacillus thuringiensis for expression, the expression is carried out in the form of inclusion bodies, and hydrolysate is obtained by treating the inclusion bodies, but whether the separation and purification of the lactoferrin peptide and the expression in the form of inclusion bodies have influence on the activity of lactoferrin is not carried out is unknown; yi Junbo et al clone the LfcinB gene fragment into a secretion type expression vector pPIC9K to obtain a recombinant plasmid, linearize electric transfer Pichia pastoris, and after methanol induction, the LfcinB is expressed in the yeast, but the LfcinB gene fragment needs to be concentrated by 10 times to see a remarkable band on SDS-PAGE gel; liu Chun et al fuse lactoferrin peptide and collagen in Pichia pastoris for expression, but the large molecular weight of collagen in the fusion mode leads to the fact that the lactoferrin peptide only accounts for 6.5% of the total molecular weight of the fusion protein, and meanwhile, the lack of means for preparing the lactoferrin peptide from the fusion protein leads to the fact that the fusion protein can only be applied integrally, so that the value of the lactoferrin peptide is reduced.
Collagen (COL) is a biopolymer which is widely present in animal connective tissue, has a highly repetitive triple helix structure, has good acid degradation properties, and has a high expression level in pichia pastoris. Werten et al expressed recombinant gelatin (the product of collagen partial hydrolysis) in Pichia pastoris with single copy expression levels up to 3-6g/L. In summary, it is possible to use collagen as carrier protein to increase the expression level of lactoferrin peptide, but the larger molecular weight of collagen itself, the larger the weight of collagen in fusion protein, results in relatively lower yield of lactoferrin peptide, and the more repeated structure increases the difficulty of preparing lactoferrin peptide by subsequent acidolysis. Therefore, reasonable selection of collagen peptide is of great importance for expression of fusion protein, and the following three points are required to be satisfied: 1. low molecular weight 2, good hydrophilicity 3 and strong cationic neutralizing capacity of lactoferrin peptide while retaining high expression capacity of collagen.
The invention preferably takes a short peptide consisting of 178 th amino acids of 608 th to 785 th amino acids in collagen as a target, and mutates hydrophobic amino acids into hydrophilic amino acids according to the characteristic repeated structural rule of the collagen and the preference of pichia pastoris amino acids, and simultaneously reduces isoelectric points as far as possible, and specifically comprises the following steps: the 614 th isoleucine is mutated to aspartic acid, the 633 th leucine is mutated to glycine, the 636 th isoleucine is mutated to proline, the 658 th phenylalanine is mutated to glycine, the 706 th valine is mutated to proline, the 721 th phenylalanine is mutated to glutamic acid so as to improve the hydrophilicity of the collagen short peptide, and the mutated hydrophilicity is-1.09 and the isoelectric point is 4.83. The hydrophilicity of the fusion protein is-1.05, the isoelectric point is 9.78, and the strong cationic property of the lactoferrin peptide in the expression process is greatly reduced. The invention uses pichia pastoris GS115 as a host to construct a recombinant pichia pastoris strain for secretory expression of preferred collagen-like short peptide-acidolysis peptide-bovine lactoferrin peptide-6 HIS fusion protein, and provides a method for preparing lactoferrin peptide from the fusion protein.
(III) summary of the invention
The invention aims to provide a collagen short peptide mutant fusion protein and application thereof in preparing lactoferrin peptide, and solves the problems of low yield and difficult purification of the lactoferrin peptide in the prior art.
The technical scheme adopted by the invention is as follows:
the invention provides a collagen short peptide mutant fusion protein (HlfcinB), which takes a collagen short peptide mutant as carrier protein, connects acidolysis peptide at the C end, connects lactoferrin peptide at the C end of acidolysis peptide, connects 6HIS at the C end of lactoferrin peptide, and constructs a novel fusion protein for expressing lactoferrin peptide; the amino acid sequence of the collagen short peptide mutant is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2; the acidolysis peptide is aspartic acid-proline-glutamic acid-tryptophan, wherein peptide bonds between aspartic acid and proline can be broken under acidic conditions, glutamic acid and tryptophan play a role in protection, and a nucleotide sequence GACCCTGAGTGG of the acidolysis peptide codes for an amino acid sequence DPEW of the acidolysis peptide.
The sequence of the lactoferrin peptide (LfcinB) is an active small peptide with antibacterial function, which is formed by 25 amino acid residues converted by decomposing full-length bovine lactoferrin (NCBI sequence number: NM_180998.2 and derived from Bos taurus) through the digestive tract, the nucleotide sequence of the encoding bovine lactoferrin peptide (LfcinB) is shown as SEQ ID NO. 5, and the amino acid sequence of the encoding bovine lactoferrin peptide (LfcinB) is shown as SEQ ID NO. 6.
The 6HIS is a histidine tag, has a structure of HIS-HIS-HIS-HIS-HIS-HIS, can be used for purifying a fusion protein nickel column, has a nucleotide sequence CACCATCATCACCACCAT and has an amino acid sequence HHHHH.
Preferably, the amino acid sequence of the collagen short peptide mutant fusion protein is shown as SEQ ID NO. 3.
The invention also provides a coding gene of the collagen short peptide mutant fusion protein, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.
The invention also provides a recombinant plasmid containing the collagen short peptide mutant fusion protein coding gene and recombinant genetic engineering bacteria constructed by the recombinant plasmid; the recombinant plasmid takes a pPIC9K plasmid as a basic vector, and the EAEA residue at the C end of the alpha signal peptide of the pPIC9K plasmid is mutated into methionine residue, so that the stability of the expressed protein is improved.
The recombinant plasmid is constructed according to the following method: (1) The nucleotide sequence of the collagen short peptide mutant fusion protein is connected to pPICC 3.5K plasmid to construct pPICC 3.5K-HlfcinB after double enzyme digestion by EcoRI and NotI;
(2) Using pPIC9K plasmid as template, amplifying pPIC9K carrier fragment obtained by mutating alpha signal peptide EAEA residue into methionine residue with the following primer;
pPIC9K-F:GGGGTATCTCTCGAGAAAAGAATG
pPIC9K-R:CATTCTTTTCTCGAGAGATACCCCT
amplifying the HlfcinB fragment by using pPICC 3.5K-HlfcinB as a template and using the following primers;
HlfcinB-F:AAGGGGTATCTCTCGAGAAAAGAATGGGTCCAACTGGTCCTGACGGTCC
HlfcinB-R:CTAGGGAATTCTACGTATTAATGGTGGTGATGATGGTGGAAAGCTCTTCTAACACAAGTAATAG
(3) And (3) seamlessly connecting the pPIC9K carrier fragment and the HLfcinB fragment in the step (2) by using a one-step cloning kit, transferring the obtained product into competent cells of escherichia coli DH5 alpha, culturing on a flat plate containing ampicillin and kanamycin, and then picking up positive cloned seed extraction plasmids to obtain the recombinant plasmids containing the collagen short peptide mutant fusion protein coding genes.
The invention also relates to a recombinant genetic engineering bacterium containing the collagen short peptide mutant fusion protein coding gene, the engineering bacterium takes pichia pastoris GS115 strain as host bacterium, the engineering bacterium is obtained by linearizing recombinant plasmid containing the fusion protein coding gene by using restriction endonuclease SacI, and then transferring the recombinant plasmid into competent cells of Pichia pastoris GS115 by an electric excitation method, and screening positive transformants.
The invention provides an application of the collagen short peptide mutant fusion protein in preparing lactoferrin peptide, which comprises the following steps: centrifuging a fermentation liquor obtained by fermenting and culturing recombinant genetically engineered bacteria containing the collagen short peptide mutant fusion protein encoding genes, collecting supernatant, extracting pure enzyme, and freeze-drying to obtain fusion protein freeze-dried powder; dissolving the fusion protein freeze-dried powder in 100-200mM HCl aqueous solution, reacting for 8-12h in a metal bath at 80 ℃, and adjusting the pH to 7.0 by using 1M NaOH to obtain lactoferrin peptide solution.
The fermentation broth is prepared by the following steps:
streaking recombinant genetically engineered bacteria on a YPD solid plate containing 100 mug/mL of G418 resistance, standing and culturing at 30 ℃ for 3 days, selecting single colonies, inoculating the single colonies on an MD culture medium (30 mL/250mL and 50mL/500 mL), and culturing at 200rpm for 16-18 hours at 30 ℃ to obtain seed liquid of a fermentation culture medium;
inoculating seed liquid into a 5L fermentation tank containing 3L BSM culture medium at 10% of inoculation amount by volume concentration, adjusting pH to 5.0, setting the temperature to 30 ℃, setting the initial rotating speed to 500rpm, and controlling the ventilation rate to be more than 20%; after the glycerol in the culture medium is consumed, the dissolved oxygen is rapidly increased (DO & gt 60 percent), and then glycerol solution is fed in, so that the culture is started until the WCW of the thalli is 150g/L; starving for 30min after the end of feeding, then feeding methanol solution for induction, and controlling the specific growth rate at 0.015h -1 Regulating the rotating speed and the ventilation quantity to control the dissolved oxygen to be more than 20%, so as to obtain fermentation liquor; the glycerol solution is prepared by adding 12mL/L trace elements (PTM 1) into a glycerol aqueous solution with the mass concentration of 50%; the methanol solution is prepared by adding 12mL/L trace element (PTM 1) into anhydrous methanol.
BSM medium: 85% H 3 PO 4 26.7mL/L，KOH 4.13g/L，K 2 SO 4 18.2g/L，CaSO 4 0.93g/L，MgSO 4 ·7H 2 14.9g/L of O, 40.0g/L of glycerol, 4.35mL/L of trace elements (PTM 1), water as a solvent and pH 5.0-5.5; the preparation method comprises the following steps: 85% H 3 PO 4 26.7mL，KOH 4.13g，K 2 SO 4 18.2g，CaSO 4 0.93g，MgSO 4 ·7H 2 14.9g of O, 40.0g of glycerol, adjusting the pH to 5.0-5.5 by ammonia before inoculation, and adding 4.35mL/L of PTM.
Trace element PTM1: h 3 BO 3 0.02g/L，CuSO 4 ·5H 2 O 6.0g/L，MnSO 4 ·H 2 O 3.0g/L，Na 2 MoO 4 ·2H 2 O 0.2g/L，CoCl 2 0.5g/L，NaI 0.08g/L，ZnCl 2 20.0g/L，FeSO 4 ·7H 2 O65.0 g/L, biotin 0.2g/L,5.0mL/L H 2 SO 4 The solvent is ddH 2 O, filtering and sterilizing, and storing at 4 ℃ in dark.
Preferably, the fermentation is divided into four stages, namely a batch fermentation stage, a fed-batch stage, a starvation stage and a methanol induction stage in sequence:
1) Batch fermentation stage
Inoculating seed solution into a 5L fermentation tank filled with 3L fermentation medium at 10% by volume, regulating pH to 5.0 with ammonia water, setting the temperature to 30deg.C, setting the initial rotation speed to 500rpm, and controlling ventilation rate to more than 20% Dissolved Oxygen (DO) until glycerol in the medium is exhausted;
2) Feed supplement feeding stage
After the glycerol in the culture medium is consumed, the dissolved oxygen is rapidly increased (DO & gt 60%), feeding and feeding are carried out by glycerol solution, the flow acceleration is 6.2mL/h/L, the feeding is carried out for 5 hours, and then the flow speed is adjusted to 12mL/h/L until the WCW of the yeast thallus reaches 150g/L;
3) Starvation phase
When the carbon source (glycerin) in the fermentation tank is exhausted, the fermentation bacteria does not consume a large amount of oxygen due to lack of nutrition source, at the moment, the dissolved oxygen in the fermentation tank can quickly rise, waiting for 30min, and after the metabolic pathway taking glycerin as a substrate in the bacteria is completely ended, performing methanol induction;
4) Methanol induction stage
After the starvation stage is finished, the methanol solution is added for induction, the induction is started to be a methanol adaptation stage, the flow acceleration is controlled to be 1.1mL/h/L, after adaptation (after the methanol feeding is started, the change of dissolved oxygen is gradually reduced to a low level (gradually adapting to the starting of the utilization of the methanol) in turn, the high dissolved oxygen (little utilization of the methanol and gradual accumulation) gradually decreases to a higher level, the fluctuation is presented (the accumulated methanol is consumed,new methanol is consumed once being supplemented), pichia pastoris methanol is completely adapted), the methanol flow acceleration is properly accelerated, the methanol flow acceleration is adjusted to be 3.6mL/h/L, the flow acceleration is properly accelerated after 24 hours of flow feeding, the flow acceleration is controlled to be 6.2-10.5mL/h/L according to the feedback condition of dissolved oxygen, and the specific growth rate is controlled to be 0.015 hours -1 And regulating the rotating speed and the ventilation quantity to control the dissolved oxygen to be more than 20 percent until the fermentation is finished.
The fusion protein freeze-dried powder is prepared according to the following steps:
1) Pretreatment of fermentation broth
Centrifuging the fermentation liquor at 12000rpm for 20min, filtering the supernatant with 0.45 μm and 0.22 μm filter membranes respectively, and collecting filtrate;
(2) Nickel affinity chromatography
Since the HLfcinB gene expressed in this experiment contains a histidine tag, it can be purified by nickel affinity chromatography using Buffer A of 20mM Tris-HCl (pH 8.0); buffer B is a 20mM Tris-HCl (pH 8.0) Buffer containing 500mM imidazole; the packing was Ni Beascase FF (column volume 20 mL), and the purification experiment was as follows:
(1) column loading and balancing: the Ni Beascase FF packing was poured into a vertically placed XK16 column and allowed to settle overnight. The settled column was first treated with ddH 2 O washes 5 column volumes, balances 3-4 column volumes with Buffer A containing 1% Buffer B, and the flow rate is 5mL/min;
(2) loading: changing the filtrate in the step (1) with Buffer A balance Buffer containing 1% Buffer B, loading the sample, setting the flow rate to be 2mL/min, setting the loading amount to be 100mL, and collecting the flow-through liquid;
(3) rebalancing: balancing the chromatographic column with Buffer A containing 1% Buffer B, and eluting after the conductivity and ultraviolet absorption value are stable;
(4) eluting: gradient eluting with Buffer A and Buffer B mixed solution containing 8%, 20%, 30%, 50%, and 100% Buffer B, monitoring eluting peak under UV280, and collecting eluting peak with absorbance; eluting 2 column volumes at each concentration, wherein the eluting speed is 2mL/min;
(5) cleaning: the column was rinsed 5 volumes with 1M aqueous NaOH and degassed ddH was used 2 O washes 3-4 column volumes；
(6) And (3) preserving: washing 5 column volumes with 20% ethanol, closing the instrument according to standard operation flow, and removing the chromatographic column;
(3) Fusion protein freeze-dried powder
And (3) freeze-drying the eluent eluted by Buffer A (namely 150mM imidazole 20mM Tris-HCl (pH 8.0)) containing 30% Buffer B in the step (2) at the temperature of minus 60 ℃ for 48 hours to obtain the fusion protein freeze-dried powder.
Compared with the prior art, the invention has the beneficial effects that: because the expression quantity of the bovine lactoferrin peptide in the prior art is low (10-30 mg/L), the invention can better guide the expression of the bovine lactoferrin peptide by modifying the collagen short peptide as carrier protein, increase the expression quantity of the bovine lactoferrin peptide by as high as 183.7mg/L, improve by 10-15 times, and can be degraded into amino acid in the subsequent acidolysis process by reasonably designed collagen short peptide and connecting acidolysis peptide to release the lactoferrin peptide, and the lactoferrin peptide can be used in the fields of medicines, foods, feeds and the like.
(IV) description of the drawings
FIG. 1 is a diagram of recombinant plasmid pPIC9K-HLfcinB of collagen short peptide mutant acidolysis peptide bovine lactoferrin peptide-6 HIS fusion protein of example 1.
FIG. 2 is a agarose gel verification gel of positive transformant of E.coli DH 5. Alpha. Transformed with the recombinant plasmid of example 1; lanes M represent markers and lanes 1-10 represent transformant colonies.
FIG. 3 is an agarose gel validation gel of positive transformants of recombinant plasmid transformed Pichia pastoris GS115 of example 2; lanes M represent markers and lanes 1-8 represent transformant colonies.
FIG. 4 is a SDS-PAGE analysis of the expression of the collagen short peptide mutant acidolysis peptide bovine lactoferrin peptide-6 HIS fusion protein of example 3, wherein lane M represents Marker, and lanes 1-8 represent uninduced control, 12h, 18h, 24h, 36h, 48h, 60h, 72h fermentation broths, respectively.
FIG. 5 is a SDS-PAGE map of the collagen short peptide mutant acidolysis peptide bovine lactoferrin peptide-6 HIS fusion protein of example 4 after purification using a nickel column, lane M represents Marker, and lanes 1 to 9 represent the electrophoresis maps of the respective components in the purification step, respectively.
FIG. 6 is a SDS-PAGE verification of small molecules of lactoferrin obtained after acidolysis of the collagen short peptide mutant-acidolyzed peptide-bovine lactoferrin peptide-6 HIS fusion protein of example 4, wherein lane M represents Marker, and lanes 1-9 represent electrophoresis of lactoferrin obtained after acidolysis, respectively.
FIG. 7 is a graph showing the inhibition of E.coli k88 by GS115 broth, lactoferrin peptide.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
pichia pastoris GS115 strain, expression vector pPIC9K, was selected for use in the present invention and was purchased from Invitrogen corporation, U.S.A.
YPD complete medium: 10g/L of yeast extract, 20g/L of peptone, 20g/L of glucose and water as a solvent.
YPD solid Medium 20g/L agar was added to YPD complete medium.
MD medium (selection medium): YNB 13.4g/L, glucose 20g/L, biotin 4X 10 -4 g/L, agarose 20g/L, and water as solvent. The preparation method comprises the following steps of: adding agarose 2g (20 g/L) into 80mL water, sterilizing at 121deg.C for 20min, cooling to 60deg.C, adding 10 XYNB 10mL (13.4 g/L), 10 Xglucose 10mL (20 g/L), 500 Xbiotin 0.2mL (4×10) -4 g/L)。
BMG medium (yeast growth medium): k (K) 2 HPO 4 3g/L，KH 2 PO 4 11.8g/L YNB (no amino yeast nitrogen source) 3.4g/L, ammonium sulfate 10g/L, glycerol 10mL/L, 4X 10 -4 g/L biotin, and the solvent is water. The preparation method comprises the following steps: 3g K 2 HPO 4 ，11.8g KH 2 PO 4 3.4g YNB (no amino yeast nitrogen source), 10g ammonium sulfate, 10mL glycerol, was completely dissolved and the volume was fixed to 1L with deionized water. Steam autoclaving at 115℃for 30min. After cooling, 2mL of 0.2% 500 Xbiotin was added to the super clean bench.
BMM medium (yeast induction medium): k (K) 2 HPO 4 3g/L，KH 2 PO 4 11.8g/L YNB (no amino yeast nitrogen source) 3.4g/L, ammonium sulfate 10g/L, methanol 10mL/L, 4X 10 -4 g/L biotin, and the solvent is water. The preparation method comprises the following steps: completely dissolve 3g K 2 HPO 4 ，11.8g KH 2 PO 4 3.4g YNB (no amino yeast nitrogen source), 10g ammonium sulfate, and deionized water was used to determine the volume to 1L. Steam autoclaving at 115℃for 30min. After cooling, 2mL of 500 Xbiotin and 10mL of methanol were added to the reaction vessel.
BSM medium (g/L): 85% H 3 PO 4 26.7mL/L，KOH 4.13g/L，K 2 SO 4 18.2g/L，CaSO 4 0.93g/L，MgSO 4 ·7H 2 14.9g/L of O, 40.0g/L of glycerol, 4.35mL/L of trace elements (PTM 1), water as a solvent and pH 5.0-5.5; the preparation method comprises the following steps: 85% H 3 PO 4 26.7mL，KOH 4.13g，K 2 SO 4 18.2g，CaSO 4 0.93g，MgSO 4 ·7H 2 14.9g of O, 40.0g of glycerol, adjusting the pH to 5.0-5.5 by ammonia before inoculation, and adding 4.35mL/L of PTM.
Trace element (PTM 1): h 3 BO 3 0.02g/L，CuSO 4 ·5H 2 O 6.0g/L，MnSO 4 ·H 2 O 3.0g/L，Na 2 MoO 4 ·2H 2 O 0.2g/L，CoCl 2 0.5g/L，NaI 0.08g/L，ZnCl 2 20.0g/L，FeSO 4 ·7H 2 O65.0 g/L, biotin 0.2g/L,5.0mL/L H 2 SO 4 ，ddH 2 O is fixed to volume to 1L, filtered, sterilized and stored at 4 ℃ in dark place.
Example 1
1. Collagen short peptide mutant
From the amino acid sequence of human type III collagen alpha I chain (NCBI sequence No. NM-000090.4, from homosapiens) a structurally stable, well-hydrophilic collagen short peptide amino acid sequence is preferred, which is located at amino acids 608-785 of the des-terminal peptide type III collagen alpha I chain sequence, together with amino acids 177, according to the collagen MODEL, using the NCBI-derived collagen amino acid sequence modeling using SWISS-MODEL website database. According to the characteristic repeated structural rule of collagen, mutating hydrophobic amino acid into hydrophilic amino acid to improve the hydrophilicity of collagen short peptide, reducing isoelectric point as far as possible, and determining specific mutation points as follows by preliminary screening mutation points: isoleucine at position 614 to aspartic acid, leucine at position 633 to glycine, isoleucine at position 636 to proline, phenylalanine at position 658 to glycine, valine at position 706 to proline, and phenylalanine at position 721 to glutamic acid. The mutant of the mutant collagen short peptide has 177 amino acids, and the amino acid sequence is shown as SEQ ID NO. 1; the length of the coding gene is 531bp, the nucleotide sequence is shown as SEQ ID NO. 2, the hydrophilicity is-1.09, and the isoelectric point is 4.83.
SEQ ID NO:1：
GPTGPDGPPGPAGQPGDKGEGGAPGGPGPAGPRGSPGERGETGPPGPAGGPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQ。
SEQ ID NO:2：
GGTCCAACTGGTCCTGACGGTCCACCTGGACCAGCCGGTCAACCAGGAGATAAAGGTGAAGGTGGTGCTCCAGGTGGTCCTGGTCCTGCTGGTCCTAGAGGTTCTCCAGGTGAAAGAGGTGAGACTGGTCCACCTGGACCTGCTGGTGGTCCAGGTGCTCCTGGTCAAAACGGTGAACCAGGTGGTAAAGGAGAAAGAGGTGCTCCAGGAGAAAAAGGAGAGGGTGGTCCACCTGGTGTTGCCGGTCCACCTGGTGGTTCTGGTCCAGCCGGTCCACCTGGACCTCAAGGTCCAAAGGGTGAAAGAGGTTCTCCAGGTGGTCCTGGTGCTGCTGGTGAACCTGGTGCTAGAGGTTTGCCTGGTCCACCTGGATCTAACGGTAACCCTGGTCCACCTGGACCTTCTGGTTCTCCAGGTAAAGACGGTCCACCTGGTCCAGCTGGTAACACTGGTGCTCCAGGTTCTCCTGGTGTTTCTGGTCCAAAGGGAGATGCCGGTCAACCTGGAGAGAAAGGTTCTCCAGGTGCTCAA。
2. Synthesis and expression of collagen short peptide mutant fusion protein
(1) Synthesis of collagen short peptide mutant fusion protein
The collagen short peptide mutant is used as carrier protein, acidolysis peptide is connected at the C end, lactoferrin peptide (the nucleotide sequence is shown as SEQ ID NO:5, the amino acid sequence is shown as SEQ ID NO: 6) is connected at the C end of the acidolysis peptide, 6HIS (the nucleotide sequence CACCATCATCACCACCAT, the amino acid sequence HHHHHH) is connected at the C end of the lactoferrin peptide, and a novel fusion protein for expressing the lactoferrin peptide is constructed, namely the collagen short peptide mutant-acidolysis peptide-bovine lactoferrin peptide-6 HIS fusion protein, namely collagen short peptide mutant fusion protein HlfcinB for short, which is subjected to codon optimization and synthesis by Nanjing Jinshi biotechnology limited company, and has the total length of 630bp, 210 amino acids, the nucleotide sequence is shown as SEQ ID NO:4, and the amino acid sequence is shown as SEQ ID NO: 3.
SEQ ID NO:3：
GPTGPDGPPGPAGQPGDKGEGGAPGGPGPAGPRGSPGERGETGPPGPAGGPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQDPEWFKCRRWQWRMKKLGAPSITCVRRAFHHHHHH。
SEQ ID NO:4
GGTCCAACTGGTCCTGACGGTCCACCTGGACCAGCCGGTCAACCAGGAGATAAAGGTGAAGGTGGTGCTCCAGGTGGTCCTGGTCCTGCTGGTCCTAGAGGTTCTCCAGGTGAAAGAGGTGAGACTGGTCCACCTGGACCTGCTGGTGGTCCAGGTGCTCCTGGTCAAAACGGTGAACCAGGTGGTAAAGGAGAAAGAGGTGCTCCAGGAGAAAAAGGAGAGGGTGGTCCACCTGGTGTTGCCGGTCCACCTGGTGGTTCTGGTCCAGCCGGTCCACCTGGACCTCAAGGTCCAAAGGGTGAAAGAGGTTCTCCAGGTGGTCCTGGTGCTGCTGGTGAACCTGGTGCTAGAGGTTTGCCTGGTCCACCTGGATCTAACGGTAACCCTGGTCCACCTGGACCTTCTGGTTCTCCAGGTAAAGACGGTCCACCTGGTCCAGCTGGTAACACTGGTGCTCCAGGTTCTCCTGGTGTTTCTGGTCCAAAGGGAGATGCCGGTCAACCTGGAGAGAAAGGTTCTCCAGGTGCTCAAGACCCTGAGTGGTTTAAGTGTAGAAGATGGCAATGGAGAATGAAGAAGTTGGGTGCTCCATCTATTACTTGTGTTAGAAGAGCTTTCCACCATCATCACCACCATTAA。
SEQ ID NO:5：
TTTAAGTGTAGAAGATGGCAATGGAGAATGAAGAAGTTGGGTGCTCCATCTATTACTT GTGTTAGAAGAGCTTTC。
SEQ ID NO:6：
FKCRRWQWRMKKLGAPSITCVRRAF。
(2) Expression of collagen short peptide mutant fusion proteins
The collagen short peptide mutant fusion protein of the step (1) is obtained by Nanjing Jinsri biotechnology Co., ltd
The HlfcinB encoding gene (the nucleotide sequence is shown as SEQ ID NO: 4) is connected to the multicloning site of pPICC 3.5K plasmid after double digestion by EcoRI and NotI, plasmid pPICC 3.5K-HLfcinB is constructed, and the gene is transferred into E.coli DH5 alpha competent cells and stored in a refrigerator at-80 ℃. After culturing on ampicillin and kanamycin-containing plates, positive clone extraction plasmids are picked up, and corresponding strain plasmids are extracted to obtain two cloning vectors pPIC9K plasmid and pPICC 3.5K-HlfcinB.
Constructing a pPIC9K-HlfcinB fusion expression cloning vector by a seamless cloning technology:
the pPIC9K vector fragment was amplified using primers pPIC9K-F and pPIC9K-R to mutate the alpha signal peptide EAEA residue to methionine residue using the pPIC9K plasmid as a template.
pPIC9K-F:GGGGTATCTCTCGAGAAAAGAATG；
pPIC9K-R:CATTCTTTTCTCGAGAGATACCCCT。
HlfcinB fragments were amplified using pPICC 3.5K-HlfcinB as template and primers HlfcinB-F and HlfcinB-R.
HlfcinB-F:AAGGGGTATCTCTCGAGAAAAGAATGGGTCCAACTGGTCCTGAC GGTCC；
HlfcinB-R:CTAGGGAATTCTACGTATTAATGGTGGTGATGATGGTGGAAAGCT CTTCTAACACAAGTAATAG。
The PCR system is as follows: primer F1. Mu.L, primer R1. Mu. L, DNA 1. Mu.L, high fidelity enzyme 25. Mu.L, water make up to 50. Mu.L.
The PCR product was digested with DPNI enzyme to remove the original template interference, the system was as follows: PCR stock 50. Mu. L, DPN I1. Mu.L, 10 XBUFFER 5. Mu.L. The digested PCR product was purified using a purification kit (available from Shanghai Biotechnology Co., ltd.) and the specific procedure was carried out according to the kit instructions.
The purified PCR products (pPIC 9K vector fragment and HlfcinB fragment) were subjected to recombinant ligation using a one-step cloning kit (purchased from Nanjinouzan Biotechnology Co., ltd.) to obtain ligation products. The specific operation of recombination connection is carried out according to the instruction of the kit, and the reaction system is as follows: 1 mu L, buffer mu L, pPIC K fragment 2 mu L, HLfcinB fragment 3 mu L of recombinase and 2 mu L of sterile water.
The ligation product was transformed into E.coli DH 5. Alpha. Commercially available from DH 5. Alpha. From Beijing gold sand Biotechnology Co., ltd.) as follows: taking 100 mu L of melted competent cells on ice, adding target DNA (connection product), gently mixing, and standing on ice for 5min; heat shock is carried out for 45s in a water bath at the temperature of 42 ℃, and the mixture is quickly put back on ice and kept stand for 2min; adding 700 mu L of sterile LB culture medium without antibiotics into a centrifuge tube, uniformly mixing, and resuscitating at 200rpm for at least 20min at 37 ℃; 100 mu L of resuscitated cells are pipetted onto LB solid medium plates containing ampicillin and kanamycin at a final concentration of 0.1mg/ml, spread evenly and blow-dried; the plates were placed in an incubator at 37℃overnight.
And selecting a single colony on a transformation plate for agarose gel electrophoresis verification of positive transformants, wherein 8 transformants in total are positive transformants in lanes 3-10, and the verification sequence length is 496bp of a general sequence and 630bp of a fusion protein gene sequence, and 1126bp. The positive transformants were extracted with plasmid pPIC9K-HLfcinB (map see FIG. 1), sequenced and the correct strain was deposited in a-80℃freezer.
The verification primers were 9K plasmid universal primers 5AOX,3AOX, the sequences were as follows:
3AOX：GCAAATGGCATTCTGACATCC；
5AOX GACTGGTTCCAATTGACAAGCTT。
example 2 construction of recombinant Pichia pastoris engineering bacteria GS115-HLfcinB;
1. linearization of the expression vector pPIC9K-HLfcinB
The plasmid pPIC9K-HLfcinB constructed in example 1 was digested with restriction endonuclease SacI at 37℃overnight, then 1% agarose gel electrophoresis was used to determine whether the cleavage was complete, and after complete cleavage, the digested solution was purified using a PCR product purification kit to recover linearized plasmids.
The enzyme digestion reaction system is as follows:
plasmid pPIC9K-HLfcinB 1. Mu.g, 10 XL buffer 2. Mu. L, sac I1. Mu.L, sterile water was added to 20. Mu.L.
2. Preparation of Pichia pastoris GS115 competent cells
(1) Pichia pastoris GS115 single colonies are picked up on YPD plates and inoculated in a test tube containing 3mL of YPD liquid medium, and shake-cultured at 30 ℃ and 220rpm overnight; (2) Inoculating 500 μl of the overnight culture into 500mL triangular flask containing 50mL fresh YPD liquid medium, shaking culturing at 30deg.C and 220rpm overnight until OD600 reaches 1.3-1.5; (3) Introducing the culture into a sterile centrifuge tube, centrifuging at 4deg.C and 5000rpm for 5min, removing supernatant, and placing on ice; (4) The cells were resuspended in 20mL of LiAc-DTT solution (100mM LiAc,10mM DTT,0.6M sorbitol, 10mM Tris-HCl, pH 7.5), shake-cultured at 30℃for 30min, centrifuged at 4℃at 5000rpm for 5min, and the supernatant was removed.
Repeating the step (4) for three times or adding 1mL of 1M sorbitol pre-cooled on ice to resuspend the thallus, transferring the thallus into a 1.5mL EP tube, centrifuging at 3000rpm for 5min, removing the supernatant, and repeating the step for three times.
The collected thalli are resuspended with 1M sorbitol precooled on ice, and the final volume is about 0.5mL; split charging into 80 μl one tube, and preserving at-80deg.C.
3. Electric transformation of Pichia pastoris
Taking out pichia pastoris GS115 competent cells prepared in the step 2 from a refrigerator at the temperature of minus 80 ℃ and placing the competent cells on ice; uniformly mixing 1 mug of the linearized plasmid prepared in the step 1 with 80 mug of the competent plasmid prepared in the step 2, transferring the mixture into a precooled electric rotating cup with the thickness of 0.2cm, lightly beating the mixture to enable the mixture to be positioned at the bottom of the electric rotating cup, and placing the mixture on ice for 5-10min; according to the operation instruction of a Bio-Rad electrotometer, setting a mode in a Pic mode, wiping off water on the outer wall of an electrotometer, and placing the electrotometer at an electric shock position, wherein the electric shock voltage is 1.5kV, the capacitance is 25 mu F, the resistance is 200 omega, and the electric shock time is 5msec; immediately adding 1mL of precooled 1M sorbitol into an electric rotating cup after electric shock is finished, lightly blowing and uniformly mixing, rapidly transferring into a 1.5mL EP tube, and carrying out stationary culture at 30 ℃ and 220rpm for 1-2h; 100-200 mu L of thalli are coated on an MD plate containing 0.1mg/ml ampicillin and 0.05mg/ml kanamycin, and are placed in a constant temperature incubator at 30 ℃ for inversion culture for 2-4 days until single colonies appear.
4. PCR identification of recombinant transformants
(1) Breaking the wall of the yeast transformant:
selecting single colony on the conversion plate, blowing and uniformly mixing in a PCR tube containing 0.1M NaOH aqueous solution until the single colony is visible turbid; heating the PCR tube in a microwave oven for 5min, rapidly cooling in liquid nitrogen for 5min, repeating the steps twice, and heating in the microwave oven for 5min; the pellet was placed at the bottom of the PCR tube by centrifugation.
(2) Identification of colony PCR:
PCR was performed using the alpha-F, 3AOX primer as the amplification primer, using the enzyme T5 Super PCR Mix (Coloney) (available from Beijing Optimago Co., ltd.) under the following PCR conditions: pre-denaturation at 98 ℃,5min, one thermal cycle; heat denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 15s,30 thermal cycles; finally, the extension is carried out for 5min at 72 ℃. And (3) detecting the PCR product by 1% agarose gel electrophoresis, wherein the amplified product is 835bp band which is the target gene, the corresponding strain is a positive transformant, the gel electrophoresis is shown in figure 3, and lanes 1-8 are all positive transformants. Single colonies corresponding to positive transformants on MD transformation plates were inoculated into 250mL triangular flasks containing 50mL YPD medium and cultured overnight at 30℃and 220rpm to obtain recombinant yeast GS115-pPIC9K-HLfcinB. The strain is preserved in a refrigerator at-80 ℃ by using a glycerol method.
The primer sequences were verified as follows:
α-F：TACTATTGCCAGCATTGCTGCT
3AOX：GCAAATGGCATTCTGACATCC
5. induction expression of recombinant yeast GS115-pPIC9K-HLfcinB
(1) Seed culture
Recombinant yeast GS115-pPIC9K-HLfcinB stored at-80℃was removed, streaked onto YPD solid plates containing 100. Mu.g/mL of G418 resistance, and allowed to stand at 30℃for 3 days. Single colonies were selected and inoculated into MD medium (30 m L/250mL, 50m L/500 mL), and cultured at 30℃and 200rpm for 16-18 hours as seed liquid for fermentation medium.
(2) Fermentation culture
The bioreactor fermentation is mainly divided into four stages, namely a batch fermentation stage, a feed-feeding fed-batch stage, a starvation stage and a methanol induction stage, and the specific steps are as follows:
1) Batch fermentation stage
Inoculating the cultured seed liquid into a 5L fermentation tank filled with 3L fermentation medium at 10% of inoculation amount, regulating pH to 5.0 with ammonia water, setting the temperature to 30deg.C, setting the initial rotation speed to 500rpm, and controlling ventilation amount to more than 20% of Dissolved Oxygen (DO) until glycerol in the medium is exhausted.
2) Feed supplement feeding stage
After the glycerol in the culture medium is consumed, dissolved oxygen rises rapidly (DO & gt 60%), feeding and feeding are carried out by using a glycerol solution (PTM 112 mL/L is added into a glycerol aqueous solution with the mass concentration of 50%), the flow acceleration is 6.2mL/h/L, the feeding is carried out for 5 hours, and then the flow rate is adjusted to 12mL/h/L until the thallus wet weight (WCW) of the yeast reaches 150g/L.
3) Starvation phase
When the carbon source (glycerin) in the fermentation tank is exhausted, the fermentation bacteria does not consume a large amount of oxygen due to lack of nutrition source, at the moment, the dissolved oxygen in the fermentation tank can quickly rise, waiting for 30min, and after the metabolic pathway taking glycerin as a substrate in the bacteria is completely ended, performing methanol induction.
4) Methanol induction stage
After the starvation stage is finished, beginning to induce the fed-batch methanol solution (PTM 112 mL/L is added into absolute methanol), wherein the induction is initially a methanol adaptation stage, the flow acceleration is controlled to be 1.1mL/h/L, after adaptation, the flow acceleration of methanol is properly accelerated, the flow acceleration is adjusted to be 3.6mL/h/L, after 24h of fed-batch, the flow acceleration is properly accelerated, the flow acceleration is controlled to be 6.2-10.5mL/h/L according to the feedback condition of dissolved oxygen, and the specific growth rate is controlled to be 0.015h -1 And regulating the rotating speed and the ventilation quantity to control the dissolved oxygen to be more than 20 percent until the fermentation is finished.
Every 12h, 1mL of bacterial liquid sample is taken, the bacterial liquid sample is centrifuged at 12000rpm at room temperature for 2min, and the supernatant is collected for SDS-PAGE electrophoresis detection. Samples were taken every 12h after induction and the fusion protein HLfcinB content and bacterial wet weight (WCW) were determined.
The sample to be detected is placed in a refrigerator at the temperature of minus 80 ℃ for standby.
Example 3 detection of recombinant Yeast fusion proteins
SDS-PAGE of the supernatant collected in example 2 is shown in FIG. 4, wherein lane M represents Marker, and lanes 1-8 represent uninduced control, 12h, 18h, 24h, 36h, 48h, 60h, 72h fermentation broth, respectively. The band of about 25kDa is collagen short peptide mutant-acidolysis peptide-bovine lactoferrin peptide-6 HIS fusion protein, the size of the fusion protein on SDS-PAGE is larger due to the special repeated structure of the collagen, the expected expression quantity of the protein is increased with time, but the collagen short peptide fusion mode also generates collagen degradation phenomenon, and the collagen short peptide is changed into multiple bands.
The quantitative determination of the expression level of the fusion protein was carried out by BCA kit (available from Shanghai Biotechnology Co., ltd.) and the specific procedure was referred to the BCA kit instructions. The total expression level of the recombinant Pichia pastoris GS115-pPIC9K-HLfcinB 72h fermentation broth fusion protein is measured to reach 2.8g/L.
EXAMPLE 4 isolation and purification of fusion proteins
1. The fermentation broth in the embodiment 2 is separated and purified to obtain fusion protein, a 6HIS tag carried by the fusion protein can be used for catching an AKTA packed nickel column for separation and purification, and the operation flow is as follows:
(1) Pretreatment of fermentation broth
The fermentation broth after 3 days of methanol induction was centrifuged at 12000rpm for 20min, and the supernatant was filtered off with 0.45 μm and 0.22 μm filters, respectively, to remove solid impurities, and the filtrate was collected.
(2) Nickel affinity chromatography
Since the HLfcinB gene expressed in this experiment contains a histidine tag, it can be purified by nickel affinity chromatography using Buffer A of 20mM Tris-HCl (pH 8.0); buffer B is a 20mM Tris-HCl (pH 8.0) Buffer containing 500mM imidazole; the packing was Ni Beascase FF (column volume 20 mL), and the purification experiment was as follows:
(1) column loading and balancing: the Ni Beascase FF packing was poured into a vertically placed XK16 column and allowed to settle overnight. The settled column was first treated with ddH 2 O washes 5 column volumes, balances 3-4 column volumes with Buffer A containing 1% Buffer B, and the flow rate is 5mL/min;
(2) loading: changing the filtrate in the step (1) with Buffer A balance Buffer containing 1% Buffer B, loading the sample, setting the flow rate to be 2mL/min, setting the loading amount to be 100mL, and collecting the flow-through liquid;
(3) rebalancing: balancing the chromatographic column with Buffer A containing 1% Buffer B, and eluting after the conductivity and ultraviolet absorption value are stable;
(4) eluting: gradient eluting with Buffer A and Buffer B mixed solution containing 8%, 20%, 30%, 50%, and 100% Buffer B, monitoring eluting peak under UV280, and collecting eluting peak with absorbance; eluting 2 column volumes at each concentration, wherein the eluting speed is 2ml/min;
(5) cleaning: the column was rinsed 5 volumes with 1M aqueous NaOH and degassed ddH was used 2 O washes 3-4 column volumes;
(6) and (3) preserving: the column was removed by washing 5 column volumes with 20% ethanol, closing the instrument according to standard procedure.
(3) Fusion protein freeze-dried powder
The eluate eluted from Buffer A (i.e., 150mM imidazole 20mM Tris-HCl (pH 8.0)) containing 30% Buffer B in step (2) was lyophilized at-60℃for 48 hours to give 0.5g of a lyophilized powder of the fusion protein.
0.05g of the fusion protein lyophilized powder was dissolved in 1mL of water, SDS-PAGE was performed, and the results are shown in FIG. 5, lanes 1-2 show that the unpurified samples, specifically, the supernatants of 24h and 72h fermentation in example 2, 3, 6, 7 and 9 were all aqueous solutions of the fusion protein lyophilized powder obtained in step (3), 4 was the flow-through solution obtained in step (2), and 5 and 8 were both concentrated flow-through solutions obtained in step (2) (the concentration method uses ultrafiltration).
It can be seen that endogenous proteins of pichia pastoris, such as AOX, etc., are greatly reduced in the purified sample compared to the unpurified sample, but cannot play a separate role in de novo degradation of collagen short peptides.
2. Acidolysis fusion protein preparation of lactoferrin peptide
(1) 0.1g of fusion protein freeze-dried powder is taken and dissolved in 1ml of 100mM HCl, the reaction is carried out for 8 hours in a metal bath at 80 ℃, the pH value is regulated to 7 by using equimolar NAOH after the powder is taken out, and the acidolysis solution of the fusion protein is obtained, and the protein content is 183.7mg/L measured by a BCA method. The results of SDS-PAGE detection and mass spectrometry identification of small molecules are shown in FIG. 6.
(2) 0.1g of the fusion protein lyophilized powder was dissolved in 1ml of 50mM HCl and reacted for 8 hours in a metal bath at 60 ℃, and after removal, the pH was adjusted to 7 by using equimolar NAOH, and the results of small molecule SDS-PAGE detection and mass spectrometry identification are shown in FIG. 6.
FIG. 6, lanes 1-4 are 80℃for 8h with 100mM HCl, lane 5 is step 1 supernatant control, lane M is marker, and lanes 6-10 are 60℃for 8h with 50mM HCl. It can be seen that the fusion protein is treated to give pure lactoferrin peptide.
3. Lactoferrin peptide Performance detection
And (3) performing bacteriostasis detection on the acidolysis solution of the fusion protein in the step (1) and the fermentation liquor in the embodiment 2, wherein the bacteriostasis detection method is performed by adopting a dynamic growth curve method, and the indicator bacterium is escherichia coli K88.
As a result, as shown in FIG. 7, the lactoferrin peptide had a certain inhibitory effect on E.coli k88 in the early stage but the inhibition was decreased with time as compared with the GS115 fermentation broth. The results show that the lactoferrin peptide prepared by the method provided by the invention has obvious antibacterial activity.
Claims (9)
1. The collagen short peptide mutant fusion protein is characterized in that the amino acid sequence of the collagen short peptide mutant fusion protein is shown as SEQ ID NO. 3.
2. A gene encoding the collagen short peptide mutant fusion protein of claim 1.
3. A recombinant plasmid comprising the gene encoding the collagen short peptide mutant fusion protein of claim 1.
4. A recombinant genetically engineered bacterium constructed from the recombinant plasmid of claim 3.
5. Use of a collagen short peptide mutant fusion protein according to claim 1 in the preparation of lactoferrin peptides.
6. The application according to claim 5, wherein the method of application is: centrifuging a fermentation liquor obtained by fermenting and culturing recombinant genetically engineered bacteria containing the collagen short peptide mutant fusion protein encoding genes, collecting supernatant, extracting pure enzyme, and freeze-drying to obtain fusion protein freeze-dried powder; dissolving the fusion protein freeze-dried powder in 50-200mM HCl aqueous solution, reacting for 8-12 hours in a metal bath at 60-80 ℃, and adjusting the pH to 7.0 by using 1M NaOH to obtain lactoferrin peptide solution.
7. The use according to claim 6, wherein the fermentation broth is prepared as follows:
streaking recombinant genetically engineered bacteria on a YPD solid plate containing 100 mug/mL G418 resistance, standing and culturing at 30 ℃ for 3 days, selecting single bacterial colonies, inoculating the single bacterial colonies to an MD culture medium, and culturing at 200rpm at 30 ℃ for 16-18 hours to obtain seed liquid of a fermentation culture medium;
inoculating seed liquid into a 5L fermentation tank containing 3L BSM culture medium at 10% of inoculation amount by volume concentration, adjusting pH to 5.0, setting the temperature to 30 ℃, setting the initial rotating speed to 500rpm, and controlling the ventilation rate to be more than 20%; when the glycerol in the culture medium is consumed, the dissolved oxygen rises rapidly, and then the glycerol solution starts to be fed, and the culture is carried out until the concentration of the thalli is 150g/L; starving for 30min after the end of feeding, then feeding methanol solution for induction, and controlling the specific growth rate at 0.015h -1 Regulating the rotating speed and the ventilation quantity to control the dissolved oxygen to be more than 20%, so as to obtain fermentation liquor; the glycerol solution is prepared by adding 12mL/L trace elements into a glycerol aqueous solution with the mass concentration of 50%; the methanol solution refers to that 12mL/L trace elements are added into absolute methanol;
BSM medium: 85% H 3 PO 4 26.7mL/L，KOH 4.13g/L，K 2 SO 4 18.2g/L，CaSO 4 0.93g/L，MgSO 4 ·7H 2 14.9g/L of O, 40.0g/L of glycerol, 4.35mL/L of trace elements, water as solvent and pH of 5.0-5.5;
trace elements: h 3 BO 3 0.02g/L，CuSO 4 ·5H 2 O 6.0g/L，MnSO 4 ·H 2 O 3.0g/L，Na 2 MoO 4 ·2H 2 O0.2g/L，CoCl 2 0.5g/L，NaI 0.08g/L，ZnCl 2 20.0g/L，FeSO 4 ·7H 2 O65.0 g/L, biotin 0.2g/L,5.0mL/L H 2 SO 4 The solvent is ddH 2 O。
8. The use according to claim 6, wherein the fermentation is divided into four phases, in turn a batch fermentation phase, a fed-batch phase, a starvation phase and a methanol induction phase:
1) Batch fermentation stage
Inoculating seed liquid into a 5L fermentation tank filled with 3L fermentation medium at 10% of inoculation amount, regulating pH to 5.0 with ammonia water, setting the temperature to 30deg.C, setting the initial rotation speed to 500rpm, and controlling ventilation amount to more than 20% of dissolved oxygen until glycerol in the medium is exhausted;
2) Feed supplement feeding stage
After the glycerol in the culture medium is consumed, the dissolved oxygen is quickly increased, then feeding and feeding are carried out by glycerol solution, the flow acceleration is 6.2mL/h/L, 5h is fed, and then the flow speed is adjusted to 12mL/h/L until the concentration of the thalli reaches 150g/L;
3) Starvation phase
After the feeding of the feed is finished, starvation induction is carried out for 30min;
4) Methanol induction stage
After the starvation stage is finished, the methanol solution is added for induction, the induction is started to be a methanol adaptation stage, the flow acceleration is controlled to be 1.1mL/h/L, the flow acceleration is adjusted to be 3.6mL/h/L after adaptation, the flow acceleration is controlled to be 6.2-10.5mL/h/L according to the feedback condition of dissolved oxygen after 24h of feeding, and the specific growth rate is controlled to be 0.015h -1 And regulating the rotating speed and the ventilation quantity to control the dissolved oxygen to be more than 20 percent until the fermentation is finished.
9. The use according to claim 6, wherein the fusion protein lyophilized powder is prepared by the steps of:
1) Pretreatment of fermentation broth
Centrifuging the fermentation liquor at 12000rpm for 20min, filtering the supernatant with 0.45 μm and 0.22 μm filter membranes respectively, and collecting filtrate;
(2) Nickel affinity chromatography
Buffer A is 20mM Tris-HCl, pH 8.0; buffer B is 20mM Tris-HCl, pH 8.0, containing 500mM imidazole; the filler is Ni Beascase FF, the column volume is 20mL, and the purification experiment steps are as follows:
(1) dress(s)Column and equilibrium: pouring the Ni Beascase FF filler into a XK16 chromatographic column which is vertically arranged, naturally settling overnight, and using ddH for the settled column 2 O washes 5 column volumes, and balances 3-4 column volumes with Buffer A containing 1% Buffer B at a flow rate of 5mL/min;
(2) loading: changing the filtrate in the step (1) by using Buffer A balance Buffer containing 1% Buffer B, and then sampling, wherein the flow rate is set to be 2mL/min, and the sampling amount is set to be 100mL;
(3) rebalancing: balancing the chromatographic column with Buffer A containing 1% Buffer B, and eluting after the conductivity and ultraviolet absorption value are stable;
(4) eluting: gradient elution is carried out by using Buffer A and Buffer B containing 8%, 20%, 30%, 50% and 100% of Buffer B respectively, monitoring elution peak under UV280, and collecting elution peak with absorbance; eluting 2 column volumes at each concentration, wherein the eluting speed is 2mL/min;
(5) cleaning: the column was rinsed 5 volumes with 1M aqueous NaOH and degassed ddH was used 2 O washes 3-4 column volumes;
(6) and (3) preserving: washing 5 column volumes with 20% ethanol, closing the instrument according to standard operation flow, and removing the chromatographic column;
(3) Fusion protein freeze-dried powder
And (3) freeze-drying the eluent eluted by Buffer A containing 30% of Buffer B in the step (2) at the temperature of minus 60 ℃ for 48 hours to obtain the fusion protein freeze-dried powder.
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| CN116904429A (en) * | 2023-08-30 | 2023-10-20 | 江南大学 | Method for efficiently expressing collagen hydrolase and application thereof |
| CN117024604A (en) * | 2023-08-09 | 2023-11-10 | 广西福莱明生物制药有限公司 | Recombinant biological defense fusion protein and application thereof |
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| CN117024604A (en) * | 2023-08-09 | 2023-11-10 | 广西福莱明生物制药有限公司 | Recombinant biological defense fusion protein and application thereof |
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| CN116904429A (en) * | 2023-08-30 | 2023-10-20 | 江南大学 | Method for efficiently expressing collagen hydrolase and application thereof |
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