CN117164674B

CN117164674B - Antifreeze protein, gene, yeast engineering bacteria and application thereof

Info

Publication number: CN117164674B
Application number: CN202311377226.9A
Authority: CN
Inventors: 陈良标; 胡瑞芹; 许强华; 胡鹏; 刘明丽; 钟文倩
Original assignee: Shanghai Water Technology Transfer Co ltd
Current assignee: Shanghai Water Technology Transfer Co ltd
Priority date: 2023-10-24
Filing date: 2023-10-24
Publication date: 2024-02-02
Anticipated expiration: 2043-10-24
Also published as: CN117164674A

Abstract

The invention belongs to the technical field of biology, and discloses an antifreeze protein and a gene sequence thereof. And discloses engineering bacteria for expressing antifreeze protein and a construction method thereof. The antifreeze protein gene containing SEQ ID No.1 sequence is used as a template for amplification to obtain the polypeptide with 5' endXhoI. Having 3' end withXbaI cleavage site and target fragment of 6 XHis tag; connecting the target fragment to an expression vector to construct a recombinant expression vector; and linearizing the recombinant expression vector, transferring the recombinant expression vector into host saccharomycetes, and screening positive transformants to obtain recombinant saccharomycetes. The obtained recombinant protein HhAFP can remarkably improve the survival rate of resuscitated cells after freezing and preserving and reduce cell damage. The invention can obtain the antifreeze protein with high activity and high yield, and provides a technical approach for the application and industrialization of the antifreeze protein.

Description

Antifreeze protein, gene, yeast engineering bacteria and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an antifreeze protein, engineering bacteria expressing the antifreeze protein, and a construction method and application thereof.

Background

In order to reduce the damage, glycerol, ethylene glycol, propylene glycol, acetamide, methanol, dimethyl sulfoxide (DMSO) and the like are usually added into cell cryopreservation liquid, and the permeable cryoprotectant can obviously relax cell membranes, so that the cells are more easily adapted to the pressure in the low-temperature preservation process. But it is toxic to cells due to its permeability and is detrimental to cell membrane proteins.

Antifreeze proteins were first found in the blood of antarctic fish, which were subsequently found in various organisms living in polar regions, such as fish, insects, plants, bacteria, fungi, and the like. The antifreeze protein is a special macromolecular protein, and can specifically lower the freezing point of the solution without affecting the melting point, so that the difference between the freezing point and the melting point is called thermal hysteresis activity, thereby preventing ice nucleus formation, controlling ice crystal growth, maintaining the liquid state of body fluid and improving the antifreeze capability of organisms to a certain extent.

The south pole ocean, which is covered by ice and snow for a long time, is the most severe living environment in the world, but still living beings adapt to the environment by producing antifreeze proteins in the body. The anti-freeze proteins found and verified to be active in antarctic fish are the eggshell protein ZPC5, the antifreeze protein LD4 and the calmodulin gene, wherein ZPC5 expressed and purified in vitro has the effect of inhibiting ice crystal growth in vitro, and LD4 protein and calmodulin gene have the effect of improving their low temperature resistance after being overexpressed in tobacco.

Except for antarctic fish, the Yunshan leaf roller is found in insectsChoristoneurafumiferana) An antifreeze protein (CfAFP) which is expressed in an E.coli expression system, and the obtained recombinant protein has the effect of obviously inhibiting the growth of ice crystals. In addition, a high-activity antifreeze protein also exists in the insect fleas living in the south poles, and researchers find that the antifreeze activity of the antifreeze protein at millimole concentration is 10-100 times of that of fish antifreeze protein. The research shows that the antifreeze protein has certain cold resistance or antifreeze effect both in vivo and in vitro, and the effect can be widely applied to the aspects of transgenic cold-resistant crop breeding, food freezing transportation, cell freezing preservation and the like.

Disclosure of Invention

The invention aims to provide an antifreeze protein (HhAFP) and a coding gene and application thereof.

The invention also provides an expression system of the antifreeze protein, yeast engineering bacteria and a method for efficiently expressing the antifreeze protein.

The technical scheme of the invention is as follows:

an antifreeze protein (HhAFP) comprising the amino acid sequence shown in SEQ ID No.1.

SEQ ID No.1:

MRVAEVAKVLWVGVCAVAAVTLVVGVQEVVKVVWVVWVWGVQAVVLVVAVVATVVVCFLVVHVVRVVTVCGRVAEVAGVAG。

Preferably, the amino acid sequence of the antifreeze protein is shown as SEQ ID No.1.

In one embodiment of the invention, the C-terminal of the antifreeze protein is further His-tagged.

The N end of the antifreeze protein is also provided with 1 or 2 signal peptide cleavage site amino acids. The amino acid of the signal peptide cleavage site is KR or R.

An antifreeze protein gene which codes for the antifreeze protein.

Preferably, the antifreeze protein gene contains a nucleotide sequence shown as SEQ ID No. 2.

More preferably, the nucleotide sequence of the antifreeze protein gene is shown as SEQ ID No. 2; alternatively, it carries a nucleotide sequence encoding a His tag at its 3' end.

Alternatively, it carries a sequence encoding 1 or 2 signal peptide cleavage site amino acids at its 5' end. Preferably Kex 2 signal peptide cleavage sites; the amino acid of the signal peptide cleavage site is KR or R.

SEQ ID No.2:

atgcgtgttgcagaagtggcaaaagtgctgtgggtgggtgtgtgtgcagtagcagcagtgaccctggtggtgggggtgcaggaggtggtgaaggtggtgtgggtcgtctgggtttggggagttcaggcagttgttcttgttgttgccgtggtggccaccgtggttgtctgctttctggttgttcatgttgttcgtgtcgtgacagtttgtggccgcgtggctgaagttgcaggtgttgccggt。

The antifreeze protein or antifreeze protein gene can be used for cell cryopreservation or frozen food preservation, and can be used for preparing cell cryopreservation liquid, frozen preservative or frozen food. The antifreeze protein gene can also be used for breeding transgenic cold-resistant plants and cultivating the cold-resistant plants.

A recombinant expression vector contains a gene for expressing the antifreeze protein.

Preferably, the recombinant expression vector contains a gene expressing a secretion signal peptide. In a preferred embodiment of the present invention, the recombinant expression vector is a pPICZ alpha A plasmid.

In a preferred embodiment of the present invention, the recombinant expression vector contains a nucleotide sequence capable of expressing the protein shown in SEQ ID No. 6.

SEQ ID No.6：

Wherein the method comprises the steps ofDouble underlineTo exocrine the signal peptide alpha-factor (1-83),wave lineIs Kex 2 signal peptide cleavage site (84-85),single underlineIs an antifreeze protein (HhAFP) sequence (86-166),dashed underlineRepresenting a His tag sequence.

In a preferred embodiment of the present invention, the recombinant expression vector contains the sequence of SEQ ID No. 5.

SEQ ID No. 5：

atgagatttccttcaatttttactgctgttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagaatgcgtgttgcagaagtggcaaaagtgctgtgggtgggtgtgtgtgcagtagcagcagtgaccctggtggtgggggtgcaggaggtggtgaaggtggtgtgggtcgtctgggtttggggagttcaggcagttgttcttgttgttgccgtggtggccaccgtggttgtctgctttctggttgttcatgttgttcgtgtcgtgacagtttgtggccgcgtggctgaagttgcaggtgttgccggtcatcatcatcatcatcattgatag。

A yeast engineering strain expressing antifreeze protein contains the nucleotide sequence of said antifreeze protein HhAFP.

Preferably, the yeast engineering bacteria, the yeast gene also contains a gene fragment encoding a secretion signal peptide Kex 2 and a signal peptide cleavage site.

The host bacteria of the yeast engineering bacteria are pichia pastoris, preferably pichia pastoris X-33.

The method for constructing the yeast engineering bacteria comprises the following steps:

(1) Amplifying the antifreeze protein gene as a template to obtain a protein with 5' endXhoI,3' end hasXbaI enzyme cutting site and target fragment of 6 XHis tag coding gene;

(2) The target fragment is connected to an expression vector to construct a recombinant expression vector;

(3) And (3) linearizing the recombinant expression vector, transferring the linearized recombinant expression vector into host saccharomycetes, and screening positive transformants to obtain recombinant saccharomycetes.

In the step (1), the amino acid sequence gene shown in the SEQ No.1 is used as a template for amplification. Preferably, in step (1), the amplification is performed using the primers shown in SEQ ID No.3 and SEQ ID No. 4.

SEQ ID No.3 （HhAFP-F）： ctcgagaaaagaatgcgtgttgcagaagtg

SEQ ID No.4（HhAFP-R）：tctagactatcaatgatgatgatgatgatgaccggcaacacctgcaacttc

The target fragment obtained by amplification has the length of 285bp and is provided with XhoI, xbaI restriction enzyme sites and a 6 XHis tag.

The expression vector in the step (2) is pPICZ alpha A plasmid. Addition of HhAFP Gene at 5' endXhoI cleavage site, adding at 3' endXbaI cleavage site and 6 XHis tag. By usingXhoI、XbaThe enzyme I carries out double enzyme digestion and connection on the target fragment and pPICZ alpha A plasmid to construct a recombinant expression vector pPICZ alpha A-HhAFP。

In step (3), usingSacThe enzyme I linearizes the recombinant expression vector and inserts it into the yeast DNA genome.

In the step (3), competent yeast host is mixed with the linearized recombinant expression vector and subjected to electric excitation treatment. Specifically, electric shock is performed after ice bath; electric shock parameters: the voltage is 1000-1500V, the capacitance is 20-30 mu F, and the resistance is 150-250 omega. Preferably, the voltage is 12000V, the capacitance is 25 muF, and the resistance is 200Ω. Sorbitol solution was added after the shock.

In step (3), positive transformants were screened with bleomycin Zeocin and methanol.

In a preferred embodiment of the invention, the sequence of SEQ ID No.5 is inserted into a yeast host.

The yeast engineering bacteria or the fermentation product thereof can be used for preparing antifreeze protein.

A method of preparing an antifreeze protein comprising the steps of: fermenting the yeast engineering bacteria in BMM culture medium containing methanol, and culturing at 27-29 deg.C for 68-75 hr.

Further, the yeast engineering bacteria are fermented in BMM culture medium containing 1.4-1.6% of methanol in volume concentration, and are cultured for 68-75h at 27-29 ℃, and methanol is added into the culture medium according to the volume ratio of 1.4-1.6% every 18-30 h. Culturing at 200-500rpm and pH=5.5-6.5.

In a preferred embodiment of the present invention, the culture is performed in BMM medium containing 1.5% (v/v) methanol at 28-29℃for 72 hours, and methanol is added to the medium every 24 hours at a volume ratio of 1.5%; and, culturing under conditions of 240-300rpm and ph=6.0.

The antifreeze protein or the fermentation product of the yeast engineering bacteria can be used for preparing cell cryopreservation liquid, frozen preservative or frozen food.

The fermentation supernatant after fermentation of the yeast engineering bacteria can be directly used for preparing cell cryopreservation liquid, frozen preservative or frozen food, or preparing cell cryopreservation liquid, frozen preservative or frozen food after purifying to obtain antifreeze protein.

The invention has the beneficial effects that a new antifreeze protein is obtained by artificially synthesizing an antifreeze protein gene sequence and expressing the antifreeze protein gene sequence. The protein can obviously improve survival rate of resuscitated cells after freezing and preserving, and reduce cell damage.

And (3) transferring the antifreeze protein gene into yeast by constructing an antifreeze protein recombinant expression vector. The strain can express recombinant antifreeze protein with high activity. The obtained recombinant protein is added into the cell cryopreservation liquid instead of DMSO, and the survival rate of the cryopreserved cells after resuscitating is detected, so that the resuscitating survival rate of the cryopreserved cells added with the recombinant protein HhAFP is higher than that of the conventional cryopreserved cells added with DMSO.

And the yield of the recombinant antifreeze protein is greatly improved by optimizing the expression condition, so that the content of the recombinant antifreeze protein in the fermentation broth of the yeast engineering bacteria reaches 527mg/L. And the recombinant antifreeze protein has high proportion and less impurity protein and other impurities.

The invention can obtain the antifreeze protein with high activity and high yield, and provides a good foundation for the application of the antifreeze protein in various aspects; the invention expresses the active antifreeze protein with high yield by pichia pastoris, and provides a technical approach for realizing industrialization of the antifreeze protein.

Drawings

FIG. 1 shows the recombinant expression vector pPICZ alpha A-HhAFPThe construction (A), the PCR (B) and the double enzyme digestion (C) are identified, wherein M: DNA molecular weight standard; 1: a PCR product; 2: empty carrier double enzyme cutting products; 3: recombinant expression vector double enzyme cutting products;

FIG. 2 shows the results of PCR identification of yeast DNA, wherein M:2000bpDNA molecular weight standard; 1-2: yeast DNA;

FIG. 3 is a SDS-PAGE analysis of yeast supernatants wherein M:180kDa protein molecular weight standard; 1: supernatant of culture of pPICZ alpha A-containing transformant; 2: containing pPICZ alpha A-HhAFPSupernatant of transformant culture;

FIG. 4 shows the result of Western blot analysis of yeast supernatants, wherein M: a 45kDa ultra-low protein molecular weight standard; 1-2: supernatant of culture of pPICZ alpha A-containing transformant; 3-4: containing pPICZ alpha A-HhAFPSupernatant of transformant culture;

FIG. 5 shows the result of SDS-PAGE analysis of purified proteins, wherein M: a 45kDa ultra-low protein molecular weight standard; and (3) FT: loading a flowing liquid; W1-W2: washing 1-2; E1-E6: eluting 1-6;

FIG. 6 shows the result of Western blot analysis of purified proteins, wherein M: a 45kDa ultra-low protein molecular weight standard; 1: purifying the protein HhAFP;

FIG. 7 shows 293T cell live cell statistics (5 replicates per data set);

FIG. 8 shows the morphology under 293T cell inverted fluorescence microscopy (A-C: hoechst33342, PI, hoechst33342/PI staining results of cells frozen with DMEM-containing culture solution; D-F: hoechst33342, PI, hoechst33342/PI staining results of cells frozen with pPICZ αA-containing yeast culture; G-I: hoechst33342, PI, hoechst33342/PI staining results of cells frozen with HhAFP-containing yeast culture; bars=100 um);

FIG. 9 is SDS-PAGE analysis of methanol concentration effect on growth of yeast transformants (A) and culture supernatants thereof (B), wherein M: a 45kDa ultra-low protein molecular weight standard;

FIG. 10 is SDS-PAGE analysis of induction time effect on yeast transformant growth (A) and culture supernatant thereof (B), wherein M: a 45kDa ultra-low protein molecular weight standard;

FIG. 11 is SDS-PAGE analysis of the effect of induction temperature on growth of yeast transformants (A) and culture supernatants thereof (B) wherein M:45kDa ultra low protein molecular weight standard.

Detailed Description

Principal materials and reagents

243bp ofHhAFPThe sequence is composed of Optimus synthesizing by a limited company; PCRPrimers were synthesized by Shanghai Bioengineering Co.

pMD19-T cloning vector and restriction endonucleaseXhoI、XbaI、SacI) DNA polymerase, DNA ligase, DNA molecular weight standards (markers) were purchased from TaKaRa. Trans1-T1 competent cells were purchased from Beijing full gold Biotechnology Co. DNA plasmid miniprep kit, DNA agarose gel recovery kit, yeast genome extraction kit were purchased from Yu Tiangen Biochemical technologies (Beijing) Co. His tag protein purification kit and enhanced CCK-8 reagent were purchased from Shanghai Biyun biotechnology Co. The pPICZ alpha A expression vector and the X-33 yeast expression strain were purchased from Yu Fenghui organisms. Bleomycin was purchased from Invitrogen. Rainbow pre-dye low molecular weight protein markers were purchased from Zhongke Ruitai Biotech Co. anti-His tag murine monoclonal antibodies were purchased from kang century biotechnology limited. DMEM high sugar medium, penicillin-streptomycin solution, DPBS buffer were purchased from Hyclone company. 0.25% pancreatin, foetal calf serum was purchased from Gibco company and DMSO was purchased from Thermo Fisher company. Hoechst33342/PI apoptosis staining kit and amino acid-free yeast nitrogen source are purchased from Beijing Soy Bao technology Co.

EXAMPLE 1 HhAFP target Gene cloning and addition of restriction enzyme sites and his tag

243bp antifreeze proteinHhAFPThe gene sequence (SEQ ID No. 2) was synthesized by the Optimus of Praeparata. Using the synthesized HhAFP sequence as a template, the addition was usedXhoI、XbaThe I cleavage site and the 6 XHis-tagged primers HhAFP-F and HhAFP-R (i.e., SEQ ID No.3, 4, see Table 1) amplify the band of interest.

TABLE 1 primer design

Primer name	Primer sequence (5)'-3')	Product Length/bp
			HhAFP-F	ctcgagaaaagaatgcgtgttgcagaagtg	30
HhAFP-R	tctagactatcaatgatgatgatgatgatgaccggcaacacctgcaacttc	51

SEQ ID No.2:

The PCR reaction system is as follows: ddH ₂ O80. Mu.L, syntheticHhAFPSequence 4. Mu.L as template, hhAFP-F (10. Mu. Mol/L) 8. Mu.L, hhAFP-R (10. Mu. Mol/L) 8. Mu.L, R-TaqDNA polymerase (0.05U/. Mu.L) 100. Mu.L, 200. Mu.L total. The PCR reaction program was set as follows: 94. pre-denatured at 3min at 94℃for 30s, annealed at 57℃for 30s, extended at 72℃for 1min, and after 34 cycles, final extension at 72℃for 10min. The PCR amplified product was purified, and the purified fragment was ligated to pMD19-T vector using DNA ligase to form pMD19-HhAFP plasmid, followed by transformation of E.coli competent cells.

And selecting positive monoclonal to carry out bacterial liquid PCR. The PCR reaction system is as follows: ddH ₂ O5.8. Mu.L, bacterial solution 0.5. Mu.L, upstream and downstream primers (10. Mu. Mol/L) 0.6. Mu.L each, r-TaqDNA polymerase (0.05U/. Mu.L) 7.5. Mu.L, total 15. Mu.L, annealing temperature 55℃and PCR procedure. The bacterial liquid was then sent to Shanghai bioengineering Co.Ltd for sequencing.

As a result of the sequencing, the size of the target band HhAFP was 285bp, and the target band HhAFP was provided with XhoI, xbaI cleavage sites and a 6 XHis tag.

EXAMPLE 2 recombinant expression vector pPICZ alpha A-HhAFPTransformation screen of the X-33 Strain

2.1 recombinant expression vector pPICZ alpha A-HhAFPConstruction of (3)

In example 1, hhAFP-F and HhAFP-R primers (SEQ ID Nos. 3, 4) were used for synthesisHhAFPAmplifying the sequence as a template, and addingXhoI、XbaI restriction enzyme site and 6 XHis tag to obtain a 273bp target band HhAFP. Connecting the target fragment HhAFP with a pichia pastoris expression vector pPICZ alpha A to construct a recombinant expression vector pPICZ alpha A-HhAFP(A in FIG. 1).

By restriction enzymesXhoI andXbai pair post-construction pMD19-T-HhAFPThe vector and empty vector ppiczαa were double digested. After enzyme digestion, the target fragment HhAFP is connected and transformed with the pichia pastoris expression vector fragment pPICZ alpha A to construct a recombinant expression vector pPICZ alpha A-HhAFP(A in FIG. 1).

The target gene is added with a 6 XHis tag, so that the detection and purification of the subsequent protein are facilitated. The 5' AOX1 promoter exists upstream of the pPICZ alpha A vector, and alpha-factor is a secretion signal peptide to carry out secretion and transportation of the expressed protein to the outside of cells. Upstream of the 5' AOX1 promoterSacI enzyme cutting site, can linearize the circular plasmid, facilitate insertion into yeast DNA genome. The recombinant expression vector has the gene for resisting Zeocin, and bleomycin Zeocin can be used for screening positive yeast transformants in subsequent experiments.

In the presence of pPICZαA-HhAFPSingle colonies were picked on E.coli screening plates and subjected to colony PCR. The selected colonies were PCR-amplified using the universal primers 5'AOX1 and 3' AOX1 of the pPICZ alpha A vector to give a 781bp band of interest. As shown in FIG. 1B, lane 1 has a distinct band around 781 bp. For recombinant expression vector pPICZalpha A-HhAFPProceeding withXhoI、XbaI double cleavage, a 273bp band (FIG. 1C) was obtained, the size of which was consistent with the theoretical value. Sequencing results confirmed pPICZ alpha A-HhAFPThe recombinant expression vector was constructed successfully.

Picking positive monoclonal to carry out double enzymesThe bacterial liquid is subjected to PCR detection, a PCR reaction system and a PCR reaction program are the same (annealing temperature is 55 ℃), and then linearized enzyme is usedSacI recombinant expression vector pPICZαA-HhAFPAnd (5) linearizing. The PCR detection result shows that the primer contains the following sequence shown as SEQ ID No. 5:

double underlineRepresenting secretion signal peptide alpha-factor sequences (1-249),wave lineRepresents Kex 2 signal peptide cleavage sites (250-255),single underlineRepresents an antifreeze protein (HhAFP) sequence (256-498),dashed underlineRepresenting His tag sequences (499-516),dot underscoreRepresenting a strong termination signal (517-522).

The protein expressed by the sequence is shown in SEQ ID No. 6:

wherein the method comprises the steps ofDouble underlineTo exocrine the signal peptide alpha-factor (1-83),wave lineIs Kex 2 signal peptide cleavage site (84-85),single underlineIs an antifreeze protein (HhAFP) sequence (86-166),dashed underlineRepresenting His tag sequences (167-172).

Screening of high-copy positive yeast transformant and recombinant protein induced expression

Recombinant expression vector pPICZαA-HhAFPAnd the empty vector pPICZ alpha A is electrically transformed into Pichia pastoris X-33 strain, and 2 HhAFP transformants and 1 pPICZ alpha A transformant are obtained after screening by Zeocin and methanol utilization rate.

The method comprises the following steps: yeast competent cells and linearized pPICZαA-HhAFPMix at 8:1 (V: V, competent cells 80. Mu.L, linearized plasmid 10. Mu.L, concentration 12.5 ng/. Mu.L), transfer into a 20mm electric beaker, shock after 5min on ice (parameters: voltage 2000V, capacitance 25. Mu.F, resistance 200. OMEGA.). Immediately after the shock was completed, 1mL of 1M sorbitol solution was added and transferred to a pre-chilled 50mL centrifuge tubeThe mixture was allowed to stand at 29℃for 2.2 h, followed by addition of 1mL of YPD medium, and culturing at 29℃for 2 hours at 200 rpm/min. The supernatant was centrifuged off, and yeast cells were collected and plated on YPDS plates containing 100. Mu.g/mL Zeocin and incubated in an incubator at 29℃until monoclonal production. And (3) selecting monoclonal and sequentially inoculating the monoclonal to MM and MD plates, comparing the growth conditions of yeasts on the MM and MD plates, and screening high-copy yeast transformants using methanol rapidly.

The yeast DNA extraction kit is used for extracting the yeast genome DNA as a template, and the 5'AOX1 and 3' AOX1 of the universal primers of the pPICZ alpha A carrier are used as primers for carrying out PCR detection on the yeast DNA, wherein the PCR reaction system and the PCR reaction program are the same as those described above (the annealing temperature is 55 ℃). In addition, the empty vector ppiczαa was electrotransformed into pichia pastoris as a negative control.

The PCR detection results are shown in FIG. 2, which shows that the HhAFP transformant has two distinct bands around 2000bp and 781 bp. Because of the existence of the primer binding site of AOX1 in the Pichia pastoris X-33 strain, a band of about 2000bp is amplified, and the 781bp is a target band containing HhAFP (FIG. 2).

Empty vector of pPICZαA and pPICZαA-HhAFPExpression was induced in BMM medium, and after obtaining yeast supernatant, the supernatant was filtered and concentrated, and SDS-PAGE analysis was performed, and the results are shown in FIG. 3. The supernatant of the culture of strain 2 had a distinct band around 15 kDa, whereas the supernatant of the culture of strain 1 (control pPICZαA) had no band.

Western blot analysis further verifies that the results, as shown in FIG. 4, lanes 1 and 2 (control pPICZ. Alpha.A) have no band, lanes 3 and 4 have a distinct band around 16 kDa, and it is presumed that the band is a recombinant antifreeze protein HhAFP with a 6 XHis tag.

EXAMPLE 3 Western blot and MALDI-TOF/TOF analysis of recombinant protein-induced expression and purification products

Positive yeast transformants were selected and cultured to OD at 29℃in 25. Mu.L BMG medium at 250rpm/min and pH 6.0 ₆₀₀ 1.5. The culture broth was centrifuged to collect the cells, inoculated into 100mL of BMM medium, and cultured at 29℃at 250rpm/min at pH 6.0 for 72 hours, and 1mL of methanol was added every 24 hours to give a volume concentration of 1%. Will be culturedThe product was collected by centrifugation and the supernatant was filtered through a 0.22 μm filter. The supernatant was then purified using His-tag protein purification kit: placing PVDF membrane in an antibody incubation box, adding a sealing solution (5% skimmed milk powder), placing the PVDF membrane in a shaking table, incubating for 2.5 hours at room temperature, adding a primary antibody (3% skimmed milk powder is used for diluting the anti-His tag mouse monoclonal antibody, the dilution ratio is 1:2500), standing overnight at 4 ℃, placing the PVDF membrane in the shaking table at room temperature, incubating for 1 h, and washing the membrane 3 times with TBST buffer solution for 5 minutes each time. Secondary antibody (3% skimmed milk powder diluted horseradish peroxidase labeled goat anti-mouse IgG, dilution ratio 1:2500) was added, and after incubation for 1.5h at room temperature, the membrane was washed 3 times with TBST buffer. Development was performed using BeyoECL Moon developer.

After SDS-PAGE analysis of the purified product, protein bands on the gel were transferred to PVDF membrane for Western blot analysis. The results are shown in FIGS. 5 and 6, respectively, with a distinct protein band around 16 kDa and no other bands. Meanwhile, the Western blot analysis result proves that the protein contains the target protein HhAFP with a 6 XHis tag. This purified product was thus confirmed to be the target protein HhAFP.

The purified product was subjected to MALDI-TOF/TOF analysis: development was performed using BeyoECL Moon developer. The protein bands on SDS-PAGE gel were sent to Shanghai, miao, new Biotechnology Co., ltd for MALDI-TOF/TOF analysis. The results showed that the sequence was identical to the theoretical sequence, SEQ ID No.1 with His tag at the C-terminal.

SEQ ID No.1:

EXAMPLE 4 detection of the anti-freezing effect of recombinant proteins in cell cryopreservation

293T cells were taken at 37℃with 5% CO ₂ After the incubator was cultured until the degree of fusion became 90%, the medium was discarded, and the cells were washed with DPBS. Cells were digested with 0.25% pancreatin, counted and grouped, 3 tubes were repeated for each group, and 5×10 frozen for each tube ⁵ The cells were then added to 1mL of the prepared frozen stock solution.

The cell grouping is as follows: (1) control group: the frozen stock solution consists of 90% DMEM culture solution and 10% DMSO; (2) negative control group: the frozen stock solution consists of 90% of yeast culture supernatant of pPICZ alpha A and 10% of DMSO; (3) HhAFP group: the frozen stock solution consisted of the concentrated HhAFP yeast culture supernatant containing 3mg/mL (about 0.2 mM) of HhAFP.

The cells were placed in a cryopreservation box and stored frozen at-80℃for 24h. After taking out the freezing box, the freezing tube is quickly thawed at 37 ℃. After removing the frozen solution by centrifugation, the 293T cells are resuscitated and subjected to adherent culture for 4 hours: adding 1mL of culture solution, uniformly mixing, adding 100 mu L of culture solution into a 96-well plate, and repeating 3 holes; 37. culturing at the temperature of 4 hours.

Cell viability was detected by CCK-8 reagent: adding CCK-8 reagent, culturing at 37deg.C for 2 hr, and detecting the absorption light OD at 450nm wavelength by enzyme labeling instrument ₄₅₀ . Then by the formula: hhAFP group cell viability (%) = [ a (HhAFP) -a (Blank)]/[A(DMEM)-A(Blank)]x100 calculation of cell viability with HhAFP added; cell viability (%) = [ a (ppiczαa) -a (Blank) of ppiczαa group]/[A(DMEM)-A(Blank)]x100 calculation of cell viability with addition of ppiczαa product; DMEM group cell viability (%) = [ a (DMEM) -a (Blank)]/[A(DMEM)-A(Blank)]x100 calculation of cell viability with DMEM medium added; wherein Blank refers to the absorbance of a well with CCK8 solution without cells. The data from each set of 5 replicates was assayed and 293T cell live cell statistics are shown in FIG. 7. As can be seen from FIG. 7, the HhAFP group had a larger number of living cells than the DMEM group and the pPICZαA group, and the difference was significant (P < 0.05).

Hoechst33342 is a nuclear dye that can penetrate cell membranes into cells to make nuclei exhibit blue fluorescence, while apoptotic cells exhibit strong blue fluorescence. PI can make necrotic cells appear red fluorescence, so that normal living cells appear weak blue fluorescence, apoptotic cells appear strong blue fluorescence, and necrotic cells appear strong red fluorescence and blue fluorescence under a fluorescence microscope. 200 μl was added to a 24-well plate and 3 wells were repeated. 37. After 4h incubation at C, the cells were observed under a normal microscope and photographed using a Zeiss microscope. The culture solution was aspirated, DPBS was added and washed once, and Hoechst33342/PI apoptosis staining reagent was added. 4. Dyeing at the temperature of 20-30min, sucking off the dyeing liquid, cleaning with DPBS, and observing and photographing under an inverted fluorescence microscope. After the experiment was repeated 3 times, statistical data were collected and single factor analysis of variance was performed using Graphpad Prism8 software.

The adherent cells were stained with Hoechst33342/PI, and the results are shown in FIG. 8 (A-C: the results of staining with Hoechst33342, PI, hoechst33342/PI of cells frozen with DMEM-containing culture solution; D-F: the results of staining with Hoechst33342, PI, hoechst33342/PI of cells frozen with pPICZ. Alpha. A-containing yeast culture; G-I: the results of staining with Hoechst33342, PI, hoechst33342/PI of cells frozen with HhAAP-containing yeast culture; bars=100 um).

From fig. 8, the HhAFP group exhibited significantly fewer cells than DMEM group and ppiczαa group, indicating that the HhAFP group had fewer necrotic cells than DMEM group and ppiczαa group.

The detection of CCK-8 reagent and Hoechst33342/PI double-staining detection prove that the cell survival rate of 293T cells frozen in the frozen stock solution containing antifreeze protein HhAFP is higher after resuscitation and has obvious difference with the control group and the negative group. From this, it was demonstrated that HhAFP has a protective effect in 293T cell cryopreservation, avoiding damage to cells by ice crystals, and reducing cell death.

Example 5 optimized expression of recombinant proteins under different conditions

Inoculating yeast transformant into BMG culture medium, culturing at 28deg.C and 250rpm under pH 6.0 to OD ₆₀₀ After 1.5, the cells were collected by centrifugation.

The cells were inoculated into 50mL of BMM medium containing 0.5%, 1%, 1.5% and 2% methanol at different volume concentrations, respectively, and the inoculum size was 100. Mu.L of the bacterial liquid, and the cells were cultured at 28℃and 250rpm at pH 6.0 for 72 hours, and the supernatants were collected every 24 hours and supplemented with methanol according to the corresponding volume ratios. On the other hand, induction expression was performed by setting an induction temperature of 10℃and an induction time of 72 hours, and the supernatant was collected.

Protein concentration was determined using BCA method and SDS-PAGE analysis was performed to determine optimal expression and yield of recombinant protein HhAFP.

As shown in FIG. 9, the amount of protein expressed in the expression medium having a methanol concentration of 0.5%, 1.0%, 1.5% and 2.0% was different, and the target protein band was the deepest (72 hours of cultivation) at a methanol concentration of 1.5%, at which time OD was the same as that of the protein band ₆₀₀ 2.22, total proteinThe concentration was 1.34mg/mL. OD after increasing methanol concentration ₆₀₀ The total protein concentration and the target protein band were not significantly changed, but the large molecular weight of the hybrid protein band was deepened, so that the methanol concentration was determined to be 1.5%.

After 0h, 24h, 48h and 72h of expression culture, it was found that the protein bands were deepest when cultured for 72h, as shown in FIG. 10, at which time OD ₆₀₀ 2.23 and total protein concentration of 1.55mg/mL.

As shown in FIG. 11, the protein bands were darker at 28℃when the methanol concentration was 1.5% and the culture temperatures were 10℃and 28℃indicating that the protein expression was higher at 28℃than at 10℃when the total protein concentration was 1.29mg/mL and the expression level of recombinant protein HhAFP was 527mg/L.

By combining the results, when the expression culture temperature is 28 ℃, and the methanol concentration in the expression culture medium is 1.5%, after 72 hours of expression culture, the target protein obtained in the supernatant is more, the impurity protein is less, and the expression quantity of the recombinant protein HhAFP is 527mg/L through protein concentration measurement.

Claims

1. An antifreeze protein is characterized in that the amino acid sequence is shown as SEQ ID No.1, or the C end of the sequence shown as SEQ ID No.1 is provided with a His tag, or the N end of the sequence shown as SEQ ID No.1 is provided with 1 or 2 signal peptide cleavage site amino acids.

2. An antifreeze protein gene encoding an antifreeze protein of claim 1.

3. Antifreeze protein gene according to claim 2, characterized in that it comprises the nucleotide sequence shown in SEQ ID No. 2.

4. A recombinant expression vector comprising a gene encoding the antifreeze protein of claim 1.

5. A yeast engineering strain for expressing antifreeze protein, characterized in that the yeast gene contains a nucleotide sequence for encoding the antifreeze protein of claim 1.

6. The yeast engineering bacterium according to claim 5, wherein the yeast gene further comprises a gene fragment encoding a secretion signal peptide and Kex 2 signal peptide cleavage site.

7. A method of preparing an antifreeze protein comprising the steps of:

fermenting the yeast engineering bacteria of claim 5 or 6 in BMM culture medium containing methanol, and culturing at 27-29 ℃ for 68-75h.

8. Use of an antifreeze protein gene according to claim 2 or 3, a yeast engineering bacterium according to claim 5 or 6 or a fermentation product thereof for preparing an antifreeze protein.

9. Use of an antifreeze protein according to claim 1, an antifreeze protein gene according to claim 2 or 3, a yeast engineering bacterium according to claim 5 or 6 or a fermentation product thereof for the preparation of a cell cryopreservation solution, a frozen preservative or a frozen food.