CN113462713A - Method for improving expression level of glucagon-like peptide hexa-linked peptide in pichia pastoris - Google Patents
Method for improving expression level of glucagon-like peptide hexa-linked peptide in pichia pastoris Download PDFInfo
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Abstract
The invention discloses a method for improving the expression level of glucagon-like peptide hexa-linked peptide in pichia pastoris. The invention analyzes the difference genes of the low-level expression strain and the high-level expression strain of the pichia pastoris based on transcriptome sequencing, and screens out a new gene GESE with obvious difference of transcription level. According to the invention, the expression level of 6 XmGLP-1 in the pichia pastoris strain is obviously improved after the GESE gene in a pichia pastoris host is knocked out by using a pichia pastoris CRISPR/Cas9 gene knockout technology. Therefore, the invention provides a method for improving the expression level of glucagon-like peptide hexagons in pichia pastoris, which comprises knocking out or mutating a GESE gene in a host pichia pastoris strain. The invention further provides a glucagon-like peptide hexa-linked yeast expression vector and a GESE gene editing vector. The invention provides a basic research and a new way for improving the expression level of the foreign protein gene in pichia pastoris.
Description
Technical Field
The invention relates to a recombinant expression method of glucagon-like peptide hexa-linked, in particular to a method for improving the expression level or expression quantity of glucagon-like peptide hexa-linked in pichia pastoris, belonging to the field of recombinant expression of glucagon-like peptide hexa-linked.
Background
Diabetes is a metabolic disease characterized by hyperglycemia, and is caused by carbohydrate metabolism dysfunction caused by relative lack of insulin, and the intestinal hormone Glucagon-like peptide-1 (GLP-1) can promote insulin secretion from islet beta cells to reduce blood sugar level due to the dependence on glucose concentration, so that the diabetes is not widely concerned, but the half-life period in blood is only a few minutes, so that the medical application of GLP-1 is limited. The glucagon-like peptide hexa-linked (6 XmGLP-1) is prepared by respectively replacing amino acid residues of DPP-IV and Trypsin sensitive sites in natural GLP-1 molecules on the basis of the existing research (His)7-Ala8Mutated to His7-Ser8/Gly8,Lys26,34Mutation to Gln26，Asn34/Asp34) Cys is added at the C terminal, 6 tandem repeats are formed by end-to-end connection, His-tag is added at the C terminal, codon optimization is carried out, and the optimized sequence is transformed into pichia pastoris to be secreted and expressed in the pichia pastoris, but the expression level of the optimized sequence in the pichia pastoris is low, so that the production and purification costs of the optimized sequence are too high, and the optimized sequence cannot meet the industrial productionAnd (4) demand.
Pichia pastoris is the most widely used yeast strain in the industrial production of recombinant protein at present, compared with other expression systems, the Pichia pastoris expression system has the advantages of high-density fermentation, simple and quick operation, and simultaneously has characteristics which are not possessed by prokaryotes as eukaryotes, such as post-translational processing and modification of protein, including disulfide bond formation, glycosylation, lipoylation and the like. For some exogenous recombinant proteins with cytotoxicity, the proteins can be positioned in peroxisomes of pichia pastoris, so that the pichia pastoris is not damaged by toxicity, the exogenous proteins can be prevented from being degraded by protease, and for the recombinant proteins secreted to the outside of cells, the secretory proteins of the pichia pastoris are fewer, so that the operation can be simplified in the later stage of separation and purification of the exogenous proteins. Is extremely suitable for industrial large-scale fermentation production. However, as the wide application of pichia pastoris expression systems shows, not all exogenous protein genes can be efficiently expressed in pichia pastoris, and the traditional methods such as strong promoter, codon optimization, copy number increase, gene fusion expression, fermentation condition optimization and the like have very limited or even no effect on improving the expression level of most exogenous proteins in pichia pastoris.
Because the 6 xmGLP-1 has low expression level in pichia pastoris, the production and purification cost is overhigh, the defect seriously limits the wide application of the 6 xmGLP-1 in medicine, and needs to be improved.
Disclosure of Invention
One of the objects of the present invention is to provide a method for increasing the expression level of glucagon-like peptide hexa-linker (6 × mGLP-1) in Pichia pastoris;
the other purpose of the invention is to provide a glucagon-like peptide hexa-conjoined gene yeast expression vector;
it is a further object of the present invention to provide a GESE gene editing vector.
The fourth purpose of the invention is to apply the glucagon-like peptide hexagons gene yeast expression vector and the GESE gene editing vector to improve the expression level of glucagon-like peptide hexagons (6 XmGLP-1) in pichia pastoris.
The above object of the present invention is achieved by the following technical solutions:
the invention provides a method for improving the expression level of glucagon-like peptide hexa-linked (6 XmGLP-1) in pichia pastoris, which comprises the following steps: constructing a glucagon-like peptide hexa-linked yeast expression vector; transforming the constructed glucagon-like peptide hexa-linked yeast expression vector into a host pichia pastoris strain to induce the glucagon-like peptide hexa-linked yeast to carry out recombinant expression in the host pichia pastoris strain; wherein, when the glucagon-like peptide hexa-linked peptide is induced to carry out recombinant expression in the host pichia pastoris strain, the GESE gene in the host pichia pastoris strain is knocked out or mutated.
As a preferred specific embodiment, the invention artificially synthesizes and optimizes the DNA sequence of the 6 XmGLP-1 gene according to the yeast preference codon, and the nucleotide sequence is shown as SEQ ID No. 1; the nucleotide sequence of the GESE gene is shown as SEQ ID No. 1.
As a preferred embodiment of the invention, the host Pichia pastoris strain is preferably Pichia pastoris expression strain GS 115.
As a preferred embodiment of the present invention, the GESE gene in the host pichia pastoris strain is knocked out or mutated by using CRISPR/Cas9 gene editing technology, for example, a GESE gene editing vector is constructed for a target sequence of the GESE gene, the GESE gene editing vector is transformed into a host pichia pastoris expression strain containing a glucagon-like peptide hexon gene, and the GESE gene in the host pichia pastoris expression strain is knocked out; or constructing a GESE gene knockout carrier, and knocking out or mutating the GESE gene in the host pichia pastoris expression strain.
The invention provides a construction method of a glucagon-like peptide hexa-conjoined gene yeast expression vector as a reference embodiment, which comprises the following steps: glucagon-like peptide hexa-linker gene and alpha signal peptide sequenceEcoRI andNoti double-restriction enzyme linearized vector pGAP9K (or eukaryotic expression vector of other exogenous protein as starting plasmid) is subjected to homologous recombination under the action of homologous recombinaseAnd then converting the competence of escherichia coli, selecting a positive strain to obtain a glucagon-like peptide hexagons gene yeast expression vector, wherein the vector is named as pGAP9K-6 XmGLP-1, the structural map of the vector is shown in figure 1, the vector comprises a yeast constitutive promoter GAP, a nucleotide sequence which is positioned at the downstream of the promoter and used for coding an alpha-factor secretion signal peptide, and a gene which is used for coding the glucagon-like peptide hexagons, and the 5' end of the gene which is used for coding the glucagon-like peptide hexagons protein is provided with a Kex2 gene expression product cutting recognition sequence AAAAAAGA. pGAP9K expression vector containing the promoter followed byEcoRI andNotthe enzyme cutting site I can linearize the carrier, and respectively synthesize the artificially synthesized glucagon-like peptide hexagons gene and alpha signal peptide into primers, wherein the 5' end of the alpha signal peptide primer contains the alpha signal peptide primer and the carrierEcoR25 overlapped bases at the upstream of the I site, 25 bp bases overlapped with 6 XmGLP-1 contained in the primer at the 3 'end of the alpha signal peptide, and a carrier contained in the primer at the 3' end of the exogenous gene 6 XmGLP-1EcoR25 overlapping bases upstream of the I site.
As a preferred embodiment of the present invention, the following will be mentionedSalThe linearized pGAP9K-6 XmGLP-1 expression vector can be introduced into a Pichia pastoris genome through electric shock transformation.
The invention also provides a method for constructing a gene editing vector aiming at the target sequence of the GESE gene by adopting a CRISPR/Cas9 gene editing method, which comprises the following steps:
designing a target site sequence shown as SEQ ID No.3 according to the GESE gene sequence, designing a primer according to the target site sequence to carry out PCR amplification: wherein the sequence of the primer is shown as SEQ ID No.4-SEQ ID No. 11; and transforming the PCR amplification product and the enzyme-digested and linearized CRISPR/Cas9 plasmid into escherichia coli through homologous recombination, and selecting a positive strain to obtain a gene editing vector, which is named as a BB3cH-CRISPR vector.
As a preferred specific embodiment of the invention, a BB3cH-CRISPR vector can be subjected to electric shock transformation to be transformed into a pichia pastoris expression strain GS115 containing a glucagon-like peptide six-linked gene to obtain a glucagon-like peptide six-linked high expression strain with a GESE gene knockout function; wherein, the electric shock transformation condition is preferably 1500V, 200 omega, 25 muf; culturing in YPD solid medium (containing 100. mu.g/mL hygromycin B and 50. mu.g/mL Cb) at 30 deg.C for 3 days; amplifying a target gene in a single colony by using genome PCR, selecting a strain with a sequencing overlapping peak at a target sequence position through sequencing, selecting the strain, culturing the strain in a YPD liquid culture medium (50 mu g/ml Cb) at 30 ℃ and 200 rpm until the OD600 value is 1.5-2.0, inoculating a bacterial liquid into a new YPD liquid culture medium (containing 50 mu g/ml Cb) according to one thousandth of inoculation amount, and repeating for three times; streaking the finally obtained bacterial liquid on a YPD solid culture medium (containing 50 mu g/ml Cb) plate, and culturing at 30 ℃ for 3 d; selecting a single colony to perform genome PCR, sequencing PCR products, selecting strains with correct GESE gene sequence editing, culturing and storing to obtain the 6 XmGLP-1-GS 115-GESE-CRI strain with the edited genome.
The invention also provides a process for molecular biological identification of transgenic yeast colony PCR, which comprises the following steps: after obtaining a single colony of the transgenic yeast, selecting the thallus to 20 mu L of 0.02M NaOH at 100 ℃ for 5 minutes, taking 2 mu L of supernatant as a template to detect the editing condition of a target gene in a 20 mu L PCR reaction system, and detecting whether the expression level of the glucagon-like peptide hexa-linked protein in GS115 is improved or not by Western blot and ELISA.
The invention screens a new gene GESE with obvious transcription level difference based on the analysis of the difference gene of a low-level expression strain (6 XmGLP-1-GS 115) and a high-level expression strain (T4-GS 115) by transcriptome sequencing (RNA-Seq). Currently, there is no research report on gene GESE (PAS _ chr1-3_ 0003), and the gene has no information annotation on the gene in GO and NCBI databases. According to the invention, after the GESE gene in a pichia pastoris host is knocked out by using a pichia pastoris CRISPR/Cas9 gene knockout technology, the expression level of 6 xmGLP-1 in a pichia pastoris GS115 is found to be remarkably improved, the finding has important values for industrialization of the 6 xmGLP-1 and expansion of the industrial application range of the pichia pastoris, and a basic research and a new way are provided for improving the expression level of a foreign protein gene in the pichia pastoris.
Drawings
FIG. 1 plasmid map of yeast expression vector pGAP9K-6 XMGLP-1 containing 6 XMGLP-1, 6 XMGLP-1 gene constructs expression cassette with pGAP promoter.
FIG. 26 XmGLP-1-GS 115 genome editing used CRISPR/Cas9 vector BB3 cH-CRISPR.
Fig. 3 gene sequence alignment before and after CRISPR editing.
Fig. 4 post CRISPR editing sequencing peak plots.
FIG. 5 Western blot identifies 6 XmGLP-1 expression before and after editing of target gene, GLP: expressing the 6 XmGLP-1 gene in a pichia pastoris host, wherein the GESE gene in the pichia pastoris host is not knocked out or mutated; GESE-CRI: expression of 6 XmGLP-1 gene in a Pichia host, wherein the GESE gene in the Pichia host is knocked out by gene editing.
FIG. 6 ELISA detection target gene editing after 6 xmGLP-1 expression level change, CK: pGAP9K empty vector transformed GS115 strain as negative control; GLP: the expression level of the 6 XmGLP-1 gene in a pichia host, wherein the GESE gene in the pichia host is not knocked out or mutated; GESE-CRI: the expression level of the 6 XmGLP-1 gene in a pichia host, wherein the GESE gene in the pichia host is knocked out by gene editing; ELISA data were statistically analyzed and plotted using GraphPad, which is extremely significant for P < 0.01.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 construction of expression vector containing 6 XmGLP-1 foreign protein Gene
Expression vector (pGAP 9K) containing the promoter followed byEcoRI andNotthe enzyme cutting site I can linearize the carrier, and respectively synthesize artificially synthesized 6 XmGLP-1 and alpha signal peptide into primers, wherein the 5' end of the alpha signal peptide primer contains the alpha signal peptide and the carrierEcoRI siteUpstream 25 overlapped bases, the primer at the 3 'end of the alpha signal peptide contains 25 bp bases overlapped with 6 XmGLP-1, the primer at the 3' end of the exogenous gene 6 XmGLP-1 contains and a carrierEcoR25 overlapped basic groups at the upstream of the I site, amplifying exogenous gene 6 XmGLP-1 and alpha signal peptide sequence by high fidelity enzyme, and reacting with the recovered gelEcoRI andNoti, incubating the double-restriction enzyme linearized vector at 50 ℃ for 15 min, transforming escherichia coli DH5 alpha competence after homologous recombination under the action of a homologous recombinase, and selecting a positive strain to obtain a pGAP9K-6 xmGLP-1 vector, wherein figure 1 is a plasmid map of a yeast expression vector pGAP9K-6 xmGLP-1 containing 6 xmGLP-1.
Example 2 construction of vector BB3cH-CRISPR containing GESE target sequence CRISPR/Cas9
According to the sequence of a target gene GESE, a target site sequence shown in SEQ ID No.3 is designed in an E-CRISP Design network, and a primer is synthesized according to the target site sequence, wherein the primer sequence is shown in Table 1.
TABLE 1 primer sequences
And transforming the amplified product and a CRISPR/Cas9 plasmid subjected to BpiI enzyme digestion linearization into escherichia coli competent DH5 alpha through homologous recombination, plating in an LB solid culture medium (containing 50 mu g/mL of hygromycin B), performing inverted culture at 37 ℃, and selecting a positive strain through colony PCR. FIG. 2 is a map of the 6 XMGLP-1-GS 115 genome editing CRISPR/Cas9 vector BB3 cH-CRISPR.
Example 3 transformation of Yeast Strain GS115 by electroporation
(1) The vector pGAP9K-6 XmGLP-1 is linearized by digestion with SalI, added with 1/10V of 3M sodium acetate and 2.5V of absolute ethanol, mixed evenly, centrifuged at 12,000 rpm for 5 min, washed twice with 75% ethanol, dried and dissolved with 10. mu.L of ddH2O for later use.
(2) Single GS115 colonies were picked and cultured at 30 ℃ and 200 rpm in 5 mL of liquid YPD (containing 50. mu.g/mL Cb)600Centrifuging at 5000 rpm for 3 min to 1.4, collecting thallus, washing thallus twice with precooled sterile water, washing thallus once with precooled 1M sorbitol solution, and subjecting the thallus to 100 μ L precooling 1M sorbitol solutionResuspend the solution to obtain GS115 competence. Mixing 80 mu L of GS115 competence with 10 mu L of plasmid flick tube wall, adding into a 2 cm electric shock cup, shocking on ice for 10min at 1500V, 200 omega and 25 mu f, adding 300 mu L of precooled 1M sorbitol solution, mixing uniformly, sucking into a sterile 2.0mL EP tube, standing at 30 ℃ for 1h, adding 300 mu L of liquid YPD solution, culturing at 30 ℃ and 200 rpm for 1h, plating 200 mu L of each plate on YPD solid culture medium (containing 500 mu g/mLG418 and 50 mu g/mL Cb), and performing inversion culture at 30 ℃ until a single colony grows out.
(3) Selecting single colony thallus to 20 mu L of 0.02 mol/L NaOH for resuspension, incubating for 5 min at 100 ℃, taking 2 mu L of supernatant as a template to perform PCR identification in a 20 mu L PCR system, performing gel electrophoresis, and selecting pGAP9K-6 XmGLP-1-GS 115 positive strain.
Example 4 CRISP vector BB3cH-CRISPR electric shock method for transforming pGAP9K-6 XmGLP-1-GS 115 strain and screening
(1) And inoculating BB3cH-CRISPR coliform strain in 10 mL of LB liquid culture medium (containing 50. mu.g/mL hygromycin B), culturing overnight, and extracting plasmid for later use.
(2) And preparing the strain pGAP9K-6 XmGLP-1-GS 115 competence (the preparation method is the same as the GS115 competence preparation).
(3) Taking 10 mu L (about 1-2 mu g, no linearization is needed) of BB3cH-CRISPR plasmid, adding 80 mu L of pGAP9K-6 XmGLP-1-GS 115 competence, flicking the tube wall uniformly, adding the mixture into a 2 cm electric shock cup, carrying out ice shock for 10min, carrying out electric shock (the electric shock condition is the same as that of GS115 transformation), carrying out subsequent operation on the same plate as that of GS115 transformation, coating the plate on 100 mu L of each plate in YPD solid culture medium (containing 100 mu g/mL hygromycin B), and culturing at 30 ℃ until a single colony grows up.
Example 5 detection of CRISPR/Cas9 Gene editing Strain
(1) And picking single colony thallus by the toothpick, resuspending the single colony thallus in 20 mu L of 0.02 mol/L NaOH solution, carrying out PCR detection in a 20 mu L PCR system by taking 2 mu L of supernatant as a template and using primers synthesized at about 100 bp positions upstream and downstream of a target gene target sequence, and sequencing a PCR product.
(2) Comparing the sequencing result with the target gene sequence, selecting a sample with overlapped peaks and base change at the target sequence, and transferring the single colony to YPD liquidThe culture medium (containing 50. mu.g/mL Cb) was cultured at 30 ℃ and 200 rpm without a screening pressure until A600The value is 1.5-2.0, one in a thousand is inoculated into a new YPD liquid culture medium (containing 50 mu g/mL Cb) and cultured until A600When the value is 1.5-2.0, inoculating one thousandth of YPD liquid culture medium (containing 50 mu g/mL Cb) again, and culturing until A600The value is 1.5-2.0, and the bacterial liquid is streaked on a YPD solid culture medium (containing 50 mu g/mL Cb) plate and cultured at 30 ℃ until a single colony is grown.
(3) Colony PCR, PCR product sequencing, and single colony with correctly edited target gene target sequence is amplified and maintained. In this case, the yeast genome may be extracted, subjected to genome PCR, and further sequenced to detect whether the target gene is correctly edited.
FIG. 5 shows the results of ELISA detection of the change in expression level of 6 XmGLP-1 before and after target gene editing, and it can be seen from the test results that the expression level of 6 XmGLP-1 gene in Pichia pastoris host is significantly improved after the gene editing knockout of GESE gene in Pichia pastoris host.
Sequence listing
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<400> 10
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaag 59
<210> 11
<211> 58
<212> DNA
<213> Artifical sequence
<400> 11
atattatcag atgtacacta aaagcagtcc aaagctgtcc cattcgccat gccgaagc 58
Claims (10)
1. A method for increasing the expression level of glucagon-like peptide hexa-linker in Pichia pastoris, comprising: constructing a glucagon-like peptide hexa-conjoined gene yeast expression vector; transforming the constructed glucagon-like peptide hexagons gene yeast expression vector into a host pichia pastoris strain, and inducing the glucagon-like peptide hexagons to carry out recombinant expression in the host pichia pastoris strain; the method is characterized in that when the glucagon-like peptide hexa-linked peptide is induced to carry out recombinant expression in a host pichia pastoris strain, a GESE gene in the host pichia pastoris strain is knocked out or mutated.
2. The method according to claim 1, wherein the nucleotide sequence of the glucagon-like peptide hexon gene is shown as SEQ ID No. 1; the nucleotide sequence of the GESE gene is shown as SEQ ID No. 2.
3. The method according to claim 1, wherein the host pichia strain is pichia pastoris expression strain GS 115.
4. The method of claim 1, wherein the step of knocking out or mutating the gene for GESE in the host Pichia strain comprises: constructing a GESE gene editing vector aiming at a target sequence of a GESE gene by adopting a CRISPR/Cas9 gene editing method, transforming the GESE gene editing vector into a host pichia pastoris expression strain containing a glucagon-like peptide hexon gene, and knocking out the GESE gene in the host pichia pastoris expression strain; or constructing a GESE gene knockout carrier, and knocking out or mutating the GESE gene in the host pichia pastoris expression strain.
5. A glucagon-like peptide hexa-conjoined gene yeast expression vector is characterized in that the construction method comprises the following steps: the glucagon-like peptide hexa-conjoined gene, the alpha signal peptide sequence and a linearization vector pGAP9K are transformed into escherichia coli competence after homologous recombination under the action of homologous recombinase, and a positive strain is selected to obtain the glucagon-like peptide hexa-conjoined gene yeast expression vector.
6. The glucagon-like peptide hexon gene yeast expression vector of claim 5, wherein the nucleotide sequence of the glucagon-like peptide hexon gene is shown in SEQ ID No. 1.
7. A GESE gene editing vector is characterized in that the construction method comprises the following steps: designing a target site sequence shown as SEQ ID No.3 according to the GESE gene sequence, designing a primer according to the target site sequence to carry out PCR amplification: and transforming the PCR amplification product and the enzyme-digested and linearized CRISPR/Cas9 plasmid into escherichia coli through homologous recombination, and selecting a positive strain to obtain a GESE gene editing vector.
8. The GESE gene editing vector of claim 7, wherein the nucleotide sequence of the GESE gene is represented by SEQ ID No. 2; the nucleotide sequence of the primer is shown as SEQ ID No.4-SEQ ID No. 11.
9. The glucagon-like peptide hexagons gene yeast expression vector of claim 5 and the use of the GESE gene editing vector of claim 7 for increasing the expression level of glucagon-like peptide hexagons in Pichia pastoris.
10. Use according to claim 9, characterized in that it comprises: glucagon-like peptide hexa-conjoined gene yeast expression vectorSalI, after linearization, the glucagon-like peptide is introduced into a host pichia pastoris genome through electric shock transformation to obtain a glucagon-like peptide six-connection pichia pastoris expression strain; the GESE gene editing vector is transferred into glucagon-like peptide six-linked pichia pastoris through electric shock transformationObtaining a glucagon-like peptide hexa-linked high-expression pichia pastoris strain with the GESE gene knocked out from the expression strain; inducing the glucagon peptide hexa-conjoined gene to be recombined and expressed in host pichia pastoris.
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