CN115029330A - Expression and purification method of wild type IMPDH II protein - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to an expression and purification method of wild type IMPDH II protein. The invention discloses an expression and purification method of wild type IMPDH II protein, comprising the following steps: s1, cloning or synthesizing to obtain a recombinant nucleic acid fragment containing a wild type IMPDH II sequence; s2, constructing a recombinant vector, and transforming the recombinant vector to a host cell to obtain a genetic engineering bacterium; s3, fermentation culture: after the gene engineering bacteria are activated, carrying out amplification culture, and then carrying out fermentation culture by adopting a self-induction culture medium; s4, purifying IMPDH II protein. The method can efficiently express the human wild type IMPDH II protein with 8x His label and TEV enzyme cutting site, can obtain more than 350mg of protein with the purity of more than 90 percent per liter of self-induction culture medium, has the specific activity of about 2, is placed in a storage Buffer, and can be stored for 14 days at the temperature of 4 ℃ to keep the activity unchanged.

Description

Expression and purification method of wild type IMPDH II protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an expression and purification method of wild type IMPDH II protein.

Background

Inosine-5 '-monophosphate dehydrogenase (inosine 5' -monophosphophosphate dehydrogenase, IMPDH, ec1.2.1.14) is the rate-limiting enzyme in guanine biosynthesis. The enzyme is mainly related to cell proliferation and the like, and the inhibition of the enzyme activity can inhibit the proliferation of tumor cells, so the enzyme is an important antitumor drug target. The IMPDH with the expression activity can be applied to high-throughput screening of inhibition, and is beneficial to screening of antitumor drugs and research of action mechanisms of the drugs.

There are two main subtypes in humans: IMPDH I and IMPDH II, wherein IMPDH II type is specifically distributed in lymphocytes, and inhibition of its activity prevents lymphocyte proliferation; therefore, the inhibitor can be applied to the regulation of body immunity; the use of inhibitors thereof includes rejection control after organ transplantation. The expressed active IMPDH II protein can be applied to the screening of immunosuppressants, and is beneficial to the screening of rejection control drugs and the research of action mechanisms of the drugs.

The nucleic acid sequence and protein sequence of wild-type IMPDH II have been reported in public [ Natsumeda, Y., et al, 1990J BioZ. chem.265: 5292-5295; collar, F.R.and Hubermann, E.1988J.biol.chem.263: 15769-15772; and U.S. Pat. No.5,665,583(SEQ ID NO:63), expression of wild-type IMPDH II is largely dependent on prokaryotic and eukaryotic expression systems. In the expression of active wild type IMPDH II, the expression engineering strains mainly used by a prokaryotic expression system comprise: IMPDH-deficient Escherichia coli H712 and conventional Escherichia coli; the patent EP 1559779A 1 discloses that IMPDH II and its variant can be expressed by IMPDH enzyme deficient Escherichia coli H712, and natural IMPDH II and its variant can be obtained by expression, but the method is applied to IMPDH deficient expression strain E.coli H712; the conventional escherichia coli expression wild type IMPDH II exists in an inclusion body form especially when the wild type IMPDH II is provided with a His tag, so that the process is complex, the protein activity is low, most of the currently marketed prokaryotic expression IMPDH II is the inclusion body, can only be used for SDS-PAGE experiments, and has no biological activity. The eukaryotic expression system is mainly used for HeLa stable transformant cell expression, and the relative specific activity of the commercially available eukaryotic expression IMPDH II is also lower and the price is more expensive.

The ability to mass-produce and purify wild-type IMPDH II is also important for clinical and research applications, such as identification and design of new IMPDH II inhibitors, sensitivity for cancer and immunosuppressive therapy and detection of IMPDH II and inhibitors, and thus a method for obtaining native IMPDH II protein with high expression level, easy purification, high activity, strong stability, and low cost is urgently needed for popularization and application.

Disclosure of Invention

The invention aims to provide a method for obtaining natural IMPDH II protein, which has high expression quantity, easy purification, high activity, strong stability and low cost.

The invention discloses an expression and purification method of wild type IMPDH II protein, comprising the following steps:

s1, cloning or synthesizing to obtain a recombinant nucleic acid fragment containing a wild type IMPDH II sequence;

s2, constructing a recombinant vector, and transforming the recombinant vector to a host cell to obtain a genetic engineering bacterium;

s3, fermentation culture: after the gene engineering bacteria are activated, carrying out amplification culture, and then carrying out fermentation culture by adopting a self-induction culture medium;

s4, purifying IMPDH II protein;

the self-induction medium in step S3 consists of the following components:

alpha-lactose: 2 g/L; glucose: 0.5 g/L; glycerol: 5 g/L; KH (Perkin Elmer) ₂ P0 ₄ ：3.4g/L；

MgSO ₄ : 0.49 g/L; peptone: 10 g/L; na (Na) ₂ HPO ₄ 3.55g/L；Na ₂ SO ₄ ：0.71g/L；

NH ₄ Cl:2.67g/L；pH 7.4。

In the existing recombinant protein Escherichia coli fermentation expression, an inducer, generally ITPG, is required to be added to be capable of inducing the expression of the recombinant protein in the genetic engineering bacteria; however, the addition of the inducer ITPG is not only costly, but also causes inactive expression of the recombinant protein, thereby affecting the activity of the recombinant protein. The invention adopts a low-temperature self-induction method, has low cost, solves the problem that inclusion bodies are easy to form in IMPDH II recombinant expression in the prior art, does not cause the inactive expression of IMPDH II, and can obviously improve the activity of IMPDH II protein.

In a preferred embodiment, the recombinant nucleic acid fragment contains a His tag located at the N-terminus of the IMPDH II nucleic acid sequence to facilitate the use of nickel affinity chromatography in later purification;

in a preferred embodiment, the recombinant nucleic acid fragment comprises a nucleic acid sequence of a TEV enzyme cleavage site intermediate a His tag sequence and an IMPDH II nucleic acid sequence to facilitate cleavage of the His tag by the TEV enzyme after purification of the recombinant protein;

in a preferred embodiment, the nucleic acid sequence of the recombinant nucleic acid fragment is as set forth in SEQ ID NO. 1.

In a preferred embodiment, the recombinant vector is pET-21a (+), pET-24a (+), pET-28a (+), pET-30a (+) or pET-Duet;

in a preferred embodiment, the host cell is BL21(DE3) or arcttcexpress (DE3) pRare2 BL21, preferably arcttcexpress (DE3) pRare2 BL 21;

in a preferred embodiment, the self-induced culture conditions are a temperature of 10-20 deg.C, a pH of 7-8

In a preferred embodiment, the self-induced culture condition is that the culture is firstly cultured at 37 ℃ to the OD of the bacterial liquid ₆₀₀ 3.0-4.0, then continuously culturing at 12 ℃ until the bacterial liquid OD ₆₀₀ Is 14-19.

In a preferred embodiment, in the step S4, the protein purification step:

a. weighing thallus

b. The wet cells were added to 50ml of Buffer A in an amount of 1g to suspend the cells and the cells were disrupted by using a homogenizer

c. Protein purification Using AKTA Pure protein purification Instrument and His trap HP Pre-column

d. Concentration of proteins by dialysis and ultrafiltration

The solutions used were as follows:

lysis solution: 50mM KH ₂ PO ₄ 300mM KCl,20mM imidazole, 0.8M urea, pH 8.0;

eluent: 50mM KH ₂ PO ₄ 300mM KCl,500mM imidazole, 0.8M urea, pH 8.0;

dialyzate: 50mM KH ₂ PO ₄ 300mM KCl,1mM DTT,0.8M urea, 1mM EDTA,1mM PMSF, pH 8.0;

the method of the invention can efficiently express the human wild type IMPDH II protein with 8x His label and TEV enzyme cutting site by adopting a low-temperature self-induction mode, can obtain more than 350mg of protein with the purity of more than 90% per liter of self-induction culture medium, has the specific activity of about 2, is placed in a storage Buffer, and can be stored for 14 days in the environment of 4 ℃ to keep the activity unchanged.

Drawings

FIG. 1 shows the structure of pET28a-IMPDH II recombinant plasmid.

FIG. 2 is the identification of low temperature self-induced protein expression.

In the figure, a strip 1 is sampled before low-temperature induction, a strip 2 is sampled after low-temperature induction, a strip 3 is sampled after Buffer A heavy suspension, a strip 4 is sampled after a homogenizer is broken, a strip 5 is sampled after the homogenizer is broken by centrifugation supernatant, a strip 6 is re-centrifuged and precipitated heavy suspension after the homogenizer is broken, a strip 7 is identified by re-centrifuged supernatant after the homogenizer is broken by centrifugation and precipitated heavy suspension, and the low-temperature self-induced culture condition can efficiently and soluble express the wild type IMPDH II protein with 8 XHis and TEV enzyme cutting sites

FIG. 3 is an electrophoretogram showing the purification results of low temperature self-induced products of IMPDH II protein.

In the figure, bands 1, 2, 3, 4, 5,6, 7 and 8 are the flow-through peaks of the loading and washing impurities in the protein purification process, bands 9 and 10 are the elution peaks of 500mM imidazole, bands 11 and 12 are 2ug of the loading after concentration, and bands 13 and 14 are 5ug of the loading after concentration.

Detailed Description

The invention relates to the same substance, namely IMPDH II and IMPDH2, the nucleic acid sequence and the protein sequence of which are disclosed and reported in Natsumeda, Y., et al, 1990J BioZ Chem.265: 5292-5295; collar, F.R.and Hubermann, E.1988J.biol.chem.263: 15769-15772; and U.S. Pat. No.5,665,583(SEQ ID NO:63)

1. Recombinant nucleic acid molecules and host vector systems

The invention provides nucleic acid molecules, such as DNA molecules (rDNA) encoding wild-type IMPDH II. As described herein, an rDNA molecule is a DNA molecule that undergoes molecular manipulations in vitro: methods for producing rDNA molecules are well known in the art, e.g., see Sambrook et al, molecular cloning (1989).

The nucleic acid molecules of the invention may be recombinant molecules, each comprising a wild-type IMPDH II nucleotide sequence, e.g., the wild-type IMPDH II nucleotide sequence may be ligated to an expression vector to produce a recombinant expression vector;

the term "Expression vectors" (Expression vectors) or recombinant Expression vectors refers to vectors that have added Expression elements (e.g., promoters, RBS, terminators, etc.) to the basic backbone of the cloning vector to enable the Expression of a gene of interest, including but not limited to viral and non-viral vectors (plasmids, cosmids). The preferred expression vector also includes an expression control element, such as a promoter sequence, which can initiate transcription of the inserted IMPDH nucleotide sequence and can be used to regulate expression (transcription and/or translation) of the IMPDH sequence in a suitable host cell, such as E.coli. Prokaryotic expression control elements, but are not limited to inducible promoters, constitutive promoters, secretion signals, enhancers, transcription terminators and other transcriptional regulatory elements, as well as other expression control elements involved in translation.

Preferred expression vectors also include at least one selectable marker gene that encodes a gene product that is resistant, such as ampicillin, tetracycline or kanamycin. Typically, the vector also includes a plurality of endonuclease restriction sites, which facilitate insertion of the foreign DNA sequence.

Preferred expression vectors are those compatible with prokaryotic host cells.

As is well known in the art, prokaryotic expression vectors are available from a variety of commercial sources, and are typically used to express foreign genes in E.coli, such as pET-21a (+), pET-24a (+), pET-28a (+), pET-30a (+) or pET-Duet expression vectors;

a fusion gene is another example of a recombinant nucleic acid molecule comprising a fused wild-type IMPDH II nucleotide sequence, e.g., the wild-type IMPDH II nucleotide sequence may be fused to a His tag to facilitate the expression of an isolated and/or purified tag sequence of IMPDH II (Marshak, D.R., et al, 1996in: variants for Protein Purification and characterisation pp 396)

The invention also provides host cells containing the nucleic acid molecules disclosed herein. The present invention provides a host-vector system comprising a suitable host cell into which the vector, plasmid, phage or cosmi has been introduced, which host cell comprises a nucleotide sequence encoding IMPDH II.

The host cell may be a prokaryotic cell or a eukaryotic cell. For example, many commercially available strains of E.coli are particularly useful for the expression of foreign proteins. Examples of suitable eukaryotic host cells include yeast cells, plant cells, insect cells or animal cells, such as mammalian cells. One preferred embodiment provides a host vector system comprising a recombinant pET28+ vector comprising the nucleotide sequence IMPDH2 introduced into an E.coli BL21(DE3) host cell;

the recombinant DNA molecules of the invention may be introduced into an appropriate cellular host by known methods which generally depend on the type of vector used and the host system used. For example, electroporation and salt treatment methods are commonly used to transform prokaryotic host cells, see, e.g., Cohen et al, Proc Acad Sci USA (1972)69: 2110; and Maniatis et al, (1989) in molecular cloning, A laboratory Manual, Cold spring harbor laboratory, Cold spring harbor, N.Y..

The vector of choice may be an expression vector for the expression and production of IMPDH II in a bacterial host cell. For example, vectors that directly express high levels of fusion proteins that are easy to purify; specific initiation signals may also be required to effectively translate the modified IMPDH II sequence. These signals include the ATG initiation codon and adjacent sequences

In some preferred embodiments, the IMPDH II sequence comprising the initiation codon and upstream sequences is inserted into an appropriate expression vector without the need for additional translational control signals. However, if only the coding sequence or part of the coding sequence or portion is inserted, exogenous transcriptional control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcription elements and initiation codons can have a variety of origins, both natural and synthetic. By including an enhancer suitable for use in a cell system, the expression efficiency can be improved

For long-term, high-yield recombinant protein production, stable expression is the first choice. For example, a cell line stably expressing IMPDH II can be transformed using an expression vector comprising a viral replication source or endogenous expression element and a selectable marker gene. After introduction of the vector, the cells can be grown in rich medium for 1-2 days and then switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, the presence of which enables successful growth and recovery of the cell;

any selection system may be used to restore transformed Cell lines, including but not limited to herpes simplex virus thymidine kinase (Wigler, M.et., 1977Cell 11:223-32) and adenine phospholipid protein transferase (Lowy, I.et., 1980, Cell 22:817-23) genes for TK-Minus or APRT-Minus cells, respectively. Similarly, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection. For example, DHFR that confers resistance to methotrexate (Wigler, M.et al, 1980Proc Natl Acad Sci 77: 3567-70); NPT confers resistance to the aminoglycoside neomycin and G-418 (Colbere-Garapin, F.et. al, 1981J Mol Biol 150:1-14) and ALS or PAT, which confer resistance to chlorosulfonation and phosphoprotein acetyltransferase, respectively

Additional selection genes are also contemplated, such as trpB, which allows the cell to use indole instead of tryptophan, or hisD, which allows the cell to use histidine instead of histidine (Hartman, S.C. and r.c. Mullingan 1988 Proc Natl Acad Sci 85: 8047-51). Recently, the use of visible markers has gained popularity, such as anthocyanins, B-glucuronidase and its substrates, GUS and luciferase and its substrates, luciferin, and the like, are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression due to a particular vector system (Rhodes, C.A., et al, 1995Methods Mol Biol 55: 121-.

2. IMPDH II protein preparation

The wild-type IMPDH2 of the present invention can be produced by recombinant methods or by chemical synthetic methods.

Recombinant methods are preferred if high yields are desired. In general, the production of a recombinant modified IMPDH polypeptide will involve the use of a host/vector system, which typically includes the following steps.

First, a nucleic acid molecule encoding a wild-type IMPDH2 is obtained, said IMPDH2 sequence, as disclosed in the polynucleotide sequence of SEQ ID No: 2.

The nucleic acid molecule encoding IMPDH2 is then preferably inserted into an expression vector operably linked to suitable expression control sequences, as described above, to produce a recombinant expression vector comprising the sequences via IMPDH 2.

The expression vector is then introduced into a suitable host by standard transformation procedures, and the resulting transformed host is cultured under conditions which permit the production of wild-type IMPDH2 protein in vivo. For example, if expression of the IMPDH2 gene is under the control of an inducible promoter, suitable growth conditions will include a suitable inducer. The recombinant vector may integrate the IMPDH2 sequence into the host genome. Alternatively, the recombinant vector may maintain the IMPDH2 sequence extrachromosomally as part of an autonomously replicating vector. The IMPDH2 protein thus produced is isolated from the growth medium or directly from the cells;

one skilled in the art can readily adapt a suitable host/expression system known in the art for use with the wild-type IMPDH2 coding sequence to produce wild-type IMPDH2(Cohen et al, 1972Proc. Acad. Sci. USA 69: 2110; and Maniatis et al, 1989Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)).

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1 construction of recombinant expression vector for human wild-type IMPDH II

1.1 Experimental materials

1.2 Gene design Synthesis

Designing a gene sequence of 8 XHis tag and TEV protease cutting site on the upstream of IMPDH II gene, designing Nco I cutting site at the start codon sequence, designing Xho I cutting site at the stop codon site, connecting the synthesized gene to pUC57 plasmid vector, wherein the gene sequence is shown as SEQ ID NO. 1: a synthetic recombinant gene whose expression product comprises three parts (as shown in FIG. 1): 8 × His tag, TEV enzyme cleavage site and IMPDH II. The synthetic recombinant gene seq1 was inserted between Nco I and Xho I restriction enzymes of plasmid pET28a (+).

1.3 restriction ligation

a. And (3) receiving the synthesized plasmid vector containing the IMPDH II gene, performing enzyme digestion by using restriction enzymes Nco I and Xho I to obtain a gene sequence containing the wild-type IMPDH II gene, performing enzyme digestion on pET28a + with the same restriction enzyme, and recovering a gene fragment and a double-enzyme digestion linearized plasmid by using a gel recovery kit.

b. The gene fragment of interest was ligated into the pET28a + plasmid vector using T4 ligase.

c. The ligation products were transformed into E.coil Top10 competent cells by heat shock at 42 ℃, plated on solid LB plates containing kanamycin antibiotics, and cultured overnight at 37 ℃

d. The single clone was picked up in 3ml of liquid LB medium containing kanamycin antibiotic, cultured overnight at 37 ℃ and 220rpm, the plasmid was extracted using a plasmid extraction kit, and the plasmid was sequenced by the sequencer.

e. Correctly sequenced pET28a (+) -IMPDH II plasmids were transformed into Arcticexpress (DE3) pRare2 BL21 competent cells by heat shock at 42 ℃, plated on solid LB plates containing kanamycin antibiotics, and cultured overnight at 37 ℃.

f. The single clone was picked up in 3ml of liquid LB medium containing kanamycin antibiotic, cultured overnight at 37 ℃ and 220rpm, and the strain was preserved.

Example 2 optimization of expression and purification conditions

2.1 materials of the experiment

2.2 Experimental part

2.2.1 expression

a. Taking out strain at-20 deg.C at 16:00 afternoon, and then placing in LB ^G+C+K+ Streaking on plates and culturing at 37C overnight. Antibiotic concentrations were as follows:

table one: information on the concentration of antibiotics required

Name of antibiotic	Stock solution concentration (mg/ml)	Working concentration (ug/ml)
			Gentamycin	40	40
Chloramphenicol	34	34
			Kanamycin	50	50

b. The next day at about 10 am, from LB ^G+C+K+ Selecting 3 single clones on a plate, respectively inoculating the single clones into 32 ml TB self-induction culture media, culturing at 37 ℃ and 220RPM for 8 hours, taking out 10ul of reserved samples in a super clean bench, marking as S0-1, S0-2 and S0-3, then adding 4 xSDS-PAGE loading Buffer according to the proportion of 1:1, boiling for 3min, and storing at 4 DEG C

c. The rest culture solution is put back into the incubator at 37 ℃ for continuous culture

d. At 10 o' clock on the third day, 10ul of overnight culture medium was taken out and recorded as S1-1, S1-2, and S1-3, and then 4 XSDS-PAGE loading Buffer was added at a ratio of 1:1 and boiled for 3 min.

SDS-PAGE, loading 15ul per well. The samples were loaded from left to right in the following order: marker, S0-1, S0-2, S0-3, S1-1, S1-2 and S1-3.

f. And (5) after electrophoresis is finished. The electrophoresis results were analyzed to see which sample had IMPDH II expression, and then the tube of the culture medium with the higher expression level was added to 1L of the auto-induction medium (5L of the shake flask with a spoiler), and the antibiotic concentration in the medium was as shown in Table II. The final OD600 was 0.04. Then, the antifoaming agent was added at a dose of 1L medium plus 4ml of 5% antifoaming agent, TABLE II: information on the concentration of antibiotics required

AntibioticName (R)	Stock solution concentration (mg/ml)	Working concentration (ug/ml)
			Gentamycin	40	40
Chloramphenicol	34	34
			Kanamycin	50	100

g. The flask was incubated at 37 ℃ on a shaker at 120 rpm. The temperature may be different at different positions, which may affect the growth rate of the cells.

h. OD600 was measured after culturing for 8 to 10 hours in general. When OD600 is 3.0-4.0, the flask is cooled to 12 ℃ with ice water, while the temperature of the shaker is adjusted to 12 ℃.

The culture was continued at 120rpm for 7 days at 12 ℃ and OD600 would be between 14 and 19.

Centrifuging at 4 ℃ for 10min at j.8000rpm to collect the bacteria, and weighing the weight of the bacteria

2.2.2 expression characterization

The resulting cells were harvested by adding 5g of wet cells to 40ml of buffer A and suspending the cells in 2.2.1, followed by disruption of the harvested cells using a homogenizer and centrifugation. Separating the centrifugal supernatant and the centrifugal precipitate, adding equal volume of Buffer A into the centrifugal precipitate again for re-suspension, and centrifuging after re-suspension. Then, SDS-PAGE was performed to identify the results as shown in FIG. 2

2.2.3 protein purification

a. Weighing thallus

Cells frozen at-40 ℃ were transferred to 4 ℃ and the bacteria were allowed to thaw overnight before the first day of work before purification. A500 ml empty beaker was weighed, and then the thawed mycelia were transferred to a centrifuge tube, and about 5g of wet mycelia were weighed on a balance.

b. Bacterial heavy suspension

The oscillator power was maximized by adding 5g of cell to 250ml of lysate per 1g of cell, and the cell was resuspended by shaking to ensure that no clumps were present and that the resuspension solution was a uniform milky suspension when opened.

c. Cell cracking of homogenizer

a. Firstly, opening a circulating condensation system, and preparing to break bacteria after the temperature is reduced to 4 ℃.

b. The homogenizer is set up as: crushing at 1000Pa and 5 deg.C for 4 times.

c. The bacterial lysate was centrifuged at 12000rpm for 60min at 4 ℃.

d. Separating the centrifuged supernatant and centrifuging the precipitate

d. Protein purification

The AKTA Pure protein purification instrument is opened, and an IMPDH2 Ni column purification method is set according to the following information.

The method comprises the steps of firstly cleaning a system by using deionized water, respectively putting an S1 pipeline of a sample Pump, an A1 pipeline of Pump A and a B1 pipeline of Pump B into Buffer A, Buffer A and Buffer B, and cleaning the sample Pump, the Pump A and the Pump B by using corresponding buffers according to the flow rate of 5ml/min, wherein each Pump is cleaned by at least 20ml volume. His trap HP pre-packed column was attached to column-site-2, taking care not to introduce air bubbles. The Hitrap HP column was then equilibrated with Pump A, line A1, at a flow rate of 2ml/min, for a volume of 20 ml.

a. Solution pipeline setting:

s1: sample to be purified

S2：Buffer A

Buff deionized water

A1：Buffer A

A2: deionized water

B1：Buffer B

B2: 20% ethanol

b. Pos2 as column position

c. The flow rate of the system is 2ml/min, the flow rate of the sample loading is 2ml/min, and the flow-through liquid is collected by a 45 ml/tube.

d. Flow-through collection at sample loading, 45 ml/tube, 60mM wash, 15 ml/tube, 150mM wash, 15 ml/tube, 300mM wash, 2 ml/tube, 500mM wash, 2 ml/tube, set up with the following program:

detecting the collected fractions by SDS-PAGE;

f. ultrafiltering and concentrating the protein containing target protein and no foreign protein, and dialyzing the concentrated protein at 4 deg.C for 3 times, each time for 4 hr;

SDS-PAGE identifies the protein purification results, the results are shown in FIG. 3;

lysis buffer (buffer a): 50mM KH ₂ PO ₄ ,300mM KCl,20mM imidazole,0.8M Urea,pH8.0；

Eluent (buffer B): 50mM KH ₂ PO ₄ ,300mM KCl,500mM imidazole,0.8M Urea,pH8.0；

Dialyzate: 50mM KH ₂ PO ₄ ,300mM KCl,1mM DTT,0.8M Urea,1mM EDTA,1mM PMSF,pH8.0。

Example 3IMPDH II Activity assay

When IMPDH II protein was purified, the OD280 was used to calculate the concentration of purified protein (extinction coefficient (. epsilon.mg/ml-0.466)) and the Δ OD340 was measured in real time per unit mass of protein (3.2ug protein) in Hitachi Biochemical apparatus per unit reaction time (3.8 min).

IMPDH II catalyzes the oxidation of creatinine monophosphate to XMP, while reducing NAD to NADH. Thus, the catalytic activity of IMPDH II can be calculated by measuring the light absorption of NADH at 340 nm. The catalytic activity of IMPDH II can be calculated by measuring the amount of NADH formed by IMPDH II catalyzing IMP in unit time and unit mass on Hitachi 7180 biochemical instrument.

Specific activity (pmol/min/ug) ═ Δ OD ₃₄₀ *Vol*10 ¹² /(6220*M*T)

Where 6220 is the molar extinction coefficient of NADH, Vol is the reaction volume in L, and the volume on a biochemical instrument is 200ul, i.e., 0.2X 10 ^-3 L; m is the mass of IMPDH II added, in ug, usually 3.2 ug; t is the reaction time in minutes, usually 3.8 minutes. Since Δ OD340 measured by hitachi 7180 biochemical analyzer is a value multiplied by 10000, the above formula can be transformed as:

Specific Activity(pmol/min/ug)＝(ΔOD ₃₄₀ x 0.2x10 ^-3 x10 ¹² )/(6220x3.2x3.8)

＝2640xΔOD ₃₄₀

the activities of purified IMPDH II were as follows:

serial number	Protein name	Date of purification	Viability assay date	Purification volume (ml)	Purification concentration (mg/ml)	Purity (SDS-PAGE)	Specific activity
								1	IMPDHII-WT	20200923	20200924	5	11.23	95％	1.67
2	IMPDH II-WT	20210813	20210814	0.7	7.19	95.95％	1.999
								3	IMPDH II-WT	20211127	20211128	4.72	3.51	95.00％	1.969
4	IMPDH II-WT	20220119	20220120	10.7	2.9	96.91％	1.932

Example 4 stability assay for IMPDH II

Purified IMPDH II was stored in a refrigerator at 4 ℃ and sampled every 1, 3, 5, 7, 14 days to test the activity according to the method of example 3, and the data are as follows:

and (4) conclusion: the activity of the purified IMPDH II is almost not reduced in an environment of 4 ℃.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

<110> Shanghai Jian Plough pharmaceutical science and technology GmbH

Shanghai Yunze Biotechnology Co., Ltd

<120> expression and purification method of wild type IMPDH II protein

<141> 2022-06-06

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acggactcac agcacagcag ctcttcaact gcggagacgg cctcacctac aatgactttc 180

tcattctccc tgggtacatc gacttcactg cagaccaggt ggacctgact tctgctctga 240

ccaagaaaat cactcttaag accccactgg tttcctctcc tatggacaca gtcacagagg 300

ctgggatggc catagcaatg gcgcttacag gcggtattgg cttcatccac cacaactgta 360

cacctgaatt ccaggccaat gaagttcgga aagtgaagaa atatgaacag ggattcatca 420

cagaccctgt ggtcctcagc cccaaggatc gcgtgcggga tgtttttgag gccaaggccc 480

ggcatggttt ctgcggtatc ccaatcacag acacaggccg gatggggagc cgcttggtgg 540

gcatcatctc ctccagggac attgattttc tcaaagagga ggaacatgac tgtttcttgg 600

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aggcaaatga aattctgcag cgcagcaaga agggaaagtt gcccattgta aatgaagatg 720

atgagcttgt ggccatcatt gcccggacag acctgaagaa gaatcgggac tacccactag 780

cctccaaaga tgccaagaaa cagctgctgt gtggggcagc cattggcact catgaggatg 840

acaagtatag gctggacttg ctcgcccagg ctggtgtgga tgtagtggtt ttggactctt 900

cccagggaaa ttccatcttc cagatcaata tgatcaagta catcaaagac aaatacccta 960

atctccaagt cattggaggc aatgtggtca ctgctgccca ggccaagaac ctcattgatg 1020

caggtgtgga tgccctgcgg gtgggcatgg gaagtggctc catctgcatt acgcaggaag 1080

tgctggcctg tgggcggccc caagcaacag cagtgtacaa ggtgtcagag tatgcacggc 1140

gctttggtgt tccggtcatt gctgatggag gaatccaaaa tgtgggtcat attgcgaaag 1200

ccttggccct tggggcctcc acagtcatga tgggctctct cctggctgcc accactgagg 1260

cccctggtga atacttcttt tccgatggga tccggctaaa gaaatatcgc ggtatgggtt 1320

ctctcgatgc tatggacaag cacctcagca gccagaacag atatttcagt gaagctgaca 1380

aaatcaaagt ggcccaggga gtgtctggtg ctgtgcagga caaagggtca atccacaaat 1440

ttgtccctta cctgattgct ggcatccaac actcatgcca ggacattggt gccaagagct 1500

tgacccaagt ccgagccatg atgtactctg gggagcttaa gtttgagaag agaacgtcct 1560

cagcccaggt ggaaggtggc gtccatagcc tccattcgta tgagaagcgg cttttctaac 1620

tcga 1624

Claims (10)

1. A method for purifying the expression of wild-type IMPDH II protein, which comprises the following steps:

s1, cloning or synthesizing to obtain a recombinant nucleic acid fragment containing a wild type IMPDH II sequence;

s2, constructing a recombinant vector, and transforming the recombinant vector to a host cell to obtain a genetic engineering bacterium;

s3, fermentation culture: after the gene engineering bacteria are activated, carrying out amplification culture, and then carrying out fermentation culture by adopting a self-induction culture medium;

s4, purifying IMPDH II protein;

characterized in that the self-induction medium in step S3 consists of the following components:

alpha-lactose: 2 g/L; glucose: 0.5 g/L; glycerol: 5 g/L; KH (Perkin Elmer) ₂ P0 ₄ ：3.4g/L；

MgSO ₄ : 0.49 g/L; peptone: 10 g/L; na (Na) ₂ HPO ₄ 3.55g/L；Na ₂ SO ₄ ：0.71g/L；

NH ₄ Cl:2.67g/L；pH 7.4。

2. The expression purification method of claim 1, wherein the recombinant nucleic acid fragment comprises a His tag located N-terminal to the IMPDH II nucleic acid sequence.

3. The method of claim 1, wherein said recombinant nucleic acid fragment comprises a nucleic acid sequence of a TEV enzyme cleavage site intermediate a His tag sequence and an IMPDH II nucleic acid sequence.

4. The method for expression purification according to claim 1, wherein the recombinant vector is pET-21a (+), pET-24a (+), pET-28a (+), pET-30a (+) or pET-Duet.

5. The method for purifying expression according to claim 4, wherein the recombinant vector is pET-28a (+).

6. The expression purification method according to claim 1, wherein the host cell is BL21(DE3) or Arcticexpress (DE3) pRare2 BL 21.

7. The expression purification method according to claim 1, wherein the host cell is ArcticExpress (DE3) pRare2 BL 21.

8. The method for purifying expression of claim 1, wherein the self-induced culture conditions are a temperature of 10-20 ℃ and a pH of 7-8.

9. The method for expression purification according to claim 1, wherein the self-induced culture conditions are culture at 37 ℃ to OD of bacterial solution ₆₀₀ 3.0-4.0, then continuously culturing at 12 ℃ until the bacterial liquid OD ₆₀₀ Is 14-19.

10. The expression purification method according to claim 1, wherein in the step S4, the protein purification step:

a. weighing thallus

b. The wet cells were added to 50ml of Buffer A in an amount of 1g to resuspend the cells and disrupt the cells using a homogenizer

c. Protein purification Using AKTA Pure protein purification Instrument and His trap HP Pre-column

d. Concentration of proteins by dialysis and ultrafiltration

The solutions used were as follows:

lysis solution: 50mM KH ₂ PO ₄ 300mM KCl,20mM imidazole, 0.8M urea, pH 8.0;

eluent: 50mM KH ₂ PO ₄ 300mM KCl,500mM imidazole, 0.8M urea, pH 8.0;

dialyzate: 50mM KH ₂ PO ₄ 300mM KCl,1mM DTT,0.8M urea, 1mM EDTA,1mM PMSF, pH 8.0.

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