CN1990859B - Method for Fusion Expression of Nuclear Receptor Ligand Binding Domain Protein on Phage Surface - Google Patents

Abstract

The invention discloses a phage, which is composed of a capsid and a phage nucleic acid located in the capsid. The surface of the phage capsid is fused with an exogenous nuclear receptor or a ligand binding region of the nuclear receptor. The phage of the present invention can be used for screening substances that bind to nuclear receptors or ligand binding regions of nuclear receptors, has the characteristics of high screening sensitivity and high screening efficiency, and can be screened with only a very low protein expression level.

Description

Method for Fusion Expression of Nuclear Receptor Ligand Binding Domain Protein on Phage Surface

technical field

The invention relates to the field of biotechnology, and relates to a phage whose capsid surface is fused with an exogenous nuclear receptor or nuclear receptor LBD protein and a method for expressing the fusion expression of the nuclear receptor or nuclear receptor LBD protein on the surface of the phage.

Background technique

Phage surface display technology has been widely used in the study of protein structure and function since its establishment. Foreign proteins, random peptides, cDNA or genome fragments are displayed on the surface of phage through fusion expression with phage coat gene to form protein library or polypeptide library. Theoretically, the biopanning process can be used to screen proteins or polypeptides that interact with target proteins, receptors, cell surface receptors, and small chemical molecules (drugs). This technology is mainly used in the following aspects: 1. Screen specific binding antibodies, or modify antibodies through binding screening technology to improve antibody affinity and binding specificity; 2. Study protein-protein interactions to determine receptors or ligand-binding proteins or polypeptides; 3. Determination of functional determinants or antibody determinants; 4. Directed evolution of enzymes or proteins, etc. However, the proteins expressed by phage display in the past are all soluble proteins, and foreign proteins that are difficult to express in soluble have not been involved. So far, there is no relevant report on the display and expression of poorly soluble proteins on the surface of phage capsids.

Nuclear receptors (nuclear receptors) are a superfamily of transcription factors. Through comparison of the cDNA and encoded amino acid sequences of the discovered family members, it is found that they are similar in structure and quite conservative in evolution. Nuclear receptors and ligands After binding to the body, it is activated and acts on the specific response element HRE (hormone respond sequence) of its target gene, thereby mediating specific gene transcription.

Nuclear receptors have common features in structure, generally including six functional areas A to F: A/B area contains a ligand-independent transcriptional activation area called AF-1 (Activation Function-1), through and auxiliary Activator, co-repressor and other transcription factors interact to regulate the target gene; the C region is the most conserved DNA binding domain (DNA binding domain, DBD), and the homology between various nuclear receptor DBD reaches 90% Above, it consists of two zinc finger structures, which mediate the binding of receptors and target genes; the D region is the hinge region, connecting the DNA binding region and the ligand binding region; the E region is the ligand binding domain (Ligand Binding Domain, LBD), It is a long hydrophobic region consisting of about 250 amino acids, which contains a nuclear localization region and a receptor-dependent transcriptional activation region (AF-2), which is relatively large. The homology between different nuclear receptor LBDs is about 15%. Although the homology is smaller than the DNA binding region, according to the analyzed LBD spatial structure data, the three-dimensional structures of all nuclear receptor LBDs are roughly the same, consisting of 12 Each α-helix has the functions of ligand binding, receptor dimerization, and cofactor binding. In addition, the F region is located carboxy-terminal to the nuclear receptor. Peroxisome Proliferator-Activated Receptors (PPARs) are members of the nuclear receptor family and play an important role in cell energy metabolism, cell proliferation and differentiation, and inflammatory response. There are three subtypes of PPARs in mammals: α, β, and γ. Like other members of the nuclear receptor superfamily, PPAR also consists of six domains (A-F). After PPAR binds to its ligand, it forms a heterodimer with another activated nuclear receptor retinoid X receptor (RXR), and binds to the specific PPAR response element PPRE (Peroxisome Proliferator Response Elements) on the target gene , thereby initiating the transcription of the target gene.

Troglitazone can effectively enhance insulin sensitivity by activating the PPARγ receptor in the nuclear receptor family and inhibiting TNF-a signal transduction. It is the first thiazole approved by the FDA as a treatment for type II diabetes Alkanediones, but the drug was withdrawn from the market due to adverse reactions such as liver toxicity. Pioglitazone (pioglitazone) and rosiglitazone (rosiglitazone) are currently approved and marketed drugs for the treatment of type II diabetes, they can also effectively reduce insulin resistance, but these drugs also have serious adverse reactions , such as leading to weight gain, may aggravate the condition of patients with heart failure, etc.

Therefore, the research and screening of nuclear receptor ligands is of great significance for the development of better drugs for the treatment of diabetes, obesity and other metabolic diseases. The traditional techniques and methods for in vitro screening molecular models of small molecule compounds based on the affinity analysis of receptors and ligands all need to obtain a sufficient amount of soluble purified protein, and need to separate and identify the binding ligand or protein. However, for poorly soluble proteins such as nuclear receptors or nuclear receptor ligand-binding domains (LBDs), these two issues have become formidable obstacles in the development of molecular models for drug screening.

Although people are currently trying to use various methods to screen small molecule ligands for nuclear receptors, since the ligand-binding domain (LBD) of nuclear receptors is a poorly soluble protein and it is difficult to obtain sufficient quantities of soluble purified protein, it is still difficult to establish a very effective screening molecular model. Therefore, there is an urgent need to develop a method in the prior art to obtain recombinant proteins that can be used to screen nuclear receptors or their ligands, so as to meet the requirements of ligand screening for protein purity and solubility, so as to find better treatments as soon as possible. Drugs for metabolic diseases such as diabetes and obesity.

Contents of the invention

The purpose of the present invention is to provide a phage with exogenous nuclear receptor or nuclear receptor ligand binding region fused on the surface of the capsid.

The purpose of the present invention is also to provide the use of the phage whose capsid surface is fused with exogenous nuclear receptor or nuclear receptor ligand binding region.

The purpose of the present invention is also to provide a method for screening nuclear receptor ligands using the phage fused with the exogenous nuclear receptor or nuclear receptor ligand binding region on the surface.

In the first aspect of the present invention, a phage is provided, which is composed of a capsid and a phage nucleic acid located in the capsid. The surface of the phage capsid is fused with an exogenous nuclear receptor or a ligand binding region of the nuclear receptor.

In a preferred embodiment of the present invention, the capsid protein pD of the phage is fused with an exogenous nuclear receptor or a ligand binding region of a nuclear receptor.

In another preferred example, the nuclear receptor or nuclear receptor ligand-binding region is a ligand-dependent transcription factor distributed in the cytoplasm or nucleus, and affects Gene transcription; the nuclear receptor or nuclear receptor ligand binding region usually consists of an N-terminal ligand-independent transcriptional activation region, a DNA binding region composed of a conserved zinc finger structure, a hinge region, a relatively conserved nuclear The localization region and the ligand-dependent transcriptional activation region are composed of the ligand-binding region; the N-terminal transcriptional activation region and DNA-binding region are relatively independent in spatial structure from the C-terminal ligand-binding region.

In a preferred example of the present invention, the nuclear receptor is selected from the following group: ① steroid hormone receptor; ② thyroxine receptor; ③ orphan nuclear receptor.

In another preferred example, the nuclear receptors of the steroid hormone receptor family include but are not limited to: glucocorticoid (GR), mineralocorticoid (MR), androgen receptor (AR), estrogen receptor (ER ) and progesterone receptor (PR).

In another preferred example, the nuclear receptors of the thyroid hormone receptor family include, but are not limited to: thyroxine receptor (TR), retinoic acid receptor (RAR) and vitamin D receptor (VDR).

In another preferred example, the orphan nuclear receptors refer to nuclear receptors whose corresponding physiological ligands have not been found, including but not limited to: RXRs, LXRs, FXRs, PPARs, COUP-TFs, ERRs, HNFs and other subgroups family.

In another preferred example, the PPARs include but not limited to: PPARα, PPARβ, PPARγ, more preferably, the PPARs are PPARγ.

In another preferred example, the phage capsid is composed of a head and a tail, and the nuclear receptor or the ligand binding region of the nuclear receptor is located at the capsid head.

In another preferred example, the nuclear receptor binding domain is the ligand binding domain (LBD) of PPARγ.

In a preferred embodiment of the present invention, the phage is selected from the group consisting of lambda phage, T7 phage, T4 phage, filamentous phage M13, fd and f1.

In another preferred embodiment, the phage is a virulent phage, such as λ, T7, T4 phage.

In another preferred embodiment, the phage is phage lambda, more preferably, the phage is phage lambda containing capsid protein pD.

In a preferred example of the present invention, the phage nucleic acid encodes a phage capsid protein and an exogenous nuclear receptor or a ligand binding region of a nuclear receptor.

In the second aspect of the present invention, the use of the phage is provided for screening substances that bind to nuclear receptors or ligand binding regions of nuclear receptors.

In another preferred example, the substance that binds to the nuclear receptor or the ligand binding region of the nuclear receptor is selected from the group consisting of ligands, antibodies, polypeptides or small molecule compounds.

In a third aspect of the present invention, there is provided a method for screening a substance that binds to a nuclear receptor or a ligand-binding region of a nuclear receptor, comprising the steps of:

(a) contacting the candidate substance with the first phage and the second phage respectively, wherein the first phage is the phage fused with the nuclear receptor or the ligand binding region of the nuclear receptor on the capsid described above, so The second phage is a phage of the same species as the phage that does not have a fused exogenous nuclear receptor or a ligand binding region of a nuclear receptor on the capsid surface;

(b) observing the combination of the candidate substance with the first phage and the second phage,

Wherein, the candidate substance that binds to the first phage but not to the second phage is a substance that binds to the nuclear receptor or the ligand binding region of the nuclear receptor.

In a preferred embodiment of the present invention, the substance that binds to the nuclear receptor or the ligand binding region of the nuclear receptor is selected from ligands, antibodies, polypeptides or small molecule compounds.

In another preferred example, in step (b), the combination of the candidate substance and the first phage and the second phage is observed by the following method: after the phage and the candidate substance are combined, the phage is recovered by competitive elution or determined by directly infecting the host bacterium Bound phage titers.

In the fourth aspect of the present invention, a nucleic acid molecule is provided, which contains the following elements: promoter, operator, replication site, screening marker site, phage coat protein coding region, phage exogenous protein coding region; and, all The above-mentioned exogenous protein coding region of phage encodes exogenous nuclear receptor or ligand binding region of nuclear receptor.

In another preferred example, there are promoters (tac promoter), operon (lac operator), and replication sites and antibiotic selection markers required for plasmid amplification in the upstream of the phage exogenous protein coding region and the phage coat protein coding region (Ampicillin) et al.

In another preferred example, the phage coat protein coding region encodes a phage coat protein.

In another preferred example, the coding region of the exogenous protein of the phage is connected with the coding region of the pD protein.

In a preferred embodiment of the present invention, a fusion protein is also provided, including a first part and a second part, wherein the first part is the coat protein pD of the bacteriophage; the second part is the nuclear receptor or the nuclear receptor the ligand-binding region.

In another preferred example, the nuclear receptor or the ligand-binding domain of the nuclear receptor is PPARγ or the ligand-binding domain of PPARγ (PPARγLBD).

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Fig. 1 shows the schematic diagram of pCGMT-LBD, p171-LBD, pET-hPPGLBD plasmid construction.

Fig. 2 shows the electrophoresis profile of the PCR amplification product of the PPARγLBD fragment cloned on p171Bio3, wherein, control represents a blank control.

Figure 3 shows the EcoRI restriction map of plasmid p171Bio3 and the newly constructed plasmid p171-LBD.

Figure 4 shows the protein electrophoresis profile of IPTG-induced PPARγLBD in the host strain BL21(DE3). S represents the supernatant of the lysate, and P represents the centrifuged pellet of the lysate. The protein mainly existed in the form of inclusion body, and accounted for more than 90% of the precipitate after sonication of the bacteria.

Figure 5 shows the Western blot analysis of the expression of the fusion proteins LBD-gp3 and pD-LBD.

Figure 6 shows Western blot showing that part of the pD-LBD fusion protein is expressed in the cytoplasm of the host bacteria in a soluble form.

Figure 7A shows that anti-PPAR LBD antibody analysis shows that PPAR LBD is assembled into lambda phage; Figure 7B shows that anti-pD antibody analysis shows that PPAR LBD assembles into lambda phage.

Figure 8 shows that λp171-LBD phage can bind to the microplate wells coated with anti-PPARγ LBD antibody, indicating that PPARγ LBD is displayed and expressed on the surface of λ phage.

Detailed ways

After extensive and in-depth research and experiments, the inventors found that the insoluble nuclear receptor or its ligand-binding region can be displayed on the surface of the phage capsid, thereby taking the lead in obtaining the fusion of nuclear receptor ligand binding on the capsid. area of phages. The phage can be used to screen substances that bind to nuclear receptors or their ligand binding regions.

At the beginning of the research, the inventor faced the difficulty of soluble expression of nuclear receptor protein, and the difficulty of phage display to express insoluble protein without any prior art reports for reference and phage display to limit the size of foreign protein assembly. Long-term research and repeated experiments have solved the problems of determining the site of display expression, local expression assembly and solubility enhancement of foreign proteins. During the experiment, the inventors selected the nuclear receptor ligand binding region as the preferential display expression, and preferred filamentous phage coat protein gp3 and lambda phage coat protein pD suitable for macromolecular protein display expression as carrier proteins, by comparing the expressed The solubility of exogenous recombinant proteins found that lytic phages such as λ are more suitable for the display and expression of insoluble nuclear receptors, so that insoluble nuclear receptors or their ligand binding regions are displayed on the surface of phage capsids, and fusion Expression of recombinant proteins in the nuclear receptor or nuclear receptor ligand binding domain of the phage coat. The recombinant protein fused and expressed on the surface of the phage can be used to screen substances combined with the nuclear receptor or its ligand binding region.

As used in the present invention, the "nuclear receptor" protein refers to a class of regulatory factor proteins with common structural characteristics and high homology. The nuclear receptor protein is closely related to the pathogenesis of metabolic diseases such as diabetes and obesity, so it is a useful drug target.

As used in the present invention, the "ligand binding region of a nuclear receptor" refers to a structural region of a nuclear receptor that has the ability to bind a ligand of a nuclear receptor. The three-dimensional structures of all nuclear receptor LBDs are roughly the same, consisting of 12 α-helices, and all have the functions of binding ligands, receptor dimerization and binding cofactors. In the preferred embodiment of the present invention, the nuclear receptor family (particularly the ligand binding region of PPARγ) is selected as an example. Due to the similarity in structure, members of other nuclear receptor families (or their ligands) body binding region) can also be displayed on the surface of the phage.

As used in the present invention, the "substance that binds to a nuclear receptor or a ligand-binding region of a nuclear receptor" includes any substance that binds to a nuclear receptor or a ligand-binding region of a nuclear receptor, including but not limited to: Ligands, antibodies, peptides or small molecule compounds, etc.

It should be understood that the figures such as the amount of ingredients and reaction conditions shown in the examples or experimental materials and methods, or the figures used in the description of this application are all approximate values. Therefore, unless otherwise specified in the text, the above numerical parameters in this specification are approximate values, which can be changed according to the desired result of the present invention. Moreover, these parameters are not used to limit the principle equivalent to the patent scope of the present invention, but are better data obtained under normal operating techniques.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

The main advantages of the present invention are:

(1) The nuclear receptor or its ligand binding region is displayed and expressed on the capsid surface of the phage, thereby constructing a phage fused with the nuclear receptor or its ligand binding region on the capsid.

(2) The nuclear receptor or its ligand binding region can be displayed and expressed on the surface of the phage, and the ability of the phage to infect host bacteria can be directly used as a detection index for screening the binding of the nuclear receptor to other substances. Moreover, using the phage of the present invention to screen substances that bind to nuclear receptors or their ligand binding regions has the characteristics of high screening sensitivity and high screening efficiency, and can be screened with only a very low protein expression level.

(3) Fusion expression of the nuclear receptor or its ligand binding region and the capsid protein of the phage can enhance the solubility of the expressed fusion protein, and overcome the difficulty of nuclear receptor or its ligand binding region because it belongs to insoluble protein Difficult to apply to the problem of screening its binding substances.

I. Materials and Experimental Methods

The following materials and methods are used in the specific embodiments of the present invention, and the materials and methods provided below are not intended to limit the present invention.

Strains and plasmids

Escherichia coli DH5α and BB4 were from Stratagene, and BL21(DE3) was from Navogen.

DH5α: F ^- , φ80d lacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rK ^- mK ⁺ ), phoA, supE44, λ ^- , thi-1, gyrA96, relA1

BB4: supF58, supE44, hsdR514, galK2, galT22, trpR55, metB1, tonA, ΔlacU169/F'[proAB+, lacI ^q , lacZΔM15Tn10(tet ^r )]

BL21(DE3): F-, ompT, hsdSB(rB-mB-), gal(λcI857, ind1, Sam7, nin5, lacUV5-T7gene1), dcm(DE3)

The human PPARγ2 gene was cloned from a liver cDNA library (Gnebank number: NM_015869); pGEX-2T was a product of Amersham Pharmacia; the plasmid pET-21a(+) was from Navogen; the construction of plasmid p171 Bio3 could be found in literature (Santi E, Capone S, Mennuni C, Lahm A, Tramontano A, Luzzago A, Nicosia A: Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics. J. Mol. Biol. 2000, 296: 497-508.); See pCGMT phagemid construction Literature (Gao C, Lin CH, Lo CH, Mao S, Wirsching P, Lerner RA, Janda KD: Making chemistry selectable by linking it to infectivity.Proc.Natl.Acad.Sci.U.S.A 1997,94:11777-11782); Lysogenic phage λD1180 (λDam15 b538 cIts857 nin5 Sam100) was prepared with reference to literature (Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, Fujita S, Hayakawa T, Takeda K, Hasegawa M, Nakanishi M: Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J. Biol. Chem. 2001, 276: 26204-26210).

Enzymes and Chemical Reagents

The chemical reagents and biochemical reagents required in the experiment were purchased from Shanghai Huashun Bioengineering Co., Ltd., which is the product of Amresco. The adjuvant required for animal immunization is a product of Sigma; the PVDF membrane (Immobilon-P) of Western blot is a product of Millipore; HRP-goat anti-mouse IgG is a product of Calbiochem.

Various restriction endonucleases, T4 DNA ligase, and DNA molecular weight standards were purchased from TaKaRa Bioengineering Co., Ltd.; dNTP and pfu DNA polymerase used for PCR were purchased from Shanghai Shennengcai Biotechnology Company, PCR primer synthesis and sequence determination Completed by Shanghai Shenergy Gaming Biotechnology Company.

Main equipment

PTC-150 PCR instrument (MJ Company of the United States); low-temperature high-speed centrifuge TL-100 (Beckman Company of the United States); shaking culture shaker (Shanghai Qite Analytical Instrument Co., Ltd.); electric constant temperature incubator (Shanghai Jinglu Experimental Equipment Co., Ltd. Company); JY-882 Ultrasonic Cell Pulverizer (Shanghai Xinzhi Biotechnology Research Institute); DY-A Electrophoresis Instrument (Shanghai Sebas Biotechnology Development Co., Ltd.); DY-24D Small Electrophoresis Tank; DY-1 Transfer Electrophoresis Tank (Jiangsu Xinghua Analytical Instrument Factory).

Culture Media and Buffer Solutions

For the medium configuration required for bacterial culture and phage titer determination, refer to the Molecular Cloning Experiment Guide (SambrookJ., Russell DW: Molecular Cloning: A Laboratory Guide, Cold Spring Harbor, NY.: Cold Spring Harbor Laboratory Press; 2001.); For the configuration of required TE, PBS buffer solution, SDS-PAGE electrophoresis related solution, Western blot detection related solution, and other required solutions, refer to the molecular cloning experiment guide.

main experimental method

Routine PCR, enzyme digestion, ligation, preparation of competent cells, plasmid transformation and extraction refer to the Molecular Cloning Experiment Guide (Sambrook J., Russell DW: Molecular Cloning: A Laboratory Guide, Cold Spring Harbor, NY.: Cold Spring Harbor Laboratory Press; 2001.).

1. Construction and verification of recombinant plasmids

1. PCR amplification of PPARγLBD fragment

Using the cDNA of PPARγLBD (GenBank accession number NM_015869) as a template, two primers, PPLBD_Fwd1 and PPLBD_Rev1, were used for PCR amplification to amplify the PPARγLBD fragment, which was ligated with plasmid pET-21a(+), and the resulting recombinant plasmid was named pET-hPPGLBD. Using PPLBD_Fwd2 and PPLBD Rev2 as primers for PCR amplification, the resulting PCR fragment of PPARγ LBD was ligated with plasmid p171Bio3, and the resulting recombinant plasmid was named p171-LBD.

Among them, primer PPLBD_Fwd1: 5'-AG GGATCC GTGGGGATGTCTCATAATGC-3'(BamHI); primer PPLBD_Rev1: 5'-ACGC GTCGAC GTACAAGTCC TTGTAGAT-3'(SalI); primer PPLBD_Fwd2: 5'-AG AC TAGT GTGGGGATGTCTCATAATGC-3' (SpeI ); Primer PPLBD_Rev2: 5'-TGTT GCGGCCGC TACAAGTCCTTGTAGATC-3' (NotI). In addition, the underlines in the above primer sequences refer to the enzyme cleavage sites shown in brackets.

2. Ligation, transformation and verification of PCR amplified fragments and phage vectors

Different restriction sites were introduced at both ends of the PCR fragment by PCR primers. After the PCR product was double-digested, it was ligated with the same double-digested vector plasmid to transform the competent cells of the host bacteria. For specific methods, refer to the Molecular Cloning Experiment Guide. Select the clones grown on the plate, inoculate them in 3ml LB medium, and culture them overnight at 37°C, then extract the plasmids with the kit and verify by enzyme digestion. Recombinants that were verified to be connected correctly by enzyme digestion were stored at -70°C.

2. Expression and analysis of exogenous proteins in E. coli

Transform host bacteria containing exogenous protein plasmids, shake in 50ml LB (ampicillin 60μg/ml, 0.1% glucose) at 37°C until the _OD600 is about 0.4-0.5, add IPTG to a final concentration of 1mM to induce exogenous protein expression, Shake culture at 30°C for 8 hours.

Collect the cells induced by IPTG by centrifugation at 8000rpm for 10 minutes, suspend in ultrasonic buffer solution (50mM Tris pH8.0, 150mM NaCl, 1mM EDTA), sonicate the cells in an ice bath, centrifuge at 10000rpm at 4°C for 15 minutes, and separate the sonicated products soluble fraction and precipitates.

The components after centrifugation were separated by 10% SDS-PAGE denaturing polyacrylamide gel electrophoresis. When bromophenol blue electrophoresis reaches the bottom of the separation, take out the gel after the electrophoresis is completed, and transfer the protein at a constant current of 500mA for 2 hours under cooling conditions, and then transfer the protein to the PVDF blot membrane.

After the electrophoresis transfer was completed, the PVDF membrane was taken out, and the blocking solution (2%BSA, TBS) was blocked overnight, and the TBST buffer (TBS, 0.05% Tween 20) was rinsed 3 times at room temperature, each time for 10 minutes; See below) or anti-pD serum (see below for preparation method) (diluted in blocking solution 1:1000) as the primary antibody, gently shake and incubate at 37°C for 1 hour; repeat rinsing three times, each time for 10 minutes; add HRP-goat anti-mouse IgG Secondary antibody (diluted in blocking solution 1:5000), shaken and warmed at 37°C for 1 hour; after repeated rinsing steps, add HRP substrate reaction solution to develop color at room temperature _. After rinsing with water, dry in the shade and store at 4°C.

3. Preparation of polyclonal antibodies anti-LBD and anti-pD

Escherichia coli BL21(DE3) transformed with plasmid pET-hPPGLBD was induced by IPTG to express PPARγLBD, and lysed by ultrasonic waves. The PPARγLBD protein mainly existed as insoluble inclusion bodies, and the PPARγLBD protein could be isolated by centrifugation. Escherichia coli BB4 transformed with plasmid p171Bio3 was used to induce the expression of pD protein with IPTG, and the ultrasonic lysate of the bacteria was separated by SDS-PAGE gel. .

Mice were immunized with purified PPARγ LBD protein and pD protein, respectively. A total of five immunizations were performed, blood was collected from the corners of the eyes of the mice, and serum was collected. After the titer was determined, the aliquots were stored at -70°C. Antibody immunization methods and titer determination refer to the molecular cloning experiment guide.

4. Display, expression and analysis of foreign proteins on the surface of phage

In a preferred example of the present invention, the nuclear receptor or its ligand binding region is fused to the capsid of bacteriophage λ, and the specific display, expression and analysis steps are as follows:

1. Preparation of lysogen BB4 (λD1180)

Prophage λD1180 (λDam15 b538 cIts857 nin5 Sam100) was constructed and prepared from the aforementioned references (Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, Fujita S, Hayakawa T, Takeda K, Hasegawa M, Nakanishi M : Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J. Biol. Chem. 2001, 276: 26204-26210). The obtained λD1180 phage was diluted ¹⁰⁴ times, and 1 μl was mixed with 200 μl fresh log-phase Escherichia coli BB4. After standing at 30°C for 30 minutes, a small amount of bacterial liquid was streaked on the plate with an inoculation needle, and cultured at 32°C. The next day, single clones were picked with a toothpick, inoculated on two plates respectively, and corresponded one by one. One plate was placed at 32°C, and the other plate was placed at 42°C for 12 hours, and the colonies that grew at 32°C and did not grow at 42°C were selected, which were BB4 lysogens integrated with prophage λD1180, called BB4(λD1180).

2. Preparation and transformation of competent lysogen BB4 (λD1180)

The preparation of BB4(λD1180) competent was similar to other strains, but the bacterial growth and culture temperature were 32°C. Plasmid p171Bio3 and recombinant plasmid p171-LBD are transformed into BB4 (λD1180) competent. The transformation process is similar to other bacterial transformation processes, but there is no need to heat shock at 42°C after ice bath, and it is directly added to LB liquid culture based on 32°C recovery. After plating, culture at 32°C.

3. Lambda phage amplification and titer determination

For the cultivation, amplification and titer (plaque forming unit, plaque forming unit) determination of lambda phage, refer to the Molecular Cloning Experiment Guide.

4. Lambda Phage Binding Assay

Mouse anti-PPARγ LBD polyclonal antibody (anti-LBD serum, see above for preparation method) was diluted 100 times with coating solution, and then coated with 100 μl per well, 6 wells in total. Simultaneously, the serum of non-immunized mice was also diluted 100 times with the coating solution as a control, added to 3 microplate wells, and coated overnight at 4°C. On the next day, 200 μl of blocking solution PBST (2% BSA, 0.05% Tween-20) was added to each well to block at 37° C. for 2 hours. Then, 1×10 ⁸ λp171Bio3 phage was added to the three wells coated with anti-LBD, and the same amount of λpD-LBD phage (λ phage fused with LBD on the pD protein) was added to the other three wells. 1×10 ⁸ λpD-LBD phage was also added to the wells coated with mouse serum, and incubated at 37°C for 1 hour. The phage solution was removed, and each well of the microplate was washed three times with 200 μl of washing solution (PBS, 0.05% Tween-20, 10 mM MgSO ₄ ), and finally washed once with PBS solution (10 mM MgSO ₄ ). Then add log phase bacteria BB4 and incubate at 37°C for 30 minutes. The number of phages bound to the wells of the microtiter plate was determined using the lambda phage titer assay described above. The obtained results were statistically analyzed, and the t test (student's paired t test) was used to determine whether there was a significant difference between the two groups of data. When p<0.05, it was considered that there was a statistically significant difference between the two groups.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

II. Specific examples

Example 1

Construction of foreign protein PPARγLBD expression plasmid

The schematic diagram of plasmid construction is shown in Figure 1. Plasmid p171Bio3 contains the gene encoding λ phage capsid protein pD, and the PCR amplified fragment of PPARγLBD is connected to the 3' end of the pD protein gene. Using primers PPLBD_Fwd2 and PPLBD_Rev2, the PPARγ LBD fragment was amplified, and the gene fragment was about 900bp. Agarose electrophoresis showed that the size of the PCR product fragment was consistent with the expectation (Figure 2), and the constructed plasmid was p171-LBD. The p171-LBD plasmid was verified by EcoRI digestion, and the fragmentation and size of the restriction enzyme were consistent with the expectation (Figure 3), indicating that the exogenous protein PPARγ LBD gene fragment was ligated into the plasmid p171Bio3

The PPARγ LBD amplified by primers PPLBD_Fwd1 and PPLBD_Rev1 was connected to the plasmid pET-21a(+) through BamHI and SalI restriction sites, and the constructed plasmid was pET-hPPGLBD. The same amplified PCR fragment was ligated with pCGMT, and the constructed plasmid was pCGMT-LBD.

Example 2

Expression of PPARγ LBD in Escherichia coli and Preparation of Anti-LBD and Anti-pD Polyclonal Antibodies

After the pET-hPPGLBD plasmid was transformed into Escherichia coli BL21 (DE3), IPTG induced the expression of PPARγ LBD, and the supernatant (S) and precipitate (P) of the supernatant (S) and precipitate (P) of the sonicated solution were analyzed by SDS-PAGE, and the results were shown in Figure 4. There is a significant protein band at the molecular weight of about 31kDa in the precipitate of bacterial cell lysate (the 4th lane), while the amount of soluble recombinant protein at the corresponding position in the supernatant lysate is relatively small (the 3rd lane), indicating that PPARγ LBD is in the In Escherichia coli, it is mainly expressed in the form of insoluble inclusion bodies. PPARγ LBD reached more than 95% in the whole inclusion body protein, and the inclusion body protein was centrifuged and used to immunize mice.

After the p171Bio3 vector plasmid was transformed into BB4, the pD protein gene expression on the plasmid was induced by IPTG. After SDS-PAGE electrophoresis separation, the pD protein was recovered by repeated freezing and thawing after tapping the gel, and the extracted protein was used to immunize mice.

After using the purified PPARγ LBD and pD protein to immunize the mice multiple times, the anti-LBD and anti-pD polyclonal antibody sera obtained respectively were tested for titer by indirect ELISA method, and then used for future use.

Example 3

The lambda phage display system is more suitable for the display and expression of PPARγLBD protein than the filamentous phage gp3 system

PPARγLBD was fused and expressed at the N-terminal of the gp3 protein encoded by the phagemid pCGMT and the C-terminal of the pD protein encoded by the plasmid p171Bio3, and the fusion proteins LBD-gp3 (55kDa) and pD-LBD (46kDa) were expressed after IPTG induction. The cells were disrupted by ultrasonic waves after the protein expression was induced by IPTG. The ultrasonic lysate was subjected to SDS-PAGE electrophoresis, and Western blot detection was performed using anti-LBD polyclonal antibody (Figure 5). T, S, and P in the figure represent the total protein of the fusion protein, the protein in the supernatant of the lysate and the protein in the precipitate of the lysate, respectively. It can be seen that the soluble gp3-LBD fusion protein is mainly distributed in the precipitate and exists in the form of insoluble inclusion body. However, the pD-LBD fusion protein induced under the same conditions exists in the supernatant and precipitate of the ultrasonic lysate, and a part of the pD-LBD protein exists in a soluble form. Because the soluble foreign protein is conducive to its assembly and display in phage particles.

Therefore, it can be seen from this that the lambda phage display system is more suitable for the display and expression of PPARγ LBD protein than the filamentous phage gp3 system.

Example 4

Part of the PPARγ LBD fused with pD protein is expressed in the cytoplasm of the host bacteria in a soluble form

Further Western blot analysis was performed on the expression of pD-LBD fusion protein in the host bacteria under induced and non-induced conditions.

The results are shown in Figure 6, which shows that the transformed bacteria of the vector p17 1Bio3 as a blank control have no expression of pD-LBD protein under induction and non-induction conditions. The bacterial clone transformed with the recombinant plasmid p171-LBD had soluble expression of the nascent protein pD-LBD at 46kDa under non-inducing conditions (track 6), but no band appeared at the corresponding position in the lysed precipitate (track 7). After IPTG induction, the expression of pD-LBD fusion protein was significantly increased in the lysate supernatant (lane 8). At the same time, due to the limitation of pD protein to improve the solubility of exogenous protein, a large amount of pD-LBD fusion protein after IPTG induction Expression of the fusion protein, the fusion protein mainly exists in the cytoplasm of the host bacteria in the form of insoluble inclusion body (track 9). The soluble pD-LBD accounts for about 30% of its total expression. This result further illustrates that the pD protein can promote the fusion protein to remain in solution to a certain extent. Although most of the expressed fusion protein still exists in the form of inclusion bodies (lane 9), this small part of the fusion protein remains in solution protein is sufficient for phage display. S and P in the figure represent the protein in the supernatant of the cell lysate and the precipitate of the cell lysate, respectively.

Example 5

PPARγ can be assembled into lambda phage capsid protein

The vectors p171Bio3 and p171-LBD were respectively transformed into lysogenic bacteria BB4 (λD1180), heat-induced lysate assembly and release of bacteriophage λ, and the λp171Bio3 and λp171-LBD obtained by PEG precipitation were transferred to PVDF membrane after SDS-PAGE electrophoresis, and the anti-LBD polyclonal Antibody (Figure 7A) and anti-pD polyclonal antibody (Figure 7B) detection. In Fig. 7A, there is an obvious band at 46kDa in λp171-LBD, but there is no corresponding band at λp171Bio3 as a control, the protein of this band should be pD-LBD assembled into λ phage. In addition to the pD-LBD protein band at 46kDa, there was also a pD protein at 11kDa in λp171-LBD (Fig. 7B), which was derived from the lysogen in the host bacterium. The pD protein encoded by the gpD gene in the genome of prophage λD1180 in BB4.

The ratio of pD protein and pD-LBD fusion protein reflects the ability of exogenous protein PPARγ LBD to assemble into λ phage. Usually, each wild-type λ phage contains 405 copies of pD protein. Through the quantitative analysis of the two bands in Figure 7B, it can be concluded that the ratio of pD-LBD to pD is about 0.4, so pD fused with foreign protein The number of pD accounts for 28% of the total number of pDs, so it can be calculated that each lambda phage assembles approximately 115 PPARγLBD molecules.

Example 6

Fusion expression of PPARγ LBD on λp171-LBD phage can be recognized and captured by anti-LBD antibody

The location of foreign protein assembly into lambda phage is determined by the interaction between phage assembled with PPARγLBD and its antibody. Anti-PPARγLBD was used to coat the wells of the microtiter plate, respectively combined with λp171Bio3 and λp171-LBD phages, and at the same time, the wells of the microtiter plate coated with mouse serum without antibody were used as a control (Figure 8). The quantity of λp171-LBD captured in the wells of the anti-LBD-coated microtiter plate was four times that of λp171Bio3, and statistical analysis showed that there was a significant difference between the two (P<0.01). The difference between the two stems from the assembly of PPARγLBD protein in λp171-LBD, indicating that there is a strong combination of anti-LBD serum and PPARγLBD protein. At the same time, the amount of λp171-LBD captured by the microplate wells coated with antibody-free mouse serum decreased significantly (P<0.01), indicating that the strong combination of anti-LBD serum and PPARγ LBD protein mainly came from the anti-LBD antibody and PPARγLBD in the antibody serum between fusion proteins.

λp171-LBD can be captured by antibody, indicating that PPARγ LBD is expressed on the surface of λ phage. At the same time, it also shows that the PPARγLBD assembled in the lambda phage is displayed and expressed on the outer surface of the lambda phage, and maintains the structural characteristics that can be recognized and bound by antibodies.

Example 7

Qualitative and quantitative detection of recombinant proteins based on display expression using PPARγ LBD assembled on the surface of bacteriophage λ

First phage: λ phage with PPARγ LBD assembled on the capsid surface.

Second phage: λ phage without PPARγLBD assembled on the capsid surface.

The candidate substance is contacted with the above-mentioned first phage and the second phage respectively, and the combination of the candidate substance with the first phage and the second phage is observed.

Separately analyze the binding results of the candidate substance to the first and second phages. If a candidate substance can bind to the first phage but not to the second phage, it is the ligand binding region of the nuclear receptor or nuclear receptor combined substances.

discuss

(1) The solubility of the poorly soluble foreign protein is improved after fusion expression with pD

Escherichia coli is still the main protein expression system due to its advantages of convenient operation and easy cultivation, but when eukaryotic proteins are expressed in Escherichia coli, insoluble protein inclusion bodies are often formed, which is a non-soluble protein during the expression process. A protein intermediate formed by correct folding. Protein function research cannot be carried out directly using inclusion bodies. The usual method is to renature the isolated inclusion body protein first. However, there is no certain rule to follow for protein renaturation. Therefore, protein renaturation is difficult under the current technical conditions. There are still great difficulties. Another way to bypass the problem of protein renaturation is to reduce the inclusion body generation during protein expression and improve protein solubility. The usual method is to improve the solubility of the protein by optimizing protein expression conditions or expressing fusion with proteins that can improve solubility, such as glutathione S-transferase (Glutathione S-Transferase, GST), maltose binding protein (Maltose Binding Protein, MBP), Thioredoxin (Trx), etc.

Nuclear receptors and their ligand-binding domains (such as PPARγ LBD) contain an interface that binds to hydrophobic small-molecule ligands, and their overall content of hydrophobic amino acids is relatively high, so inclusion bodies are more likely to form during expression , The inventor's test results have also confirmed this point, when PPARγ LBD is expressed in Escherichia coli, most of the expressed protein is present in the bacteria in an insoluble form. Even after fusing GST protein, the solubility of PPARγ LBD was not greatly improved. However, the fusion of λ phage capsid protein pD and PPARγLBD can significantly improve the solubility of the latter, indicating that the use of pD protein is a better choice.

(2) Maintaining protein solubility is a prerequisite for assembly into phage

Although phage display technology does not require protein purification, exogenous proteins maintain a certain solubility when expressed in host bacteria, which is also a prerequisite for exogenous proteins to be displayed and expressed on the surface of phages. Although it is not required that all of the foreign protein remains soluble, at least a portion of it needs to be soluble. Previous studies have shown that improving the solubility of exogenous proteins, including fusion expression with solubilizing proteins or co-expression of protein chaperones, can improve the efficiency of exogenous protein assembly on the surface of phage. From this point of view, the choice of pD protein as the carrier protein is suitable for the display and expression of those proteins that are easy to form insoluble inclusion bodies during the expression process.

(3) Protein size limitation is not the main limiting factor in the lambda phage display system

The inventors' results showed that larger foreign proteins can be displayed and expressed on the surface of phage. Other reports have also shown that large molecular weight proteins, such as β-galactosidase, β-lactamase, and protein toxins, can be assembled into phage λ in high copies. These results indicate that phage λ is limited in the size of the proteins displayed Relatively wide. Therefore, the λ phage display system is more suitable for displaying large molecular weight proteins.

(4) λ phage display system is a multivalent display system

Lambda phage is a multivalent display system, which is more suitable for screening ligands with low affinity. For the screening of high-affinity ligands, the widely used monovalent filamentous phage gp3 system can be selected, and the expression of pD protein fused with foreign protein can also be regulated by regulating the promoter. By changing the wild-type pD protein and the fused pD protein The ratio of the number of molecules, so as to regulate the valence of exogenous protein assembled into λ phage.

(5) Other nuclear receptors or nuclear receptor ligand binding regions can also be displayed and expressed on the surface of phage

Other nuclear receptors are very similar in structure. The size of the ligand-binding region is about 250 amino acids, which is not much different, and because they are all bound to fat-soluble ligands, they have strong hydrophobicity. When expressed in E. coli Both tend to form insoluble inclusion bodies. For the display and expression of foreign proteins on the surface of phage, the main limitation comes from the solubility limitation and molecular size limitation of foreign protein. According to the embodiments of the present invention, PPARγ LBD can be displayed and expressed on the surface of phage, so it can be inferred that for other Similar nuclear receptors or nuclear receptor binding regions can also be displayed on the surface of phage.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. phage, constitute by capsid and the bacteriophage nucleic acid that is positioned at capsid, it is characterized in that, described phage is a lytic phage, merge on the capsid surface of described phage the nuclear receptor of external source or the ligand binding domain of nuclear receptor, described lytic phage is selected from: lambda particles phage, T7 phage.

2. phage as claimed in claim 1 is characterized in that, the capsid protein pD of described phage goes up to merge the nuclear receptor of external source or the ligand binding domain of nuclear receptor.

3. phage as claimed in claim 1 is characterized in that, described nuclear receptor is selected from down group:

1. steroid hormone receptor;

2. pth receptor;

3. orphan nuclear receptor.

4. phage as claimed in claim 1 is characterized in that described phage is a lambda particles phage.

5. phage as claimed in claim 1 is characterized in that, described bacteriophage nucleic acid coding phage capsid protein and the nuclear receptor of external source or the ligand binding domain of nuclear receptor.

6. the purposes of phage as claimed in claim 1 is characterized in that, is used to screen the ligand binding domain bonded material with nuclear receptor or nuclear receptor.

7. the method for the ligand binding domain bonded material of a screening and nuclear receptor or nuclear receptor is characterized in that, comprises step:

(a) candidate substances is contacted with second phage with first phage respectively, wherein said first phage is the described phage of claim 1, and described second phage is ligand binding domain and the phage same kind of the described phage of claim 1 that does not have the external source nuclear receptor or the nuclear receptor of fusion on the capsid surface;

(b) observe the situation that combines of candidate substances and first phage and second phage,

Wherein, be incorporated into first phage but debond is exactly a ligand binding domain bonded material with nuclear receptor or nuclear receptor in the candidate substances of second phage.

8. method as claimed in claim 7 is characterized in that, described ligand binding domain bonded material with nuclear receptor or nuclear receptor is selected from part, antibody, polypeptide or micromolecular compound.

9. method as claimed in claim 7, it is characterized in that, observe combining of candidate substances and first phage and second phage in the step (b) by the following method: phage and candidate substances in conjunction with after, reclaim phage or measure the bonded phage titre by the competition wash-out by directly infecting the host bacterium.

10. a nucleic acid molecule is characterized in that, it contains following element: promotor, operon, replication site, selection markers site, bacteriophage coat protein coding region, phage foreign protein coding region; And, the nuclear receptor of described phage foreign protein coding region encoding exogenous or the ligand binding domain of nuclear receptor; Described phage is a lytic phage.

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