Northwestern screening of protein expression libraries is performed by constructing IPTG-inducible fusion protein expression libraries (the N-terminus of the fusion protein is encoded by the vector sequence, and the C-terminus is encoded by the cDNA library cloned into the vector), then inducing the expression of fusion proteins, immobilizing the proteins on nitrocellulose membranes, and then screening the proteins by labeled RNA probes, and finally obtaining the cDNAs that can specifically bind to the target RNA molecules. The cDNA was screened with a labeled RNA probe to obtain the cDNA that can specifically bind to the target RNA molecule.This experiment was obtained from the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Northwestern Screening of Protein Expression Library Experiments
Principle
Northwestern screening of protein expression libraries is performed by constructing IPTG-inducible fusion protein expression libraries (the N-terminus of the fusion protein is encoded by the vector sequence, and the C-terminus is encoded by a cDNA library cloned into the vector), then inducing the expression of the fusion protein, and immobilizing the protein on nitrocellulose membranes and screening with labeled RNA probes to obtain cDNAs that can bind specifically to the target RNA molecule. The protein is immobilized on a nitrocellulose filter membrane and screened with a labeled RNA probe to obtain a cDNA that binds specifically to the target RNA molecule.
Materials and Instruments
Expression library Host bacteria Antibiotics Move -Materials and equipment For more product details, please visit Aladdin Scientific website.
SM buffer IPTG blocking buffer rinse buffer binding buffer elution buffer Ficoll PVP BSA Torula yeast core RNA
NZY medium LB medium NZY top agar NZY plate cellulose nitrate membrane RNA probe
1. Expression libraries: λ phage vectors are mostly used to construct expression libraries, and the vectors can be selected according to the needs. We can buy pre-prepared expression libraries (Stratagenc, Clontech, etc.), or we can prepare our own libraries. Vectors such as λgt11, λZAP or λORF8 have a lacZ gene in front of the polyclonal site, which enables the fusion protein to be expressed by TPTG. Improved vectors such as λTriplEx allow translation of all three reading frames, ensuring protein expression. In addition, histones can be fused to β-galactosidase for blue-white screening of recombinants, and the addition of the 5'-UTR of the E.coli ompA gene to λTriplEx vectors improves the stability of the recombinants.
2. Host bacteria: the host bacteria must correspond to the library vector. For example, E.coli XL I-blue can be used as the host of λTriplEx and λZAP vectors, and the add-on F' of E.coli XL I-blue contains the △M15 lacZ and lac Iq genes: the expression of △M15 lacZ gene can be used to screen recombinant clones for blue-white spot through α-complementary; lacIq gene encodes the lac repressor, which represses the lac gene when TPTG is not present; and the 5'-UTR of lacIq gene can improve the stability of the recombinant. The lacIq gene encodes a lac suppressor that represses transcription from the lacZ promoter in the absence of the inducer TPTG.
3. NZY medium: 5 g NaCl, 2 g MgSO4-7H2O, 5 g yeast extract, 10 g NZ amine (enzymatic hydrolysis product of casein), volume to 1 L, autoclaved and stored.
4. LB medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, volume to 1 L, adjust pH 7.4, autoclaved and stored.
5. NZY top agar: add 0.7% agarose to NZY medium and autoclave.
6. NZY plate: add 15 g of agar powder into 1 L of NZY medium, autoclaved and spread the plate (about 70 ml/150 mm Petri dish, about 30 ml/100 mm Petri dish).
7. Antibiotics: ampicillin: 50 μg/ml; tetracycline: 12.5 μg/ml.
8. SM buffer: 5.8 g NaCl, 2 g MgSO4-7H2O, 50 ml 1 mol/L Tris-HCl (pH 7.5), 5 ml 2% gelatin, volume to 1 L, autoclaved.
9. 0.5 mol/L IPTG, X-Gal (2.5 g X-Gal dissolved in 10 ml of dimethylformamide), 20% maltose, all filtered for sterilization.
10. cellulose nitrate membrane (supported nitnKdliilose, BA-S85, Schleicher & Schuell, Inc. is recommended).
11. 32P-labeled RNA probe: conventional preparation, purification of the probe by vertical electrophoresis with acrylamide gel containing 7 mol/L urea, elution with HSCB buffer [ 25 mmol/L Tris-HCl (pH 7.5), 400 mmol/L NaCl, 0.1% SDS ] overnight.
12. Blocking buffer: 5 mg/ml Torula yeast core RNA (Sigma), 10 mmol/L Tris-HCl (pH 7.8), 150 mmol/L NaCl.
13. Rinse buffer: 10 mmol/L Tris-HCl (pH 7.8).
14. binding buffer: 10 mmol/L Tris-HCl (pH 7.8), 1 mmol/L EDTA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone (PVP), 0.02% bovine serum albumin, and pre-tested concentration of NaCl (usually around 50 mmol/L). /L).
15. Elution buffer: 10 mmol/L Tris-HCl (pH 7.8), 1 mmol/L EDTA, 0.02% Ficoll, 0.02% PVP, 0.02% bovine serum albumin and a pre-tested concentration of NaCl (usually around 100 mmol/L).
16. 2% Ficoll (10X storage solution): 2 g of Ficoll (type 400) is dissolved in 100 ml of distilled water, sterilized at 110°C for 15 min, and stored at 4°C for 1 month.
17. 2% PVP (10X stock solution): 2 g PVP (molecular mass 40,000 Da) dissolved in 100 ml distilled water, sterilized at 110°C for 15 min, stored at 4°C for 1 month.
18. 2% BSA (10X stock solution): 2 g of high purity bovine serum albumin dissolved in 100 ml of distilled water, filtered and sterilized, stored at -20℃.
19. 50 mg/ml Torula yeast core RNA (10X storage solution): 5 g of Torula yeast core RNA was dissolved in 100 ml of distilled water, proteinase K (0.25 mg/ml) was treated at 37℃ for 1 h. Phenol was extracted with chloroform and ethanol was used to remove the contaminated nuclease.
Methods
1. Preservation and operation of λ phage library
Most of the purchased λ phage libraries are stored in 7% DMSO, which has no effect on phage activity, so they can be operated directly without removal. For long-term storage, they should be divided into 50 μl portions and stored at -80℃ to avoid repeated freezing and thawing. If the library is to be diluted, SM buffer should be used; the working samples can be stored at 4℃ for 2 months.
Before performing the experiment, it is better to perform a titer test on the purchased library. A good cDNA library should be ≥108 pfu/ml and should contain ≥104 different clones to ensure the diversity of proteins expressed. It is also required to contain at least 75% recombinant clones.
2. Host bacteria preparation
Inoculate the monoclonal host bacteria into 50 ml of LB culture medium containing 10 mmol/L MgSO4, 0.2% maltose ( MgSO4 and maltose can help phage enter into the host bacteria), and incubate at 30℃ with 120 r/min overnight until the OD600 reaches 2.0. Lower temperature and aeration can ensure that there is no overgrowth of the overnight culture.
Centrifuge at 2000 r/min for 5 min to collect the organisms, remove the supernatant, and resuspend the organisms in 10 mmol/L MgSO4 solution to achieve an OD600 between 0.5 and 1.0. It is important to avoid the use of a vortex shaker, but to ensure that the cells are completely resuspended. Resuspended bacteria can be stored at 4°C for up to 1 week without significant loss of activity.
3. Phage expression library coated plates
(1) Add 1 ml of MgSO4 resuspended bacterial suspension and appropriate amount of phage library (about 5X104 pfu) to each of 20 reaction tubes and incubate at 37℃ for 15~20 min to allow phage to enter into the host bacteria.
(2) Add 9 ml of melted NZY top agar into each reaction tube one by one and spread 150 mm NZY plates immediately (melted NZY top agar should be cooled down to 45 ℃ before use; each reaction tube should be coated with NZY top agar immediately after the addition of NZY top agar; NZY plates should be warmed up at 37 ℃, and rotate the plates as soon as possible after the addition of NZY top agar to make the bacterial layer uniformly distributed; the total number of screening in 20 reaction tubes should be about 106 pfu. (The total number of plates screened in 20 reaction tubes is about 106 phages). Leave the plate at room temperature for 10 min to solidify the top layer of agar, then invert the plate and incubate at 37 ℃ for 3~5 h until tiny phage spots appeared. Be careful not to stack the plates during incubation to ensure that each plate is at the same temperature and that phage spots appear at the same time.
4. IPTG-induced protein expression and protein transmembraneization
(1) Use a ballpoint pen to mark the nitrocellulose filter membrane on the surface not in contact with the plate. During incubation of the plate, the membrane was immersed in 10 mmol/L IPTG for 30 min and then blotted on 3 MM filter paper for 5 min to ensure that the membrane was in a slightly wet state when it was spread onto the plate. Wear gloves and use sterilized forceps during the procedure.
(2) Spread the IPTG-treated membrane on the plate with phage spots and incubate at 37°C for 4 h to induce the expression of recombinant protein.
(3) Mark the membrane and the plate (you can use a big pin to mark different positions) and remove the membrane carefully, taking care not to bring down the top layer of agar, you can leave the plate and the membrane in the refrigerator for 15 min before removing the membrane. The membrane should be placed on a 3 mm filter paper and blotted for 5 min, then immersed in the blocking buffer and stored at 4℃.
5. RNA probe preparation
(1) Prepare the probe by in vitro transcription with T7/T3/SP6 RNA polymerase. Usually [ α-32P ] UTP is used for labeling, and nearly 1/10 of the uracil nucleotides in the synthesized RNA are labeled with the isotope.
(2) After transcription, the RNA is extracted with phenol and the probe is purified by vertical electrophoresis on a 50% acrylamide gel containing 7 mol/L urea. The probe position was determined by autoradiography, the gel was cut and recovered, and the RNA probe was eluted with HSCB buffer overnight, extracted with phenol:chloroform and quantified.
6. Co-incubation of probe and membrane
(1) Nitrocellulose membrane with phage protein was pre-hybridized in blocking buffer and incubated in shaker at 37℃ for 1 h ( 40~50 r/min ) to seal the non-specific binding site. Both the blocking buffer and hybridization buffer were very viscous, so it was necessary not only to avoid foaming, but also to ensure the homogeneous interaction with the membrane. The containers and instruments used in the operation should be pre-treated to avoid nuclease contamination.
(2) Wash the membrane twice with 500 ml of rinsing buffer and immediately transfer it to 100 ml of binding buffer with probe RNA. Incubate for 1 h at room temperature on a shaker (40~50 r/min).
7. membrane washing
Wash the membrane with 250 ml of elution buffer and repeat several times until the background level of radioactivity is minimized. Protein cross-linked membranes corresponding to unphage-transfected bacterial plates can be used as negative controls.
8. Radiation autoradiography to pick positive clones
The membrane is blotted on 3 MM filter paper, wrapped in plastic wrap, and radioautoradiographed at -70°C. The exposure time depends on the concentration of the probe. The exposure time depends on the concentration of the probe and the intensity of the signal (for a 107 cpm/ml probe, a positive result can be observed after 8-16 h of exposure). In addition, it is possible to repeat the membrane transfer operation during the protein transfer process to obtain two membranes corresponding to the same culture plate for hybridization.
Compare the fixed X-ray film with the membrane and the culture plate to find out where the positive spots correspond to the phage spots on the plate. Pick the positive spots from the plate and elute them with 1 ml of SM buffer and 2 drops of chloroform to obtain phage.
9. Confirmation of positive clones
To confirm the reliability of the positive clones obtained, a secondary screening was required. The phage particles obtained from the overnight elution of the plates were re-laid to a density of about 100 mm NZY agar plates with 200 clones, as before. The number of positive clones on the nitrocellulose membrane from the second screening should be increased by picking individual positive clones. If desired, a third screen can be performed, this time with approximately 50 clones per plate.