An in vitro translation system for African Xenopus oocytes
An in vitro translation system for African Xenopus oocytes
The African Xenopus oocyte in vitro translation system is capable of sustained modification and processing of most exogenous mRNAs due to its ability to translocate, excise signal peptides, and glycoproteolyze. Because of its high membrane stability, the localization of transcripts can be analyzed by sucrose density gradient centrifugation or protease protection reactions.
Operation method
An in vitro translation system for African Xenopus oocytes
Materials and Instruments
Adult female African clawed toad Peptidase tRNA Protease K Move I. Materials and equipment Caveat 1) As with any in vitro translation system, care should be taken to avoid RNase activity in the oocyte in vitro translation system.2) Eggs from different females vary in quality and should be tested for activity prior to use. The ability of the eggs to be fertilized is a more effective method.3) Occasionally, a batch of eggs may not cleave well. The distinguishing mark of this occurrence is the grayish color of the extract after the first centrifugation in the preparation process, instead of amber. Such extracts do not lend themselves well to in vitro evaluation and should therefore be discarded.4) For cryopreservation, the extracts should be dispensed in l00ul/tube or less into ice pre-cooled centrifuge tubes and placed in liquid nitrogen for 1 min. After freezing, the extracts should preferably be stored in liquid nitrogen, but can be kept alive for several months if stored at 1.070℃.5) If the frozen extracts are used for translation reactions, avoid operations such as T: i.e., add the required reagents to the thawed extracts. Then divide the reactants into _ and perform the translation reaction separately. This is because after such manipulation, the concentration of the extract will be diluted and this will greatly affect its reactivity.6) The purity of RNNaseA from different sources and batches varies, so each batch should be titrated to determine the optimal concentration of RNNaseA to be used. 0.1-5ug/ml is the recommended concentration of RNNaseA, too low a concentration will not remove endogenous RNA and too high a concentration will degrade the exogenous mRNA that has been added.7) In the in vitro translation system of African Xenopus oocytes, most of the mRNAs derived from higher eukaryotic organisms can be translated well, but the mRNAs from prokaryotic organisms cannot. In addition, natural mRNAs are more efficiently translated than in vitro synthesized mRNAs.8) The amount of mRNA used for in vitro translation depends greatly on its source and activity. In general, the amount of in vitro synthesized mRNA is about 50ug/ml, and the amount of poly(A)+mRNA is 100~200ug/ml, which is not beyond the ability of the extract to be modified in vitro. If this dosage is exceeded by 1, the effective secretion of secretory proteins will be significantly reduced, and the efficiency of N-glycosylation will be drastically decreased. However, even with a very high amount of mRNA, the signal peptide was not found to be sheared.9) The maximum methionine content in the extract was about 35 umol/L (±10%), so the protein yield of the reaction can be estimated from the [35S]~methionine scoring ratio in the product of the TCA-precipitation reaction. When the reaction is performed with monoclonal mRNA, the methionine content of the product can be calculated accurately; however, when poly(A)1 mRNA is used, the methionine content is generally calculated as a percentage of the total protein yield. However, when poly(A)1 mRNA is used, the methionine content is generally estimated to be 2%. If the goal of the translation reaction is to obtain as much protein as possible, then the protein yield can be increased by adding excess amino acids by adding approximately 5% by volume of extracts containing 700umol/L methionine and 2 mmol/L of all other amino acids.10) For in vitro translation reactions: [35S] Methionine is often used, not only because it is a convenient source, but also because it is present at lower levels in the extract, which produces a better signal. However, when the methionine content of the target protein is very low or not at all, other amino acids can be used in the reaction instead of methionine. However, this assumes that these amino acids are limited in abundance in the oocytes of the African Xenopus oocyte, as is the case with methionine, e.g. cysteine, leucine, histidine and proline.11) It is preferable to perform the sucrose density gradient separation reaction or the protease protection reaction as soon as the translation reaction has been completed.12) In the protease protection assay, some signal peptides are located in the cytoplasmic sprouting membrane-penetrating proteins and the length of the peptide chain is reduced by the shearing action of the added proteinase K. The peptide chain length of the signal peptide is reduced by the shearing action of the added proteinase K. For more product details, please visit Aladdin Scientific website.
Centrifuge Cold chamber
(i) Preparation of oocyte extracts from African clawed toad (Xenopus laevis)
1) Adult female African Xenopus laevis: several pcs.
2) ModifiedBarth,X(MBS): 1.28 g of NaCl was added to each liter of solution to reach a final concentration of llOmmol/L. The final concentration of NaCl was 1.28 g of NaCl per liter of solution.
3) Plasma gonadotropins and chorionic gonadotropins.
4) Solution A; Hydrochloric acid solution of 2% cysteine, pH adjusted to 7.7 with NaOH.
5) Solution B (extraction buffer): 100 mol/L KCl,0.1 mmol/LCaCl2,1 mmol/LMgCl2,50 mmol/L sucrose and 10 mmol/L HEPES-KOH (pH 7.7)
6) VersilubeVF50: density between frog eggs and solution B.
7) Cytochalasin B: at 4°C, to be stored in DMSO
8) Peptidase: at -20°C, stored in water at 10 mg/ml.
9) RNaseA: 1 mg/ml in water; dispense and store at -20℃.
10) Ribonuclease inhibitors.
11)DTT: lmol/L, store in -20℃ by portion. It should now be diluted to lOOmmol/L for use before adding to the extract.
12)tRNA: 5 mg/ml in water, dispense and store at -20℃.
13) Centrifuge.
14) Cold room.
(ii) Translation of oocyte extracts of African clawed toad (Xenopus laevis)
1) Sarcosine phosphate: 350 mmol/LL, stored at -20°C
2) [32S]-Methionine: 100 uCi/tube, store at 70°C.
3) Spermidine: 120 mmol/L, stored at -20℃.
4) Rabbit Reticulocyte Lysate/S-100 Extract: To remove the exogenous ribosomes from the reaction, S-100 fragments must be prepared. If the exogenous ribosome is not required, then nuclease-treated reticulocyte lysate can be used instead of S-100 fragments. Preparation of S-lOO.... Centrifuge 100ul of reticulocyte lysate at 100,000 g for 2 h to obtain approximately 80ul of supernatant. After loading the supernatant in 10ul volumes, it is immediately placed in liquid nitrogen for rapid cooling and then stored at 70°C for later use. Note: Do not inhale the ribosomes when removing the supernatant.
5) Centrifuge
6) Cold room
(ii) Analysis of translation products
1) 10% TritonX-100: After opening the package, the unused solution should be stored at 4℃ and protected from light.
2)PMSF
3)TritonX-100,1 mmol/LPMSF: freshly prepared.
4)2XT buffer: 100 mmol/LKCl, 10 mmol/LMgAcetate, 200 mmol/LNaCl and 40 mmol/LTris-HCl(PH7.5)D Ultrafiltration and storage at 20℃. The products can be mixed with 40% sucrose reservoir and diluted into 1XT+10% and 1XT+20% sucrose solutions, respectively, and the unused lXT-f10% and 1XT+20% matrices can be stored at 20℃.
5) Proteinase K: 25 mg/ml stored in 50% glycerol plants.
6) Na2CO3: For each alkaline sucrose density gradient separation experiment, freshly prepare a 1mol/L reservoir solution with 100 mmol/h PH of 11, and dilute the reservoir solution with water at a ratio of 1:5 to make a 200 mmol/L use solution. The reservoir is diluted 1:5 with water to form a 200 mmol/L use solution for treating membrane fragments. Alternatively, the reservoir can be mixed with a 40% sucrose solution (1 volume of 1moL/L4 volume of water and 5 volumes of 40% sucrose solution) to make a 20% sucrose density gradient.
7) 1mol/LHCL
8) ThrTyr-Asn: Because of its poor solubility in water, the reservoir solution is usually made up of DMSOS at lOOmmol/L. For use, dilute the reservoir solution accordingly.
9) Centrifuge.
10) Cold room.
II. Methods of operation
(I) Preparation of oocyte extract of African clawed toad (Xenopus laevis)
1. Preparation of basic extract
The faster the process is carried out, the better the final extract will be. Therefore, before starting the preparation of the extract, it should always be ascertained whether all buffers, tubes and centrifuge rotors have been pre-cooled to 4°C, and that all necessary materials are available. In addition, all steps after step 2) above should be performed in a cold room at 4°C on an ice bath, unless the temperature is indicated otherwise.
1) A number of large adult females are selected and each is injected with 50~1O0U of plasma gonadotropins on day 1, followed by 500~750U of chorionic gonadotropin 3~5 days later, i.e., the evening before the preparation of the extract, in order to induce K-ovulation. Afterwards, the Xenopus laevis toads were left overnight and allowed to drain their eggs into commercial salt MBS to maintain the biological activity of the eggs.
2) Approximately 30 ml of loosely bound eggs were transferred to a 250 ml wide-mouth glass and rinsed several times with high-salt MBS before being pipetted with a plastic pipette to remove cellular debris and dead oocytes. Remove as much of the supernatant buffer as possible by adding 100 ml of Solution A, and repeat the procedure 2 to 3 times. ^ Vortex the suspended eggs from time to time for 5 to 10 minutes. When the volume of mussels occupied by the eggs is significantly reduced, the outer gelatinous sheath of the eggs is gradually dissolved. At this point, rinse the eggs with Solution A, discard the solution, and repeat the process several times before transferring the eggs to Solution B, which is pre-cooled on ice.
3) Using a wide-mouth pipette, gently pipette the eggs into four 2 ml centrifuge tubes, drawing as little as possible onto Bath B. After the eggs have been allowed to stand for approximately 1 min, carefully aspirate the supernatant, cover the surface with a layer of VersihjbeVF50, and centrifuge the eggs at 500 g for 1 min at 4°C. After centrifugation, the eggs should be tightly packed together but should not break apart. At the end of centrifugation. Solution B should be in the top layer, the Vei^lubeVF50 layer in the center, and the eggs in the bottom layer.
4) Carefully remove the upper buffer and oil layer with a 200ul pipette, then place the tube in a centrifuge and centrifuge at 20,000 g for 15 min at 4°C to lyse the frog eggs. The expected product of centrifugation consists of multiple layers of cell lysis products, of which the viscous amber-colored interplant layer accounts for approximately 40% of the volume, which is the target product, i.e., the cytoplasm. A plastic pipette is inserted through the thin layer of lipid into the middle layer. The cytoplasm is transferred.
5) Remove the product from all centrifuge tubes, estimate the volume of juice and add 10 mg/ml of cytochalasin at 5ul/ml (blow gently to mix), then transfer the mixture to a new 1.5 ml centrifuge tube and centrifuge at 20,000 g for 15 min at 4°C. After centrifugation, the cytoplasm should account for the vast majority of the volume, and care should be taken to avoid aspirating the bottom of the precipitate when it is removed. When removing them, great care should be taken to avoid aspirating the sediment from the bottom.
6) Add 10 mg/ml peptidase at 1ul/ml and mix gently and thoroughly.
The extract can be used for translation reaction, frozen storage, or the endogenous mRNA can be removed as follows.
2. Removal of endogenous mRNA
As in the case of basic extracts, the removal of endogenous mRNA is best performed in a cold room. Removal of endogenous mRNA is best done in a cold room.
1) Dilute the RNaseA Reservoir to a concentration of 100X using solution.
2) Add diluted RNawA to 1.5 ml centrifuge tubes with screw caps at lul/tube, then add 100ul of extract to each tube and blow up and down to mix. Incubate at 10 ℃ for I5 min.
3) Place the centrifuge tubes on ice and add 100 mmol/LDTT at 1ul/tube, followed by RNa inhibitor at 50U/tube. Incubate at 10 ℃ for 15 min, and add 5 mg/ml tRNA at 2ul/tube. The extract with endogenous mRNA removed can be used immediately for translation reaction or frozen in liquid nitrogen as soon as possible.
(ii) Translation of exogenous mRNA in the oocyte extract of Xenopus laevis.
1) Thaw the frozen extracts at room temperature and place them on ice.
2) At the same time, the mRNA for translation should be dispensed into 0.5 ml or 1.5 ml centrifuge tubes and placed on ice.
3) For every 100ul of extract, the following components should be added separately: 10ul of reticulocyte lysate S-100, 1ul of 120 mmol/L arginine, 2.5ul of 350mol/L phosphoinositide and lO0ul of Ci[35S]-methionine. The mixture was added to the centrifuge tubes containing mRNA at the rate of 10~50ul/tube, mixed well, and incubated at 21℃ for lh.
4) If in vitro synthesized mRNA with high radioactivity is used for translation, add lOug/ml RNaseA at the end of the reaction and incubate at 21°C for 15 min to remove the radioactive background of the radioactive mRNA that will prevent further analysis.
5) If storage is required, the reaction can be terminated by freezing at this step.
6) If analyzed by gel electrophoresis or TCA precipitation reaction, 1%X-100 and lmmol/LPMSF should be added to the reaction product before adding an equal volume of 2XSDS polyacrylamide gel electrophoresis addition buffer or filtering.
(iii) Analysis of translation products
General gel electrophoresis of the translation product often reveals the secretory phenotype: if the site-disputed peptide sequence is cleaved, the
If the peptide sequences are cleaved, the molecular mass of the product is usually reduced by 2k~3kDa; if glycosylation occurs, the mobility of the product is usually relatively lower than that of the end-modified proteins produced by translation of wheat germ extracts or reticulated erythrocyte lysates. Since other causes can also contribute to the change in mobility, we can determine this by using Egg H enzyme-protected counterstaining or sucrose density gradient centrifugation.
The real cause of the change in mobility = Alkaline sucrose density gradient centrifugation can be used to determine whether the protein is free in the endoplasmic reticulum lumen or integrated into the ten membrane, whereas specific inhibition of N-glycosylation may be manifested as an increase in molecular mass on the surface.
1. Protease protection reaction
1) Remove 3X10ul of reaction solution from the transcription reaction system and place on ice. If it is necessary to use less than 10ul of reaction solution, e.g. if the product is to be analyzed by multiple methods, add 4 times the volume of 1X10ul of reaction solution. The reaction solution can be diluted by adding 4 times the volume of 1XT buffer + 10% sucrose.
2) Add 1ul of 10% TrimnX-100 to each tube to lyse the membrane present and provide a positive control for protein hydrolase.
3) Add 1ul1/ml of Proteinase K solution to 10% sucrose solution with Triton (one of the two samples was left out as a stability control during processing) and place on ice for 1 hour.
4) Dilute lOOmmol/LPMSF with 3 times the volume of 10% sucrose solution: add PMSF at 1ul/reaction to reach a final concentration of 2~2.5 mmol/L and continue incubation on ice for 15 min.
5) Add lOOul2XSDS polyacrylamide gel spiking buffer including 1% TntonX-100). Boil for 5 min, if the sample is diluted before protease processing, add a smaller volume of 2XSDS Polyacrylamide Gel Tunneling Buffer so that the volume of extract in the final mixture is not less than 10%.
6) SDS polyacrylamide gel electrophoresis analysis (which consists of -tubes of identically diluted untreated samples from the same -inverse setup system . . and used as a marker).
2. Neutral sucrose density gradient separation
Sucrose density gradient separation can be used not only for analytical purposes. It can also be used to analyze the purity of the samples prior to activity analysis.
Assuming a basic sucrose density gradient as described below, it is recommended that the starting volume of the reaction be set to a minimum of 50 ul, i.e., so that the volume can be more easily controlled.
1) Dilute the sample with 10 times the volume of 1XT buffer + 10% sucrose on ice. Leave a small portion of the sample as a marker for SDS polyacrylamide gel electrophoresis and carefully spread the remaining portion into a 1.5 ml centrifuge tube containing 1 ml of 1XT buffer sucrose solution.
2) Centrifuge at 4°C, 40,000 g for 30 min.
3) Recover the top layer containing the cytoplasmic proteins (10% sucrose solution), avoiding intermixing this layer with the 20% sucrose solution layer in order to improve the purity of the product. Remove and discard the remaining sucrose buffer, being careful not to touch the brownish membrane beads at the bottom of the tube.
4) If membrane stability is necessary for experiments such as protease protection reactions or alkaline sucrose density gradient separations, gently resuspend the membrane beads with one-half the volume of 1XT Buffer X 20% Sucrose Solution from the sucrose density gradient described above, and then dissolve the membrane beads by adding TritonX-100 and PMSF to a final concentration of 1%X-100 and Immol/LPMSF, respectively.
5) The total reaction products as well as each fraction were analyzed by SDS polyacrylamide gel electrophoresis.
3. Density gradient separation of basic sucrose
Alkaline Suchen disrupts the vesicle membrane and releases the proteins from the vesicles, but does not dissolve the lipid bilayer. After sucrose density gradient separation, the vesicular proteins will remain in the supernatant, while the membrane-bound fraction will precipitate as small beads.
1) Take 1OOul of membrane solution obtained from step 4) of "Neutral Sucrose Density Gradient Separation" in 2 of "Analysis of Translation Products", add an equal volume of 200 mmol/L Na2C03, and incubate on ice for 30 min.
2) Spread 250ul of Na2C03 solution from the previous step into a 1.5 ml centrifuge tube containing lml of 20% sucrose solution and centrifuge at 100000 g for 1 h at 4℃.
3) Remove 100~150ul of supernatant. Note: Allow for a small loss of supernatant, do not remove all of the supernatant. After removing the 20% sucrose solution, re-dissolve the membrane beads with 1% TritonX-100, 1 mmol/LPMSF.
4) Prior to SDS polyacrylamide gel electrophoresis analysis, approximately 10% of the total volume of lmol/L HCL should be added to neutralize the supernatant. Note: HC1 should be added slowly and mixed with constant stirring to prevent localized precipitation of the protein fractions. In addition, the pH of the solution should be checked continuously with pH paper to prevent overdose of HC1.
4. Specific N-glycosylation inhibition
Traditionally, N-glycosylation sites on peptide chains can be detected by endoglycosidase H, which can specifically cleave the peptide chain. Here, we will introduce a new and completely different strategy to inhibit the glycosylation of translated peptides of African Xenopus laevis extracts in vitro by incorporating competitive Thr-Tyr-Asn' peptide sequences, in order to reveal whether or not the translated peptide sequences have been N-glycosylated.
1) Prepare two series of diluted aqueous solutions of the dipeptide in the range of 1.5~50mol/L. The concentration of the dipeptide in the range of 1.5~50mol/L was determined.
2) Prepare a minimum 80UL in vitro translation reaction system on ice, which should contain mRNTA for translation into the target protein.
3) Add lul of a series of concentration gradient dilutions of the tripeptide solution to each 9ul of translation reaction solution. In addition, take one 9ul of translation reaction solution and add 1ul of DMSO in place of the tripeptide solution as a control for the effect of DMSO. Incubate these mixtures and the remaining reaction as a positive control at 21 °C for 1 h. The samples should then be analyzed by SDS poly(lacton amide) gel electrophoresis, which should include an unglycosylated protein translated from reticulocyte lysate as a marker, and the results of the electrophoresis should show that the specifically inhibited glycosylated egg is moving faster than the glycosylated protein.