Ribosome Inactivation Demonstration System Experiment
Ribosome Inactivation Demonstration System Experiment
Here we present a new strategy for linking genotype and expression in vitro for the selection of functional proteins. In this strategy, the A chain of the ricin protein is activated upon translation, so that a stable complex of ribosome, messenger mRNA and translated protein is formed without removing the stop codon. This experiment is derived from "A Guide to Modern Protein Engineering Experiments" [German] K.M. Arndt, K.M. Miller, eds.
Operation method
Ribosome Inactivation Display System
Materials and Instruments
RTA Gene Move The methods presented here outline the construction of expression vectors as well as the construction of a RIDS loop system for screening functional proteins. For more product details, please visit Aladdin Scientific website.
Binding (or rinsing) buffer Biotin agarose Elution buffer
RNeasy™mini kit
3.1 Construction of RIDS expression vectors
First, DNA encoding the T7 promoter, protein library, linker, RTA, and spacer region needs to be prepared to construct the RIDS (Fig. 13.2A). 13. 3.1.1 to 13.3.3.3 describe this process in detail. Although it is theoretically possible to prepare double-stranded DNA by polymerase chain reaction (PCR), we recommend the use of plasmids to construct this DNA because it is difficult to ensure that non-specific amplification will not be introduced by using PCR to ligate the different parts (including the T7 promoter, protein library, linker, RTA, and spacer region). In this section, we describe the process of constructing the RIDS vector. Since the use of standard re
DNA, the DNA manipulation process will not be repeated here due to space constraints.
3.1.1 Constructing the pLRS Expression Vector
First, we need to prepare the universal vector pLRS, which encodes the T7 promoter, protein library, linker, RTA, and spacer region like the DNA used to construct the RIDS (Fig. 13.2 A ). This vector is based on the pET30a vector (Novagen ) expression system. We can easily construct RIDS protein libraries by inserting cDNA libraries or random libraries into the sites of the pLRS vector.
A linker fragment was synthesized using a DNA synthesizer and inserted into the pET30a vector through the XbaI/XhoI site downstream of the T7 promoter. The linker encodes NheI, the glycine/serine enrichment sequence of phage M13 gene II, NcoI, Bam HI, Pstl, and XhoI (in order from upstream). The DNA coding for the RTA was obtained by PCR from the pUTA plasmid (a gift from Prof. J. Robertus of the University of Texas) and inserted downstream of the glycine/serine-rich sequence through the NcoI/Bam HI site. The DNA encoding the spacer region was derived from the phage M13 gene HI and was inserted downstream of the RTA via the Bam Hl/Pst l site.The sequence of the DNA (excluding the inserted RIDS protein library sequence) is shown in Fig. 13.2 B. The sequence of the DNA is shown in Fig. 13.2 B. The sequence of the DNA is shown in Fig. 13.2 B. 
The 71-mer sequence between the T7 promoter and the first ATG of the protein library is derived from the pET30a vector. a flexible linker upstream of the RTA is necessary for the primed protein to fold into the correct three-dimensional structure and to remove the spatial barrier between the protein library and the downstream RTA. Previous studies have shown that the amount of protein/mRNA selected strongly depends on the length, composition, and sequence of this linker [ 6, 16, 17]. In the current study, we used 44 amino acids containing glycine/serine-enriched sequences as linkers.
Removing the spatial barrier of RTA is very important. This is because the stabilization of the mRNA-ribosome-protein complex is achieved by the introduction of the RTA gene in RIDS. In addition to this linker, the spacer region at the 3' end of the readable frame (ORF) is also very important. This spacer region acts as an anchor to connect the ribosome and must be of the right length to occupy the long channel of the ribosome [ 18, 19] and to allow the incipient RTA to fold correctly without any spatial obstacles [ 20, 21]. It has been reported that a spacer region of at least 20-30 amino acids is required at the C-terminus to maintain the activity of enzymes displayed on ribosomes [ 22, 23]. Therefore, the amount of mRNA isolated on the ribosomal display system is affected by the length of the spacer and the secondary structure of its 3' end. We introduced a long and a short spacer sequence at the 3' end of the ORF to compare their effects on translation and selection, respectively. We found that in our system, the long spacer (404 amino acids encoded by the full-length gene III of phage M13) is inappropriate for translation and selection.
Therefore, in all our experiments, we used a fragment of 121 amino acids as a spacer.
3.1.2 Construction of pStAv-R and pGST-R vectors
In order to calibrate the proposed methodology and the potential applicability of RIDS, we introduced genes encoding streptavidin or GST in DNA libraries as model studies (Fig. 13.3 A ). These proteins are often fused to newly discovered proteins or interacting molecules, and the functional properties of the newly discovered fusion proteins have been successfully investigated [ 25-28]. Streptavidin and GST bind to biotin and glutathione, respectively, and this binding has high affinity and specificity. Therefore, isolation and confirmation of protein-ribosome-mRNA complexes may be easy.
DNA encoding streptavidin and GST were excised from pSTA (a gift from Prof. M. Sisido, Okayama University) and pGEX-4T-3 (AmershamBiosicences) plasmids, respectively. pStAv-R and pGST-R plasmids were constructed by amplifying and ligating these fragments into the XbaI/NhI sites of the pLRS plasmid, respectively, using PCR. These fragments were amplified by PCR and ligated into the XbaI/NhI sites of pLRS plasmid to construct pStAv-R and pGST-R plasmids.
The sequences of the DNA encoding streptavidin or GST are shown in Figures 13.3B and 13.3C. 
3.1.3 Construction of pStAv-mR and pGST-mR Plasmids
In order to confirm that RTA is beneficial in stabilizing the ribosomal complex, we changed the functional amino acids of RTA by point-specific mutation in the control experiments, and obtained the mutation inactivation gene of RTA. The steps were as follows: glutamic acid at position 177 was mutated to glutamine; arginine at position 180 was mutated to histidine; and glutamic acid at position 208 was mutated to aspartic acid. The mutation of these three amino acids completely inhibited the activity of RTA [ 29, 30 ]. To construct the pStAv-mR and pGST-mR plasmids, each plasmid (pStAv-R or pGST-R) replaced the RTA gene with the RTA mutant gene in a standardized way [31], with the following primers: 5'-TTG CAT CCA AAT GAT TTC AAAGC AGC AC A C T T T CCA A T A T A T TA G GGA G AA ATG-3' (underlined for the E177A and R180H mutations), and 5'-G A T CCT AGC G TA A T T AC A C T T G AG G AT CCT AGCG T A A T T AC A C TT GA-3' (underlined for the E208D mutation).
3.2 Selection of functional proteins for the RIDS cycle
In a standard recombinant DNA approach, linear DNA is generated by XhoI cleavage of the 3' end of the ORF containing either a random library or a cDNA library (in our model study, pStAv-R, pGST-R, pStAv-mR, and pGST-mR) This cleavage reaction is required to terminate transcription.
3.2.1 DNA library to mRNA transcription
In the presence of T7RNA polymerase, the DNA is transcribed and contains a cap-like structure. In our model study, DNA was transcribed with the T7 Ampliscribe™ kit at the recommendation of the vendor (Epicentre Technologies Co.).
( 1 ) 1 μg of linear DNA was added to 20 μl of transcription mixture containing 3 mmol/L cap analog, 7.5 mmol/L rATP, rCTP, rUTP; 0.75 mmol/L rGTP; 10 mmol/L reaction buffer, and AmpliScribe T7 enzyme solution, as recommended by the vendor.
( 2 ) The reaction mixture was allowed to stand at 37°C for 2~4 h to produce various mRNAs (we named these mRNAs: StAv-R, pGST-R, pStAv-mR, and GST- mR, respectively).
( 3 ) After the transcription reaction, the template DNA was completely digested with DNase I, because the residual template DNA would affect the later selection steps.
( 4 ) Purify mRNA with the RNeasy™ mini kit as recommended by the supplier (Qiagen).
3.2.2 Affinity Selection of Ribosome-mRNA-Protein Complexes and Isolation of mRNAs
RNA-encoding libraries (in our model experiments, StAv- R, pGST- R, pStAv-mR, and GST- mR) and the Flexi Rabbit Reticulocyte Lysis System (Promega; see Note 1) were prepared.
( 1 ) Add 2 μg of mRNA to 40 μl of translation mixture containing 33 μl of Flexi Rabbit Reticulocyte Lysate, 40 mmol/L KCl, 40 μmol/L Total Amino Acid Mix, and 40 U of Recombinant RNasin Ribonuclease Inhibitor (Promega), and volume to 50 μl with distilled water. Do not add DTT and Mg2+ ions (see Note 1).
( 2 ) Allow to stand at 30°C for 20 min. In our study, three mRNA systems were translated (2 μg of RNA for each system: mRNA mixed in a 1:1 ratio, and mRNA encoding streptavidin or GST (StAv-R and GST-R) as a control; see Note 2).
( 3 ) After translation, the beads are immobilized by the addition of 1 ml of appropriate binding buffer [ see 13.2 ( 13 ) ] and ligand. In our study, 10 μl of biotin agarose or glutathione bead termination solution was added (see Note 3).
( 4 ) The binding reaction is favored by standing at 4°C for 1 h with gentle stirring. Although standing at room temperature is acceptable, we recommend 4°C because nonspecific binding is sometimes detected in antisense transcription PCR (RT-PCR) (see Note 4).
( 5 ) After the binding reaction, centrifuge at 500 g for 1 min to precipitate the ligand-immobilized beads.
( 6 ) Remove the supernatant containing unbound mRNA and protein.
( 7 ) Add 200 μl of rinse solution [see 13.2 (13)] and mix gently for 30s.
( 8 ) Centrifuge at 500 g for 1 min to precipitate the ligand-immobilized beads.
( 9 ) Remove the supernatant containing unbound mRNA and protein.
( 10 ) Repeat the rinsing steps (7 to 9) two or three times.
( 11 ) Add 100 μl of Elution Buffer [see 13.2(16)] and shake vigorously for 30 min at room temperature to isolate the bound mRNA from the Precipitation Ligand Fixed Beads.
( 12 ) Purify the eluted mRNA with the RNeasy™ kit according to the supplier's recommendation and concentrate the purified mRNA with a vacuum pump.
3.2.3 Antisense Transcription
Antisense transcription reactions were performed with RT. In our model study, antisense transcription reactions were performed with ReverTra Ace (Toyobo) according to the supplier's recommendations.
( 1 ) Purified mRNA, 4 μl of 10 μmol/L RT primer, 2 μl of each 10 mmol/L dUTP, and 4 μl of 5X RT buffer were fixed to 18 μl with distilled water without RNase inhibitor and ReverTra Ace according to the supplier's recommendation.The RT primer was used to (5'-G G T T G G T A G G C T T T T T T G G C A C A G G T G G C -3' ) to recognize the upstream portion of the RTA sequence.
( 2 ) The mixture was denatured for 5 min at 65°C and quickly cooled on ice.
( 3 ) Add 1 μl RNase inhibitor and 1 μl ReverTra Ace to the mixture.
( 4 ) Reverse transcription was performed at 42°C for 1 h. The mixture was then allowed to stand at 99°C for 5 min. Then inactivate the enzyme by standing at 99°C for 5 min.
3.2.4 Polymerase chain reaction
The PCR reaction was carried out using heat stabilized DNA polymerase. In our model study, the PCR reaction was performed with KOD Dash (Toyobo) as recommended by the supplier.
( 1 ) 20 μl of cDNA, 10 μl of 10X PCR buffer, 10 μl of each 2 mmol/L dUTP, 1 μl of 10 mmol/L RT ( as a downstream primer), and 2 μl of 10 μmol/L upstream primer were fixed to 99 μl with distilled water without KOD Dash. upstream primer ( 5'-AAT TTT GTT TAA CTT GAA GGA G-3') was used to recognize the downstream portion of the T7 promoter sequence.
( 2 ) The mixture was left at 98°C for 2 min to denature and then quickly cooled on ice.
( 3 ) Add 0.5 μl KOD Dash.
( 4 ) Run the PCR program (98°C for 1 min; then 98°C for 10 s, 55°C for 2 s, 72°C for 30 s, 15~30 cycles; finally 72°C for 10 min). 98°C was left for 1 min.