Part:BBa_K5398003
The 3' intron of td gene from T4 phage
This part is a component of the td intron (3' side), an intron of the td gene from T4 phage belonging to group I introns, which can form a circular mRNA (cmRNA) to make the ribosomes repeatedly translate the extron. This year, we utilized the td intron to produce the squid ring proteins with various long tandem repeats. We explored different production and purification strategies of target protein produced by cmRNA and examined the function of protein.
Introduction
Due to special internal structure, the td intron, also called RNA cyclase ribozyme, can splice themselves out without assistance from the spliceosome or other proteins, and instead rely on a free guanosine nucleotide to initiate the splicing reaction in vivo. This process results in joining of the flanking exons and circularization of the intervening intron to produce an intronic circRNA (Fig. 1). So it is a strategy to produce circular RNAs in vivo.
Fig. 1 Mechanism of group I introns. (GOMES R M O da S et al. 2024)
Therefore, an engineering cmRNA was designed by employing the RNA cyclase ribozyme mechanism. This elaborate design of cmRNA sequence circularizes the exon to form a back-splice junction (BSJ) in a reaction catalyzed by guanosine. To ensure that the ribosomes do not translate the open reading frame (ORF) of gene of interest (GOI) from unprocessed linear mRNA, the ribosome binding sequence (RBS) and start codon ATG were placed downstream of GOI coding sequence. Consequently, the regulatory sequences were located upstream of the coding sequence only after circularization of the mRNA. To purify the resulting polypeptides, a His tag was incorporated into the GOI. If the mRNA is circularized, the ribosome could circle the cmRNA, producing a long repeating polypeptide (Fig. 2).
Fig. 2 Design of a circular mRNA based on td flanking introns.
Usage and Biology
In our project, given the positive correlation between number of repeat units and magnitude of cohesive force, we designed a circular mRNA on which the OFR of TRn5 ( BBa_K5398001) between the 3' and 5' intron of td gene from T4 phage (BBa_K5398002 and BBa_K5398003). This strategy could use short sequences to express highly repetitive squid ring teeth proteins. A self-cleaving RNA cyclase ribozyme was incorporated to form the circular mRNAs, allowing ribosomes to repeatedly translate the sequence of interest and producing proteins with different repeat numbers, thus we could obtain proteins with exceptional self-healing properties.
Characterization
Protein expression
The synthetic plasmid pET-29a(+)-cmRNA(TRn5) was transformed into E.coli BL21 (DE3) and recombinant proteins were expressed using LB medium (Fig. 3).
Fig. 3 The plasmid map of pET-29a(+)-cmRNA(TRn5).
Optimization of incubation temperature
Aim: To determine which incubation temperature is beter for protein expression using mRNA circularization.
Methods: The cells were inoculated in LB media at 37℃ for 5 h, 23℃ for 16 h and 16℃ for 20 h respectively. The cultures were induced with 1 mM IPTG and the proteins were expressed. An SDS-gel was used to assess the results.
Results:
① Proteins formed a ladder on the gel
The TRn polypeptide was composed of repeating units with a size of 16 kDa, which was formed by the ribosome traveling one round along the cmRNA. Due to uncertainty of the round number that the ribosome traveled, TRn sample was a mixture of proteins with various sizes that formed a ladder on the gel. According to the protein marker, we supposed that the sizes of the proteins ranged from about 8 to 96 kDa, indicating that the ribosome could travel along the cmRNA at least 6 rounds (Fig. 4).
② The strategy of cmRNA facilitated the solubility of TRn
It was proved that TRn is a sort of inclusion body protein expressed in E.coli from plenty of literature. In our SDS-PAGE results, though part of TRn in the precitate, a substantial portion of TRn existed in inclusion body protein supernatant, which indicated the strategy using cmRNA could improve protein solubility.
③ Incubation temperature barely influenced the TRn expression
From the SDS-PAGE of expression products of cmRNA at different incubation temperatures (Fig. 4), we found there were few differences among them. This showed the strategy employing cmRNA to express TRn had a low requirement.
Fig. 4 | SDS-PAGE of expression products of cmRNA at different incubation temperatures.
a. SDS-PAGE of cmRNA expressed at 23℃. Lane 1: marker; lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells, respectively. b. SDS-PAGE of cmRNA expressed at 37℃ and 16℃. Lane 1: marker; lanes 2-5: whole-cell lysate, supernatant, pellet and diluted pellet from induced cells at 37℃, respectively; Lane 6: marker; lanes 7-9: whole-cell lysate, supernatant and pellet from induced cells at 16℃, respectively.
Optimization of IPTG concentration
Aim: To determine which IPTG concentration is beter for protein expression using mRNA circularization.
Methods: The cells were inoculated in LB media at 37℃ for 5 h. The cultures were induced with 0.5 mM and 1 mM IPTG and the proteins were expressed. An SDS-gel was used to assess the results.
Results: From the SDS-PAGE (Fig. 5), we found that the TRn expression level at two IPTG concentration (0.5 mM and 1 mM) had little difference and the protreins also formed a ladder on the gel.
Fig. 5 | SDS-PAGE of expression products of cmRNA induced with different IPTG concentration.
Lane 1: marker; lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG, respectively; lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells with 1 mM IPTG, respectively.
Protein purification by Immobilized Metal Affinity Chromatography (IMAC)
Aim: To purify the protein by IMAC (Immobilised Metal Affinity Chromatography) using Ni-NTA resin.Methods: The cells were induced with 0.5 mM IPTG and inoculated in LB media at 37℃ for 5 h. The cultures were centrifugated to get the supernatant and pellet. Next, the following steps were used:
Results: From the SDS-PAGE (Fig. 6), we found that the TRn expression level was too low to verify by SDS-PAGE. We supposed the His tag on TRn could not function well because it was not at the C or N terminal of targeting proteins like others, which posed a challenge for protein purification.
Fig. 6 | SDS-PAGE of expression products of cmRNA purified by IMAC.
Lanes 1-6: induced cell sample at 16℃; lane 1: sample after being bound to Ni-NTA resin; lane 2: sample eluted with 20 mM Tris-HCl; lane 3-6: sample eluted with 50, 150,300 and 500 mM imidazole; lane 7: marker; Lanes 8-13, induced cell sample at 37℃; lane 8: sample after being bound to Ni-NTA resin; lane 9: sample eluted with 20 mM Tris-HCl; lanes 10-13: sample eluted with 50, 150 and 300 mM imidazole.
Protein purification using a new protocal
Aim: To purify the protein using a new protocal containing 5% acetic acid.
Methods: The cells were induced with 0.5 mM IPTG and inoculated in LB media at 37℃ for 5 h. The cultures were centrifugated to get the supernatant and pellet. Next, the following steps were used:
Results: From the SDS-PAGE (Fig. 7), we found that the TRn dissolved in 5% acetic acid still presented a ladder on the gel. And due to unpredictable and intermittent translation, the bands of TRn were a little shallow to recognize.
Fig. 7 | SDS-PAGE of expression products of cmRNA.
Lane 1: marker; lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively; lane 5: sample washed with 5% acetic acid.
Self-healing test
We obtained protein samples of TRn by freezedrying 24 h. The final yield was about 187.2 mg/L bacterial culture. Next, we dissolved protein samples in 5% acetic acid to reach 20 mg/μL, cast them into square models and dried them at 70℃ for 3 h to obtain protein films.
Fig. 8 | The freeze-dried protein sample.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 217
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
[1] LIU L, WANG P, ZHAO D, et al. Engineering Circularized mRNAs for the Production of Spider Silk Proteins[J]. Appl. Environ. Microbiol., 2022, 88(8): e00028-22.
[2] PERRIMAN R, ARES M. Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo[J]. RNA, 1998, 4(9): 1047-1054.
[3] LEE S O, XIE Q, FRIED S D. Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins[J]. ACS Cent. Sci., 2021, 7(10): 1736-1750.
[4] OBI P, CHEN Y G. The design and synthesis of circular RNAs[J]. Methods, 2021, 196: 85-103.
[5] GOMES R M O da S, SILVA K J G da, THEODORO R C. Group I introns: Structure, splicing and their applications in medical mycology[J]. Genet. Mol. Biol., 2024, 47: e20230228.
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