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The 3' intron of td gene from T4 phage

This part is a component of the td intron (5' side), an intron of the td gene from T4 phage belonging to group IE, which can form a circular mRNA (cmRNA) to make the ribosomes repeatedly translate the extron. This strategy can be used for expression of long peptides containing tandem repeats, like fibrous proteins.

The mechanism of cmRNA

Due to the special internal structure, the td intron can circularize the exon to form a back-splice junction (BSJ) in a reaction catalyzed by guanosine. To ensure that the ribosomes do not translate the 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. 1).

Fig. 1 Design of a circular mRNA based on td flanking introns.

In our project, we used this part to design a cmRNA to produce the squid ring teeth proteins with five tandem repeats (TRn5).

The constructed plasmid pET29a(+)-cmRNA was transformed into E.coli BL21 (DE3) and recombinant proteins were expressed using LB medium (Fig. 2).

Fig. 2 The plasmid map of pET29a(+)-cmRNA.

After incubation at 37℃ for 5h and 16℃ for 20h, respectively, we found that the TRn expression level in both supernatant and pellet was pretty low. 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 circular mRNA. 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. 3).

Fig. 3 SDS-PAGE of expression products of cmRNA. Lane 1: marker; lanes 2 to 5: whole-cell lysate, supernatant, pellet and diluted pellet from induced cells at 37℃, respectively; Lane 6: marker; lanes 7 to 9: whole-cell lysate, supernatant and pellet from induced cells at 16℃, respectively.

Then, we purified TRn on a HiTrap Ni-NTA column. However, the TRn expression level was too low to verify by SDS-PAGE (Fig. 4).

Fig. 4 SDS-PAGE of expression products of cmRNA purified by IMAC. Lanes 1 to 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 to 6: sample eluted with 50, 150,300 and 500 mM imidazole; lane 7: marker; Lanes 8 to 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; lane 10 to 13: sample eluted with 50, 150 and 300 mM imidazole.

To optimize the TRn expression, we reviewed plenty of literature, from which we found that TRn could easily be dissolved in 5% acetic acid (pH≈3) due to the existence of Histidine. Thus, we used a new protocol to obtain the purified TRn. Solubilized in 5% acetic acid, the bands of TRn were still seen as a form of ladder, which is a symbol of multi-circle translation of cmRNA. (Fig. 5)

Fig. 5 SDS-PAGE of expression products of cmRNA. Lane 1: marker; lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively; lane 5: sample washed with 5% acetic acid.

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.

Sequence and Features