Part:BBa_K4674010

The mGL-4A-C7 expressing cassette

This expressing cassette is regulated by lac operator. The N-terminal of mGL-4A-C7 protein is fused with a 6xHis-tag and a thrombin cleavage site.

The mGL fluorescent protein is developed for the expression in the mammalian systems, since the low soluibity or misfolding of eGFP limit the total fluorescence output of whole cells.

Cellular Brightness and Maturation

In the characterization, nGL shows 630% greater brightness than eGFP. The maturation half-time of mGL is 13.5 min, which is 207% faster than eGFP (33.0min). mGL is compatible with common EGFP filter sets and the popular 488-nm argon-ion laser line for widefield and confocal microscopy. Its excitation and emission peaks of 503/514 nm additionally make it well matched to newer solid-state 491-nm lasers and 505-nm light-emitting diodes (LEDs).

Biochemical Characterization

The QY and EC of mGreenLantern (ϕ = 0.72, ε = 101 mM−1⋅cm−1) compare favorably with those of other bright GFPs such as Clover (ϕ = 0.75, ε = 97 mM−1⋅cm−1), and its low pKa of 5.6 indicates greater acid resistance than EGFP.

mGreenLantern completely lacks a 405-nm absorbance peak, unlike EGFP and Clover, suggesting that it will be better suited for multicolor imaging with T-Sapphire.

Chemical and thermal stability

In the strongly denaturing solution of 6.3 M guanidinium HCl, pH 7.5, mGL persisted for 100 s before reaching half the fluorescence of its native control wells, while other fluorescent protein (e.g. eGFP, eYFG, and Clover) denatured instantly. The melting temperture of mGL is 87.2 °C, which is much higher than the mNeonGreen (68.0 °C), eGFP (80.3 °C), or sfGFP (86.4 °C)

Antibody recognition

The mGL protien could be recognized at least by three commercial antibodies (goat α-GFP polyclonal, Abcam, #ab6673; or goat α-GFP polyclonal, Novus, #NB1001770; or Ms α-GFP monoclonal, Life Technologies, #A-11122). The truth that 91% sequence identity of mGL shared with eGFP strongly implies that mGL is readily compatible with existing technology for EGFP affinity-related applications.

Photostability

When analyzed using normalized bleaching plots beginning at time 0 to include the Emerald-like behavior, mGreenLantern’s photostability was better than Venus and lower than Clover.

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Table 1. Spectroscopic characterization of fluorescent protein (quoted from (Campbell et. al. 2020)

C7 dodecapeptide

The sequence of C7 peptide is Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser, which is developed from the phage library screening. The ability of C7 peptide to recognize FRα is proved by FACS analysis of SKOV3 cell lines in vitro and the in vivo tumor staining. The equilibrium dissociation constant (KD) value between C7 and FRα was 0.3 μM. Importantly, the computational modeling and interaction analysis indicated that C7 binds with FRα close at the entrance of the pocket, which avoid the competition to folic acid (Xing et al. 2018)

The design of mGL-4A-C7

To efficiently label the CTCs captured by DNA tetrahedron, we first perform modeling and determine to link mGL protein and C7 dedocapeptide with 4 alaines (mGL-4A-C7). To expression the mGL-4A-C7 fusion protein, we design and cloned the composite par BBa_K4674010 into the pET15b. The fusion protein was induced by IPTG and purified by FPLC. Finally, to confirm whether the mGL-4A-C7 is functional, we perform the labeling experiments by SKOV3 cell line, which is the mimic of the captured CTC. (Due to the highly expression of FRα in SKOV3 cells)

Fig. 1: The design of mGL-4A-C7

Experimental confirmation

To examine whether the mGL-4A-C7 protein is functional in labeling CTCs as the modeling result, we builded the composite part (BBa_K4674010) for mGL-4A-C7 protein expression. After confirming the expression cassette insertion into pET-15b, we induced the mGL-4A-C7 protein expression by adding 1mM IPTG at 37°C. The Coomassie blue staining of PAGE analysis clearly showed that the induced mGL-4A-C7 protein was soluble and could be purified by Ni2+ beads.

Fig. The coomassie blue staining of SDS-PAGE analysis of mGL-4A-C7 protein induced by IPTG.

To purify a large batch of mGL-4A-C7 and eGFP-4A-C7 protein for further examination and application, we performed the FPLC analysis to separate the non-specific binding protein from target protein. The absorption spectra at 280 nm indicated that there are two main proteins containing fractions. One is from 14th to 18th fraction, and the other is from 21st to 38th fractions.

Fig. The 280 nm absorption spectra of proteins eluted from mGL-4A-C7 purification

Although the greens fluorescence clearly shown in the 21-38 fraction, we still performed the SDS-PAGE analysis, confirming the existence of mGL-4A-C7 protein.

Fig. The coomassie blue staining of SDS-PAGE analysis of fractions from FPLC elution.

After confirming the existence of target proteins, we freeze-dried the fractions containing mGL-4A-C7 or eGFP-4A-C7 proteins. The freeze drying protein was then rehydrated to the same concentration for examination.

We first examine whether the mGL exhibits a strong brightness when excited by 488 nm laser. The value of fluorescence brightness detected by standard eGFP filter indicated that, the mGL exhibits fluorescence at least twofold fluorescence brighter than eGFP at the same concentration, and protein could be stored at room temperature at least one week.

Fig. The fluorescence detection of rehydrated mGL-4A-C7 and eGFP-4A-C7 stored at room temperature using the standard eGFP filter.

Next, we examine whether the mGL-4A-C7 protein could recognize the CTC mimics, the SKOV3 cells. Accordingly, we incubated SKOV3 cells with medium containing different concentrations of mGL-4A-C7 and eGFP-4A-C7 (50μg/ml, 100μg/ml, 200μg/ml) at 37 ℃ for 2 hours. The observation of fluorescence indicated that the 50 μg/mL of mGL-4A-C7 could label the SKOV3 cells, while the 50 μg/mL of eGFP-4A-C7 showed moderate labeling. Furthermore, the extension of incubating time to 4 hours did not significantly improve the labeling. Together, we confirmed that the mGL-4A-C7 could label the SKOV3 cells, suggesting mGL-4A-C7 protein as CTC labeling reagent.

Fig. The observation of mGL-4A-C7 and eGFP-4A-C7 labeling of SKOV3 cells.

Reference

Benjamin C. Campbell, Elisa M. Nabel, Mitchell H. Murdock, and Gregory A. Petsko. mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging. Proc Natl Acad Sci U S A. 2020 Dec 1;117(48):30710-30721

Lijun Xing, Yifeng Xu, Keyong Sun, Hong Wang, Fengguo Zhang, Zhengpin Zhou, Juan Zhang, Fang Zhang, Bilgen Caliskan, Zheng Qiu, Min Wang Identification of a peptide for folate receptor alpha by phage display and its tumor targeting activity in ovary cancer xenograft Sci Rep. 2018 May 30;8(1):8426.

Sequence and Features

Assembly Compatibility:

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• 12
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