Team:Bielefeld-CeBiTec/Improve

Part improvement

Introduction

Fluorescent proteins play a crucial role as biomarkers and biosensors that can be utilized in a wide array of scenarios like the visualization of living cells, the depiction of the interaction between two proteins or to elucidate the fate of a single molecule[1]. The goal of our project is the development of an adaptable plant-based biosensor that can to detect a variety of chemicals. For detection the chemicals are bound by a reengineered periplasmic binding protein that subsequently binds a transmembrane receptor and activates a signaling cascade, resulting in the expression of reporter genes. We deployed a green fluorescent protein as a reporter gene to visualize the functionality of our signaling cascade in Escherichia coli .

However, the use of fluorescent proteins is limited by its environmental conditions. For instance, the investigation of acidic environments like endosomes, lysosomes, plant vacuoles, etc. remains challenging for conventional fluorescence proteins due to the loss of fluorescence under those conditions[2]. Especially the internalization pathway through the lysosome after endocytosis is a crucial part in many pharmaceutical applications like antibody-drug conjugates in cancer therapeutics and therefor an important target for the use of fluorescent based analysis[3]. Regarding these limitations of the application of fluorescent proteins we improved the fluorescent protein mRUBY3 (Part: BBa_M50009) by enhancing its pH stability with our improved part mCRISPRed (Part: BBa_M50009).

mRUBY3

mRUBY3 is a monomeric red fluorescent protein with an excitation maximum at 558 nm and emission maximum at 592 nm, which was evolved from mRUBY2[4]. mRUBY3 is characterized by its high brightness and photostability and comparatively established performance in mammalian cells[4] and therefore is widely used as Förster resonance energy transfer (FRET) acceptor or for localization studies [3,5]. With an pKa of 4.8 however it lacks a reliable stability in the lysosome, that has a pH of 4 to 5. Therefore, it is not suitable for applications like retracing of internalization pathways through the lysosome[3,6]. Because of mRUBY3s already favorable traits, an enhancement of the pH stability is beneficial for further applications.

For engineering a fluorescent protein´s ability to emit light upon excitation at a certain wavelength, with a definite intensity or even at given conditions, the focus is on altering the chromophore. The chromophore is formed in a post-translational self-modification of three amino acids inside the rigid β-barrel structure of the fluorescent protein. By oxidation, the amino acids are cyclized. This reaction results in a chromophore which properties are dictated by the amino acids forming and surrounding it[8].

For mRUBY3 it has been shown that through decisive changes in the surrounding of the protein’s chromophore an enhanced pH stability can be achieved. This improved fluorescent protein was named mCRISPRed (Part: BBa_M50009) . The mutations P146A, N147D, T162L, and I164R ensure, that mCRISPRed´s chromophore is locked in the cis isomerization through physical exclusion and hydrophobic interaction throughout pH 2 to 11, while the chromophore of mRUBY3 switches from cis to trans isomerization around pH 5, resulting in the loss of fluorescence[9].

Methods


To improve mRUBY3s (Part: BBa_M50009) pH stability, we changed four amino acids according to mCRISPRed (Part: BBa_M50009) and analyzed its fluorescence intensity on pH sensitivity. Therefore, we expressed both proteins in E.coli BL21 DE3, purified the proteins by immobilized metal ion affinity chromatography (IMAC) and detected the relative fluorescence intensity at different pH conditions using the TECAN Infinite M200 plate reader.
Click on the boxes below for a detailed description of the experiments!


Cloning




Cultivation and Expression



Purification




Protein quantification




Fluorescence measurement




In case the buttons dont work, here is the text for our methods.

Cloning


We ordered mRUBY3 as a gene synthesis from IDT and received mCRISPRed from the working group cellular and molecular biotechnology at Bielefeld University. Both genes were introduced into the expression vector pRSETb. For this the plasmid was digested with the restriction enzymes BamHI and EcoRI, the insert was amplified with primers creating a matching overlap and both pieces were joined together through gibson assembly.

Expression

To express both fluorescent proteins, E. coli BL21 DE3 was transformed with the aforementioned expression vectors, allowing an inducible expression and purification using His-tag affinity chromatography.
Therefore, we inoculated overnight cultures in 10 mL LB medium, supplemented with 100 μg/mL ampicillin, in a shake flask at 180 rpm and 37°C. The main cultures of 450 mL were inoculated at OD600 of 0.1 and at 180 rpm and 37°C until OD600 of 0.6 was reached. After that the cultures were then cooled on ice for 5 minutes and then induced with 0.4 mM IPTG. From this on the cultures were cultivated at 18°C and 180 rpm overnight, to reduce protease activity, misfolding and the formation of inclusion bodies.

Harvest and purification

After cultivation at 18°C the cultures are harvested through centrifugation at 8000x g for 3 min at 4°C. The supernatant was discarded and the pellet either stored at -20°C overnight or resuspended in 1x LEW buffer for purification. The cells were disrupted in the Precellys 24 Cell homogenizer during three cycles of 30 seconds and 6,500 rpm, with the samples being stored on ice for 5 minutes in between the cycles. Subsequently the lysate was centrifuged at 20000x g at 4°C for 20 minutes to separate cell debris from the supernatant. Purification of the proteins from the supernatant was then conducted using IMAC and Protino Ni-TED gravity flow columns. In this step the instructions of the manual for native protein purification from E. coli were followed. Afterwards, the eluted fractions were rebuffered and concentrated in PBS with Amicon centrifuge filters with a MWCO at 10 kDA. After rebuffering the fluorescent proteins in PBS, mCRISPRed was matured for several days at 4°C to develop its full fluorescence.

Protein quantification

To quantify protein yield for the fluorescence measurements, a bradford assay was performed for both isolated proteins. As reference, a BSA standard curve was measured with concentrations from 1 g/L to 0.2 g/L BSA. The samples were prepared in a cuvette with 980 µL RotiQuant solution and 20 µL protein sample diluted 1:2, 1:5 and undiluted. In a photospectrometer the samples were then measured at a wavelength of 595 nm.

Fluorescence measurements at different pH conditions

PBS solutions with pH values from 7.4 to 2 were prepared for the fluorescence stability experiment. Samples were prepared in a 96-Well plate and measured using the TECAN Infinite M200 plate reader. Per well, 8 µL of 20 nmol protein sample to a final concentration of 0.16 nmol was added to 192 µL of PBS, plus one well with just PBS as reference. Both fluorescent proteins were excited at their respective excitation optimum, mRuby3 at 558 nm and mCRISPRed at 460 nm, while the emission was detected at 592 nm for all wells. For each well the fluorescence was measured at nine spots with 25 flashes and all pH conditions were tested with three replicates per protein.

Results


After purification, both protein eluates were rebuffered and concentrated to a volume of 1.5 mL. The concentration was measured by bradford assay and a concentration of 81.6 μmol/L for mRuby3 and 20.4 μmol/L for mCRISPRed was determined. The samples were diluted to the same concentration, before fluorescence measurement at different pH conditions using the TECAN plate reader.
For direct comparison the results are displayed as relative fluorescence intensity with the respective fluorescence intensity maximum at pH 4.6 for mRuby3 and pH 3.6 for mCRISPRed. Throughout pH 6.5 to 3, the relative fluorescence intensity of mRuby 3 and mCRISPRed is similar. However, for conditions of pH 3 and lower the relative fluorescence intensity of mRuby3 drops rapidly from 87 % at pH 3.6 to 8 % at pH 3. For mCRISPRed the relative fluorescence intensity declines stepwise from pH 3.6 to 2, with the final detected value being at 33 %. At this pH level, no significant fluorescence intensity of mRUBY3 could be detected. One-sided Students t-test (n = 27) for unrelated samples proved the differences in relative fluorescence to be highly significant for pH 3; 2.5 and 2 (p < 0.001).

Figure 2: Relative fluorescence intensity measured for mRuby3 and mCRISPRed for different pH conditions ranging from pH 7.4 to pH 2 at the TECAN reader. Significance of differences was proven with one-sided t-test for unrelated samples (n=27) and are displayed as * for p > 0.05, ** for p > 0.01 and *** for p > 0.001.


The results show a significantly better relative fluorescence at low pH conditions for mCRISPRed in comparison to mRuby3, indicating an improved stability of the chromophore in its cis-isomeration. With our improvement of mRuby3 we enable future iGEM teams to utilize the potential of fluorescence proteins when working in acidic environments.

Figure 3: Picture of purified mRuby3 and mCRISPRed on UV-table

References


[1] Rizzo, M. A., Davidson, M. W. & Piston, D. W. Fluorescent protein tracking and detection: applications using fluorescent proteins in living cells. Cold Spring Harbor protocols 2009, pdb.top64; 10.1101/pdb.top64 (2009).
[2] Shinoda, H., Shannon, M. & Nagai, T. Fluorescent Proteins for Investigating Biological Events in Acidic Environments. International journal of molecular sciences 19; 10.3390/ijms19061548 (2018).
[3]Chalouni, C. & Doll, S. Fate of Antibody-Drug Conjugates in Cancer Cells. Journal of experimental & clinical cancer research : CR 37, 20; 10.1186/s13046-017-0667-1 (2018).
[4] Bajar, B. T. et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Scientific reports 6, 20889; 10.1038/srep20889 (2016).
[5] Marter, P., Schmidt, S., Kiontke, S. & Moog, D. Optimized mRuby3 is a Suitable Fluorescent Protein for in vivo Co-localization Studies with GFP in the Diatom Phaeodactylum tricornutum. Protist 171, 125715; 10.1016/j.protis.2020.125715 (2020).
[6] DiCiccio, J. E. & Steinberg, B. E. Lysosomal pH and analysis of the counter ion pathways that support acidification. The Journal of general physiology 137, 385–390; 10.1085/jgp.201110596 (2011).
[7] Stepanenko, O. V. et al. Modern fluorescent proteins: from chromophore formation to novel intracellular applications. BioTechniques 51, 313-4, 316, 318 passim; 10.2144/000113765 (2011).
[8] Heim, R., Prasher, D. C. & Tsien, R. Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 91, 12501–12504; 10.1073/pnas.91.26.12501 (1994).
[9] Erdogan, M., Fabritius, A., Basquin, J. & Griesbeck, O. Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells. Cell chemical biology 27, 610-621.e5; 10.1016/j.chembiol.2020.02.004 (2020).
[10] Sehnal, D. et al. Mol* Viewer: modern web app for 3D visualization and analysis of large biomolecular structures. Nucleic acids research 49, W431-W437; 10.1093/nar/gkab314 (2021).