Experiment and Results

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LAB Work

Experiment and Results

Experimental Aims

Our project addresses the lactate-rich tumor microenvironment by engineering a lactate-responsive module that enables immune cells(e.g., CAR-T cells in Car-T therapy) to secrete lactate oxidase (sLOx) and reduce excess lactate. To build this module, a lactate sensing protein is split to two fragments, which are fused to the N-terminal and C-terminal fragments of TEV-protease, respectively. High lactate induces TEV-protease reconstitution, which then cleaves a GV2ER fusion protein to release the transcription factor GV (Gal4-VP64). GV enters the nucleus and activates sLOx expression, lowering lactate concentration and enhancing immune cell anti-tumor efficacy.

Experimental Text

2.1 Design and Results

2.1.1 Design

Our ultimate goal is to design a highly responsive lactic acid sensor. After an extensive review of the relevant literature, we have formulated the following preliminary plan:

1. Our design is based on the study by Michael et al. In this study, TEV protease is divided into two inactive components, N-TEV and C-TEV. When brought close together through protein-protein interactions (PPIs), they can reassemble into active TEV protease.

2. We are using LIdR as the lactate sensor. When lactate is present in the environment, LIdR undergoes conformational changes. Therefore, we combined the split TEV protease fragments with LIdR fragments. When LIdR senses high levels of lactate, it will trigger the PPI of TEV protease, activating the downstream signaling cascade.

3. We chose GV2ER to respond to TEV activation and mediate the expression of the downstream sLOX gene. GV (Gal4-VP64) is an important transcription factor that is fused to the ERT in the cytoplasm. Between GV and ERT, we inserted a TEV protease cutting site. When the TEV protease is activated, GV can be released from the module and enter the nucleus to activate downstream gene expression.

4. Initially, we chose LOx (lactate oxidase) to target the lactate tumor microenvironment. We inserted a secretion peptide sequence upstream of LOx, enabling its secretion outside the cell as sLOx (secreted lactate oxidase). Additionally, we added the UAS promoter upstream, allowing GV to bind with UAS and activate gene expression. This function is critical in scenarios that require control over lactate levels to maintain microenvironment stability or regulate biological processes.

5. Cell Line Selection: We selected the 293T cell line, which is a commonly used eukaryotic cell line known for easy cultivation and high transfection efficiency. This enables more efficient acquisition of the desired cell phenotype or function when performing genetic manipulations (such as introducing target genes to express the lactate-sensing protein and lactate oxidase). Furthermore, the biological characteristics of 293T cells are well understood, facilitating subsequent experimental research and analysis.

By adhering to this structured plan, we aim to create a responsive lactic acid sensor that can effectively improve the tumor microenvironment and enhance the anti-tumor effects of immune cells.

2.1.2 Results

2.1.2.1 First Construction of the Lactate-Sensing Module

We split the TEV protease into N-terminal and C-terminal segments, which were then connected to the N-terminal and C-terminal of the LIdR protein.（Figure1）

Figure 1. This figure illustrates the structural design of the first-generation lactate sensor (LS1.0). It shows that the N-terminal and C-terminal fragments of TEV protease (TEV-N and TEV-C) were respectively connected to the N-terminal and C-terminal of the LldR protein, forming the initial sensor structure for lactate detection.

Therefore, we conducted the first verification experiment. We plated cells in complete medium, transfected the LS1.0 plasmid (constructed based on the LIdR sequence in our components and the pcDNA backbone) into 293T cells, set a lactic acid concentration gradient of 0, 1, and 5 mM for simultaneous stimulation, and then used the dual-luciferase reporter gene for quality detection.

The ideal mechanism of structural change should be as follows.(Figure2)

Figure 2 .This video displays the ideal structural change mechanism of the LS1.0 sensor under lactate stimulation. It visually demonstrates how lactate binding induces conformational changes in the sensor, thereby promoting the reassembly of TEV-N and TEV-C fragments to restore TEV protease activity.

However, the results showed that the TEV enzyme activity was very high even without the addition of lactic acid.(Figure3)

Figure 3. This figure presents the experimental results of the LS1.0 sensor verification using the dual-luciferase reporter gene assay. The ordinate (RLU, relative light unit) reflects luciferase enzyme activity; the results show that even without lactate stimulation (0mM), the TEV enzyme activity of the LS1.0 group is very high, indicating background activity issues in the first-generation sensor.

After team discussion, we speculate that this was because the split TEV enzyme was too close to each other on the LIdR protein, causing the TEV to become active and function even before lactic acid binding. Therefore, we improved it to develop LS2.0 - the second-generation lactate sensor.

Elevated lactic acid levels did not significantly alter enzyme production.

2.1.2.2 Second Construction of the Lactate-Sensing Module

Based on the failure of the previous generation sensor, we made the first improvement to the original sensor: we split the LIdR protein into two parts (N-terminal and C-terminal) and connected them to the N-terminal and C-terminal of the TEV protease, respectively, completing the design of a brand-new sensor.(Figure4)

Figure 4. This figure shows the structural design of the second-generation lactate sensor (LS2.0). Unlike LS1.0, the LldR protein was split into N-terminal (Lac-N) and C-terminal (Lac-C) fragments, which were then respectively connected to TEV-N and TEV-C to form a new sensor structure, aiming to reduce background TEV activity.

Subsequently, we conducted the second experimental verification, and the results are as follows.(Figure5)

Figure5. This figure presents the experimental results of the LS2.0 sensor verification using the dual-luciferase reporter gene assay. The ordinate (RLU) represents TEV enzyme activity; similar to LS1.0, the results indicate that the LS2.0 group still exhibits high TEV enzyme activity in the absence of lactate (0mM), meaning the background activity problem was not resolved.

However, during the experiment, we found that the TEV enzyme activity was still high without the addition of lactic acid. Therefore, we decided to further improve our sensor based on the above.

2.1.2.3 Third Construction of the Lactate-Sensing Module

In this improvement, we changed the connection mode of the N-terminal and C-terminal of the two proteins and constructed 8 combination modes.(Figure6)(Figure7)(Figure8)

Figure 6.This figure schematically shows the two connection modes of LldR fragments (Lac-N, Lac-C) and TEV fragments (TEV-N, TEV-C) before optimizing the LS3.x series sensors. It contrasts the original fixed connection mode (Lac-N-TEV-N and Lac-C-TEV-C) with the subsequent diversified combination design ideas.

Figure 7.This figure details the 8 different combination modes of LldR fragments (Lac-N, Lac-C) and TEV fragments (TEV-N, TEV-C) for constructing the LS3.1-LS3.8 sensors. Each combination represents a unique fusion sequence of the sensing module and TEV enzyme fragments.

Instead, we changed the connection mode of the N-terminal and C-terminal of the two proteins and constructed 8 combination modes.(Figure8)

Figure 8.This figure provides a visual diagram of the structural composition of each sensor in the LS3.1-LS3.8 series. It clearly marks the connection relationship between Lac-N/Lac-C and TEV-N/TEV-C in each sensor, serving as a design basis for subsequent background screening.

First, we conducted a background screening to investigate the response degree of each group of sensors without the addition of lactic acid. The screening results are shown in the figure.(Figure9)

Figure 9. This figure presents the background screening results of the LS3.1-LS3.8 sensors. The ordinate (RLU) reflects the TEV enzyme activity of each sensor without lactate stimulation; the results help screen out sensors with low background activity (e.g., LS3.5, LS3.6, LS3.8).

Finally, we screened out three combinations with low background, namely LS3.5, LS3.6, and LS3.8. We finally demonstrate that LS3.5 is the sensor we need with high sensitivity and stability. Our modeling results also corroborate this.Our modeling results also confirm this. For details, please visit our Model page.The results are shown in the figure.(Figure10)

Figure 10 displays the sensitivity and stability verification results of the low-background sensors that were screened. Ls3.5 exhibits the lowest background, and its signal increases with higher lactate input, whereas Ls3.6 and Ls3.8 do not show this trend.

2.2 Report System

2.2.1 GV2ER

2.2.1.1 The Structure of GV2ER

To accurately detect the functional efficacy and stability of the lactate sensor within cells, we constructed the GV2ER reporter gene plasmid.(Figure 11)

Figure 11. GV2ER:The "G" stands for the yeast Gal4 DNA-binding domain. In transcriptional regulation, this domain can specifically recognize and bind to DNA sequences containing Gal4 response elements.The "V" represents the herpes simplex virus VP16 transactivation domain, which possesses a potent function of activating transcription. When fused with the Gal4 DNA-binding domain to form "GV", once GV binds to the corresponding DNA sequence, VP16 can recruit transcription-related enzymes and protein factors, promote the initiation and progression of transcription, and thereby trigger the expression of downstream reporter genes (such as the firefly luciferase gene G5-Luci or the enhanced green fluorescent protein gene G5-EGFP).The "2ER" denotes two copies of the estrogen receptor mutant ERT2. The ERT2 domain is a mutated estrogen receptor domain that is sensitive to 4-hydroxy-tamoxifen (4-OHT) but insensitive to estrogen. In the absence of 4-OHT in cells, ERT2 traps the proteins linked to it in the cytoplasm. In GV-2ER, the two ERT2 domains are located at the N-terminus and C-terminus of GV, respectively. Acting like "molecular locks", they firmly sequester the GV transcription factor in the cytoplasm, preventing it from entering the nucleus to initiate transcription. Only after the ERT2 domains are removed via cleavage by TEV protease can GV enter the nucleus and exert its transcriptional activation function.

The lactate sensor, as a key protein for sensing changes in lactate concentration inside and outside cells, undergoes conformational changes or mediates protein-protein interactions (PPI) when bound to its ligand (lactate); these molecular events are crucial links in regulating physiological processes such as cellular metabolism and signal transduction.

By specifically capturing the molecular interaction events related to the lactate sensor through the GV2ER system, the response efficiency (effect) of the sensor to lactate and its functional maintenance capability (stability) under different time and environmental conditions can be quantitatively evaluated. This provides a reliable detection method for in-depth analysis of the physiological functions of the lactate sensor and the mechanisms of related diseases.

2.2.1.2 The Mechanism of Action of GV2ER

The entire system enables the detection of target protein-protein interactions (PPI) through a molecular cascade process: "TEV protease fragment complementation and activation → release of GV transcription factor → transcriptional activation in the nucleus → reporter gene expression".(Figure 12)

Figure 12. In this process, lactate enters the cell. Lac-N and Lac-C, along with TEV-N and TEV-C, bind to form a complex. This complex cleaves the ERT domains from GV via TEV protease activity. Freed GV then enters the nucleus, binds to the 5×UAS sequence, and initiates the expression of the downstream reporter gene sLOx.

In the absence of target protein-protein interactions (PPI), the system remains in an initial quiescent state. The GV transcription factor, flanked by TEV cleavage sites (tevS) and linked to ERT2, is "dragged" and anchored in the cytoplasm by ERT2, thereby being unable to translocate into the nucleus through nuclear pores. Consequently, downstream reporter genes (e.g.G5-Luci, G5-EGFP) remain unexpressed due to the lack of transcriptional activation signals.

When the target protein-protein interaction (PPI) occurs, the system is triggered and activated. The two proteins involved in the target PPI are fused with the inactive N-terminal TEV fragment (N-TEV) and C-terminal TEV fragment (C-TEV), respectively. When these two target proteins interact, the fused N-TEV and C-TEV fragments undergo reconstitution due to the proximity of the proteins, forming a fully active TEV protease. Serving as the signal sensing module of the entire system, this step converts the molecular event of PPI occurrence into a biochemical signal of TEV protease activation.

Subsequently, the process proceeds to a critical step: upon the formation of the active TEV protease, it specifically recognizes and binds to the tevS site located between the GV transcription factor and ERT2. Through proteolytic hydrolysis, the TEV protease cleaves the peptide bond at the tevS site, leading to the dissociation of the GV transcription factor from the ERT2 domain. At this point, the GV transcription factor, freed from the constraint of ERT2, is no longer anchored by heat shock proteins (HSPs) and gains the ability to translocate into the nucleus. With this, the system completes signal transduction and shifts from a quiescent state to an activation-ready state.

The released GV transcription factor translocates into the nucleus through nuclear pores, initiates a transcriptional cascade, and generates detectable reporter signals (e.g., fluorescence, chemiluminescence).

The signal intensity generated by reporter gene expression is positively correlated with the occurrence efficiency of the target PPI. By detecting fluorescence intensity (EGFP) or chemiluminescence intensity (Luci), the occurrence frequency and strength of the target PPI can be indirectly quantified, enabling visual and quantitative analysis of PPI.

2.2.2 Fluorescence Detection

In the lactate sensor system of this study, a dual-luciferase reporter gene was employed for quality assessment. The dual-luciferase reporter gene takes Renilla Luciferase and Firefly Luciferase as its core components.

2.2.2.1 The Structure of pGL4.35

Among them, Firefly Luciferase serves as the "functional reporter signal", and we designed the plasmid pGL4.35 [luc2P/9XGAL4UAS/Hygro] (provided by Promega).(Figure 13)

Figure 13. This figure presents the plasmid map of pGL4.35 [luc2P/9×GAL4UAS/Hygro] (provided by Promega), a core plasmid for dual-luciferase reporter gene detection in the experiment. It clearly marks multiple key functional elements, including the Ad promoter (driving gene transcription), 9×GAL4 UAS (binding to GAL4 protein to regulate gene expression), luciferase gene (serving as a reporter gene to produce detectable fluorescence), hPEST sequence (regulating luciferase stability), SV40 ori (facilitating plasmid replication in specific cells), HygR (hygromycin resistance gene for cell screening), AmpR (ampicillin resistance gene for prokaryotic system screening), and poly(A) signal (ensuring correct processing of transcription products). These elements work synergistically to enable the plasmid to be used for detecting the transcriptional activation efficiency mediated by the GV transcription factor.

The pGL4.35 [luc2P/9XGAL4UAS/Hygro] plasmid contains multiple key elements:The Ad promoter (adenovirus promoter) drives the transcription of relevant genes;

The 9X GAL4 UAS (9 copies of GAL4 upstream activation sequence) can bind to the GAL4 protein to regulate gene expression;

Luciferase (luciferase gene) acts as a reporter gene, which produces fluorescence for detection after expression;

The hPEST sequence regulates the stability of luciferase;

The SV40 ori (SV40 origin of replication) facilitates plasmid replication in specific cells, while the SV40 promoter drives the transcription of downstream genes;

The HygR (hygromycin resistance gene) confers hygromycin resistance to cells, enabling the selection of cells containing the plasmid;

The AmpR (ampicillin resistance gene) allows selection in prokaryotic systems;

The poly (A) signal (polyadenylation signal) ensures the correct processing of transcription products;

Additional elements such as the signal pause site are also included.

All these elements work synergistically, allowing the plasmid to be used in studies related to gene expression regulation and other relevant research areas.

The Mechanism of Action of Luciferase

The expression or activity of luciferase (luciferase gene) directly depends on the detection of protein-protein interactions (PPI) by the sensor. In the GV2ER system, when the target PPI occurs—inducing the reconstitution and reactivation of TEV protease fragments—the active TEV protease cleaves the membrane-anchored or cytoplasm-retained transcription factor. The released GV then translocates into the nucleus and drives the expression of the downstream Firefly luciferase gene (G5-Luci).

2.3 Lactate Degradation

Figure 14 . This figure shows the structural design of the engineered secreted lactate oxidase (sLOx). It illustrates that a signal sequence derived from the rat follicle-stimulating hormone β-subunit (rat FSHB) was fused to the N-terminus of the lactate oxidase (LOx) sequence, and a FLAG tag was also included in the structure (with the total length marked as approximately 1250 base pairs). The rat FSHB signal sequence is responsible for directing the newly synthesized sLOx protein into the cell secretory pathway, ensuring that sLOx is secreted extracellularly to exert its lactate-degrading function, rather than being retained in the cytoplasm.

Subsequently, to ensure the smooth implementation of the lactic acid detection assay—aimed at verifying the performance and stability of the sensors we had screened—we replaced the Luciferase gene (which had served as a reporter gene in the previous sensor screening experiments) with the optimized sLOx gene. Based on this modification, we designed an entirely new plasmid. (Figure 15)

Figure 15. This figure presents the map of the novel plasmid designed for verifying sLOx function. Based on the original plasmid used in sensor screening (which contained the Luciferase reporter gene), the Luciferase gene was replaced with the optimized sLOx gene. The plasmid retains key elements required for gene expression and cell screening, enabling the expression of sLOx to be regulated by the upstream lactate sensor system and facilitating subsequent verification of sLOx secretion and lactate-degrading activity.

We co-transformed the sLOx plasmid and GV2ER into HEK293 T cells, cultured them in medium containing five mmol lactic acid, and then extracted cell lysates and culture medium separately after 24 hours. Expression of LOx in both cell lysates and culture medium was detected using the flac antibody (located at the loxC site). As shown in the figure, lox can be induced by lactic acid and secreted into the culture medium.

Figure 16. This figure displays the results of the Western blot assay used to verify the secretion of sLOx protein. The blot includes two sample types: culture medium (extracellular fluid) and cell lysate (intracellular fluid), with both NC (negative control) and LS3.5 (sensor-sLOx system) groups tested. The presence of the sLOx protein band in the culture medium of the LS3.5 group (while no obvious band is detected in the NC group’s culture medium) confirms that the engineered sLOx protein was successfully secreted from 293T cells into the extracellular space.

Further results show that the expression of sLOx can significantly reduce extracellular lactate concentration. Figure 17 shows the results of our lactate degradation experiment:

Figure 17. This figure shows the results of the extracellular lactate degradation assay. The ordinate represents the relative concentration of extracellular lactate, and the abscissa shows the NC group and the LS3.5 group (sensor-sLOx system group). The results clearly indicate that compared with the NC group, the extracellular lactate concentration in the LS3.5 group is significantly lower, directly demonstrating that the secreted sLOx protein has a stable and efficient lactate-degrading function, which can reduce the local extracellular lactate concentration as expected.

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