Cancer has emerged as a global public health challenge. Current production of therapeutic antibodies often suffers from high costs, complex processes, and limitations in expression levels and stability.Consequently, developing a safe, controllable, and efficient antibody production system is crucial for advancing tumor therapeutics[1-3]. The red light-induced gene expression system, an emerging regulatory technology, has demonstrated unique advantages in non-toxic regulation and spatiotemporally precise expression. This study aims to develop an efficient and controllable nanobody production platform utilizing a red light-induced gene expression system in Escherichia coli. By integrating red light-mediated gene regulation technology with the robust protein production capacity of E. coli, we will achieve precise control over nanobody expression for the preparation of anti-tumor drugs[4-6].

Our design consists of two main cycles:

Cycle 1: Establishment and Validation of Red Light-Induced System

Part 1: Construction of red light-induced system (pRED vector)

Part 2: Development of red light-regulated GFP expression system (pRED-GFP)

Cycle 2: Nanobody Production Using the Red Light System

Part 1: Production of PD-L1 nanobody

Part 2: Production of PD-1 nanobody

In summary, Cycle 1 was designed to establish and validate both the feasibility and sensitivity of the red light-induced expression system. Cycle 2 aimed to systematically evaluate the nanobody production parameters under this optogenetic control, including yield optimization, ideal production duration, light intensity requirements, and dissolved oxygen levels. These studies provide fundamental theoretical support for our product-nanobody production kit.

Part 1 Construction of red light-induced system pRED

Design:

a red light-induced system, whose photosensory module (PSM) has the bacterial phytochrome (DrBphP) as the key gene. The primary mechanisms are: a. Perception of red and far-red light through the biliverdin chromophore. Biliverdin (BV) can be produced by heme oxygenase (HO) alone. b. Light-induced structural changes in PSM are transmitted to the effector HK domain (YF1), thereby altering its basal kinase and phosphatase activities. FixJ: an inverter cassette encoding the λ phage repressor cI and λ promoter pR. The pRED plasmid was provided by our institution's strain repository, and the target gene HO, DrBphP, YF1, FixJ were synthesized by a biotech company. The fragment was homologously recombined with a linearized plasmid to construct the pRED(Figure 1).

Figure 1. The plasmid map of pRED

Build:

We obtained fragments for constructing plasmid pRED using PCR technology, as shown in Figure 2a. We obtained fragments of approximately 4600bp and 4300bp in length, which are consistent with our expected sizes. Subsequently, we seamlessly ligated the obtained target fragments and transformed them into Escherichia coli DH5α, as shown in Figure 2b.

Furthermore, we randomly selected around 5 single colonies from the obtained agar plates, sequenced the plasmids, and obtained the sequencing results of the correct plasmid as shown in Figure 2d. Subsequently, after cultivation, we extracted the correct plasmid and transformed it into Escherichia coli BL21, as shown in Figure 2c. Through this complete process, we successfully obtained bacterial strains containing the correct plasmid pRED.

Figure 2. Construction results of plasmid pRED. a: PCR results; b: Transformation into DH5α after ligation; c: Successful transformation of correct plasmid into BL21. d.Sequencing results of selected single colonies

Part 2 Development of red light-regulated GFP expression system pRED-GFP

Design:

The construction of the pRED-GFP plasmid is based on the plasmid pRED. Specific primers were designed to linearize the pRED plasmid. The GFP gene was provided by our institution's strain repository. Through homologous recombination, the two fragments were ligated to form a plasmid, resulting in the construction of pRED-GFP(Figure 2).

Figure 3. The plasmid map of pRED-GFP

Build:

PCR technology was employed to generate fragments for the construction of plasmid pRED-GFP, with details presented in Figure 4a. A fragment of roughly 714 bp in length was obtained, matching our anticipated size. Following this, the target fragments acquired were subjected to seamless ligation, and the resulting product was transformed into Escherichia coli DH5α, as illustrated in Figure 4c.​

In addition, about 5 single colonies were randomly picked from the obtained agar plates, and their plasmids were sequenced. The sequencing results of the correct plasmid are displayed in Figure 4d. After cultivation, the correct plasmid was extracted and transformed into Escherichia coli BL21, as shown in Figure 4b. Through this entire procedure, we successfully obtained bacterial strains harboring the correct plasmid pRED.

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Figure 4. Construction results of plasmid pRED-RED. a: PCR results; b: Successful transformation of correct plasmid into BL21; c: Transformation into DH5α after ligation; d: Sequencing results of selected single colonies

Test

To validate whether our constructed strains undergo normal transcription and translation under red light induction, we conducted fluorescence intensity tests on the strains under red light and dark conditions. We utilized strains containing the GFP gene, and the experimental results demonstrate favorable outcomes in our experimental group (Figure 5). Only under red light induction did the strains exhibit significantly high fluorescence intensity, confirming the feasibility of our strain modifications.

Figure 5. Fluorescence intensity of the four strains under dark and red light conditions.

Part 1: Production of PD-L1 nanobody (pRED-PD-1 )

Design:

The construction of the pRED-PD-1 plasmid is based on the plasmid pRED. Specific primers were designed to linearize the pRED plasmid. The Anti-PD-1 was synthesized by a biotech company. Through homologous recombination, the two fragments were ligated to form a plasmid, resulting in the construction of pRED-PD-1(Figure 6).

Figure 6. The plasmid map of pRED-PD-1

Build:

We successfully amplified fragments for constructing plasmid pRED-PD-1 via PCR technology, as depicted in Figure 7a. The resulting fragment, approximately 372 bp in size, matched our expected length. Subsequent seamless ligation of the target fragments was performed, followed by transformation into Escherichia coli DH5α, illustrated in Figure 7c.

Following this, we randomly picked approximately 5 individual colonies from the agar plates, sequenced the plasmids, and obtained sequencing data confirming the correct plasmid, as displayed in Figure 7d. After cultivation, we isolated the correct plasmid and introduced it into Escherichia coli BL21, as shown in Figure 7b. This comprehensive process led to the successful acquisition of bacterial strains harboring the correct plasmid pRED.

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Figure 7. Construction results of plasmid pRED-PD-1. a: PCR results; b: Successful transformation of correct plasmid into BL21; c: Transformation into DH5α after ligation Sequencing results of selected single colonies; d: Sequencing results of selected single colonies

Part 2: Production of PD-1 nanobody (pRED-PD-L1)

Design:

The pRED-Anti-PD-L1 consists of eight components, including BBa_25OTICA0(PD-L1), BBa_25CKVE78(HO),BBa_25W1NO5 （DrBphP）;BBa_25D3H7BT(YF1);BBa_25CG7CJU(FixJ);BBa_259SXEM0(λ repressor)and BBa_25PPOVCP(pRED-Vector). We constructed the pRED red light-inducible system using homologous recombination methods. Subsequently, this vector pRED was utilized to insert the nanobody PD-L1, enabling the production of nanobodies.

The construction of the pRED-PD-L1 plasmid is based on the plasmid pRED. Specific primers were designed to linearize the pRED plasmid. The Anti-PD-L1 was synthesized by a biotech company. Through homologous recombination, the two fragments were ligated to form a plasmid, resulting in the construction of pRED-GFP-Anti-PD-L1(Figure 8).

Figure 8. The plasmid map of pRED-PD-L1

Build:

Using PCR technology, we successfully obtained fragments for the construction of plasmid pRED-PD-L1, depicted in Figure 9a. The fragment obtained was approximately 453 bp in length, aligning with our expected size. Subsequent to seamless ligation of the target fragments, they were transformed into Escherichia coli DH5α, as illustrated in Figure 9b.

Moreover, approximately 5 single colonies were randomly selected from the agar plates, and the plasmids were sequenced, yielding sequencing results confirming the accuracy of the plasmid, as presented in Figure 9c. Following cultivation, the correct plasmid was extracted and introduced into Escherichia coli BL21, as shown in Figure 9d. This comprehensive process culminated in the successful acquisition of bacterial strains carrying the correct plasmid pRED.

Figure 9. Construction results of plasmid pRED-PD-L1. a: PCR results; b: Transformation into DH5α after ligation; c: Sequencing results of selected single colonies; d: Successful transformation of correct plasmid into BL21

Functional Test

1. Protein Expression and Validation

1.1 SDS-PAGE for Anti-PD-1 and Anti-PD-L1

Two strains of Escherichia coli BL21 carrying the correct plasmids with Anti-PD-1 and Anti-PD-L1 were separately subjected to SDS-PAGE to verify the protein expression of the target genes. The target proteins Anti-PD-1 and Anti-PD-L1 have sizes of 14.6 kDa and 16.2 kDa, respectively. Protein expression was induced with 12W and performed at 25°C for 20 hours. Based on the results shown in Figure 10, we can clearly observe protein bands of the target genes Anti-PD-1 and Anti-PD-L1 in both crude and purified proteins, confirming the accuracy of our results.



Figure 10. The SDS-PAGE results for Anti-PD-1 and Anti-PD-L1

1.2 WB for Anti-PD-1 and Anti-PD-L1

To further validate the expression and correct molecular weights of our target proteins Anti-PD-1 and Anti-PD-L1, we conducted Western blot experiments on these two strains. The experimental results, as shown in Figure 11, clearly demonstrate the expression of our target proteins at lengths of 14.6 kDa and 16.2 kDa, confirming the accuracy of our protein expression.



Figure 11. The WB results for Anti-PD-1 and Anti-PD-L1

2. ELISA-Based Quantification of Nanobody Yield

2.1 ELISA analysis of production of two nanobodies under different lighting times

The duration of red light induction is a critical factor influencing the production of nanobodies by the strains. In order to determine the detection time for subsequent experiments, we first conducted experiments on two strains under different durations of red light induction. Before testing the production of nanobodies in the fermentation broth, we tested the absorbance of our antibody when it completely reacted with different concentrations of PD-1 antigen, and drew the standard curve. Through the standard curve, we can quantitatively calculate the antibody yield. Based on this, we conducted the quantification of nanobodies in the fermentation broth. We ensured the reliability of our experimental data by diluting the samples at different multiples. Through the experimental results (Figure 12), we explored the duration of light exposure at which the strain's antibody production was maximized and did not decompose. Based on this experiment, we determined that in subsequent experiments, samples would only be taken at 24 hours to measure antibody production.



Figure 12. The results of nanobody production under different light exposure durations.

2.2 ELISA analysis of production of two nanobodies under different lighting intensities

To investigate the impact of light intensity on nanobody production in the strains, we selected three intensities 3W, 6W, and 12W based on data from literature reports for simultaneous induction of the strains, aiming to assess the effect of different red light intensities on nanobody production. From the experimental results (Figure 13), it can be observed that when the red light irradiation intensity increased from 3W to 6W, the production of the two antibodies increased by 10.53% and 16.06%, respectively. However, upon further increase to 12W, there was minimal production enhancement, and the production of PD-L1 even decreased by 4.44%. Therefore, for subsequent steps, we will utilize a red light intensity of 6W as our irradiation intensity.



Figure 13. The results of nanobody production under different lighting intensities.

2.3 ELISA analysis of production of two nanobodies under different oxygen conditions

Because Escherichia coli is a facultative anaerobe, it means that it can produce under both aerobic and anaerobic conditions. However, it is not clear whether the production of nanobodies requires sufficient oxygen supply. We conducted experiments using two types of Erlenmeyer flasks, as shown in Figure 14: a regular Erlenmeyer flask and a baffled Erlenmeyer flask, with the latter providing more oxygen. This was done to provide different oxygen levels to the strains and assess the production of nanobodies. The experimental results, as shown in Figure 15, revealed that as the dissolved oxygen level in the shake flask increased, the production of antibodies PD-1 and PD-L1 increased by 17.30% and 6.56%, respectively.

We hypothesize that with the increase in dissolved oxygen, the metabolism of the bacterial strain becomes more active, leading to higher antibody production.



Figure 14. Different Erlenmeyer flasks - regular Erlenmeyer flask on the left, baffled Erlenmeyer flask on the right.



Figure 15. The results of nanobody production under different oxygen conditions.

Table 1 presents the nanobody production yields achieved under the established conditions (baffled flasks, 6W illumination, 24h induction period), with ELISA measurements showing 2.289 ng/L for anti-PD-1 and 1.9485 ng/L for anti-PD-L1 nanobodies.

Table 1 . Nanobody production yield of PD-L1 and PD-1

	PD-1(ng)	SD	PD-L1(ng)	SD
yield	2.289	0.008485281	1.9485	0.009545942

Learn

1. The proteins we expressed are intracellular. In the future, we may consider incorporating signal peptides and specific transport proteins to transport the target antibodies to the extracellular space.
2. Currently, we have only used ELISA to verify the yield and efficacy of the nanobodies. In future studies, we will employ additional validation methods such as surface plasmon resonance (SPR) and animal experiments to further characterize the nanobodies's performance.
3. While our current focus has been on PD-1/PD-L1 nanobodies, we aim to establish a generalized platform capable of producing various nanobody classes, thereby validating the system's versatility.

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