Overview

This project focuses on the potential applications of the natural protease papain and its XCP2-like isoenzymes in skincare. Based on structural modelling, affinity screening, and expression validation, we constructed multiple basic and composite components and performed expression and functional testing in the pET-28a(+) vector. We explored the possibilities of synthetic biology in the design and industrialisation of skincare proteases, providing reusable components, for instance, pET28a-papain, pET28a-XCP2-Abrus precatorius, and pET28a-XCP2-Actinidia eriantha, and methods for future iGEM teams conducting research in the field of biological skincare.

Figure 1. Overview of experimental steps

Table 1. Part contribution

Part number	Part name	Contribution type	Part type
BBa_25Q4HARH	Papain	New part	Basic part
BBa_25Z91JKF	XCP2-Abrus precatorius	New part	Basic part
BBa_253OXRSB	XCP2-Actinidia eriantha	New part	Basic part
BBa_25ZTGYBD	XCP2-Cajanus cajan	New part	Basic part
BBa_25MX6CXZ	XCP2-Cicer arietinum	New part	Basic part
BBa_25ULT0GX	pET28a-papain	New part	Composite part
BBa_K5764006	pET28a-XCP2-Abrus precatorius	New part	Composite part
BBa_25TUUN1F	pET28a-XCP2-Actinidia eriantha	New part	Composite part
BBa_25FRHJRN	pET28a-XCP2-Cajanus cajan	New part	Composite part
BBa_251N76W5	pET28a-XCP2-Cicer arietinum	New part	Composite part

Part Contribution

1. Add a Basic Part, BBa_25Q4HARH(Papain); BBa_25Z91JKF(XCP2-Abrus precatorius); BBa_253OXRSB(XCP2-Actinidia eriantha); BBa_25ZTGYBD(XCP2-Cajanus cajan); BBa_25MX6CXZ(XCP2-Cicer arietinum)

1.1 BBa_25Q4HARH(Papain)

Basic Pairs: 678bp

Origin: Carica papain

Description: Chain A, Papain

Properties: Papain is composed of 212 amino acids, a single subunit enzyme, and its secondary structure is composed of 4 alpha-helices and one beta-fold. Papain cysteine protease is the largest subfamily of cysteine proteases in the human body. Their main function is to hydrolyse the extracellular matrix (ECM), such as fibrous structural proteins. So, it has effect on exfoliation activity of the skin. (Trevisol et al., 2023)

Figure 2. Gene maps of papain

Usage and Biology: The incorporation of papain into the mask matrix resulted in a more compact structure with enhanced mechanical properties and reduced water solubility. Films containing papain with different concentration all demonstrated enzymatic activity against both casein and dermal matrix substrates, confirming papain's exfoliation efficacy. Unlike papain solutions, the immobilised enzyme in the film matrix maintained stable activity for up to 90 days. Characterisation of physicochemical properties and proteolytic activity revealed that the papain-embedded films retained significant exfoliation capacity while exhibiting ideal characteristics for cosmetic applications. These findings demonstrate that the developed film system preserves papain's enzymatic exfoliation function and possesses suitable properties for utilisation as a bioactive facial mask in dermatological and cosmetic formulations. (Trevisol et al., 2023)

Cultivation: A single colony containing the papain plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the papain coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 3. Result of gel electrophoresis of papain

1.2 BBa_25Z91JKF(XCP2-Abrus precatorius)

Length: 1083 bp

Source: Abrus precatorius

Description: cysteine protease XCP2

Properties: XCP2 is a cysteine protease derived from Abrus precatorius (rosary pea), which exhibits sequence-specific proteolytic activity. Similar to other plant-derived proteases (e.g., papain), XCP2 has potential applications in targeted protein degradation.

Figure 4. Gene maps of XCP2-Abrus precatorius

Usage and Biology: Abrus precatorius is one of the important medicinal materials that has excellent effects of clearing heat, detoxifying and promoting diuresis and it mainly used for treating sore throat. And it has a wide range of biological activities, including anti-tumor, antibacterial, insecticidal, antiparasitic, anti-parasitic, anti-inflammatory, antioxidant, immunomodulatory, anti-fertility, anti-diabetic and other pharmacological activities (Qian et al., 2022). Moreover, the isolated isoflavanquinone also shows strong anti-inflammatory potential and can be used as a candidate drug for further research on cancer treatment (Okoro et al., 2021).

Cultivation: A single colony containing the XCP2-Abrus precatorius plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the XCP2-Abrus precatorius coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 5. Result of gel electrophoresis of XCP2-Abrus precatorius

1.3 BBa_25ZTGYBD(XCP2-Actinidia eriantha)

Length: 1089 bp

Source: Actinidia eriantha

Description: cysteine protease XCP2-like

Properties: XCP2 is a cysteine protease derived from Actinidia eriantha (a kiwi species), characterized by its substrate-specific proteolytic activity. Similar to other plant cysteine proteases (e.g., papain and bromelain), XCP2 exhibits Thermostability and pH adaptability, making it suitable for industrial and biotechnological processes. It enables cysteine-type endopeptidase activity and involved in proteolysis of protein catabolic process.

Figure 6. Gene maps of XCP2-Actinidia eriantha

Usage and Biology: The Actinidia eriantha is a plant used as a heat-clearing medicine, mainly used for treatment of tumours of the digestive tract (Xiang et al., 2022). The crude extract and relatively pure components of the hairy kiwi fruit possess a variety of pharmacological activities, including anti-cancer, immunomodulatory, anti-angiogenic, neuroprotective, anti-inflammatory and antioxidant effects. In addition, more than 104 chemical substances have been identified from Artemisia annua (Wang et al., 2022).

Cultivation: A single colony containing the XCP2-Actinidia eriantha plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the XCP2-Actinidia eriantha coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 7. Result of gel electrophoresis of XCP2-Actinidia eriantha

1.4 BBa_253OXRSB(XCP2-Cajanus cajan)

Length: 1083 bp

Source: Cajanus cajan

Description: cysteine protease XCP2

Properties: A conserved catalytic triad (Cys-His-Asn) within a hydrophobic active site, enabling nucleophilic cleavage of peptide bonds. A substrate-binding cleft with specificity for hydrophobic residues at the P2 position, similar to papain. Has high Substrate Specificity. Preferentially cleaves after bulky hydrophobic amino acids, akin to papain. Can be used for tag removal or controlled protein digestion due to predictable cleavage patterns. It retains activity across a broad pH range (4.0–8.0) and moderate temperatures (<50°C), ideal for industrial processes.

Figure 8. Gene maps of XCP2-Cajanus cajan

Usage and Biology: Cajanus cajan can be used to treat various diseases such as toothache, dizziness and diabetes. These characteristics are related to the bioactive components in Cajanus cajan. It also has antioxidant, antibacterial, anti-diabetic, neuroprotective and anti-inflammatory effects. In addition, the hexane extract isolated from it enables it to act as an antioxidant.

Cultivation: A single colony containing the XCP2-Cajanus cajan plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the XCP2-Cajanus cajan coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 9. Result of gel electrophoresis of XCP2-Cajanus cajan

1.5 BBa_25MX6CXZ(XCP2-Cicer arietinum)

Length: 1095 bp

Source: Cicer arietinum

Description: cysteine protease XCP2-like precursor

Properties: A highly conserved catalytic triad that forms the active site for proteolytic cleavage. A substrate-binding pocket with preference for hydrophobic residues at the P2 position, similar to papain. It exhibits optimal activity at pH 6.0-7.5 and temperature range of 40-55°C. And it also shows 3-fold higher specific activity compared to papain against synthetic substrates (Z-Phe-Arg-AMC) and it can maintains stability in organic solvents (up to 30% methanol). XCP2-Cicer arietinum has variety of bioactivity including antioxidant activity, antifungal, antibacterial and analgesic (Begum et al., 2023).

Figure 10. Gene maps of XCP2-Cicer arietinum

Usage and Biology: It is used in the food industry to produce plant-based snacks and bioactive compounds related to type 2 diabetes (T2D). Future research opportunities and new application fields of specific bioactive compounds as new food ingredients. The application of chickpea bioactive compounds as food ingredients (for example, in the management of type 2 diabetes (Acevedo et al., 2021). Cicer arietinum is a type of bean that is rich in protein (about 20% dry weight), and their consumption is closely related to health benefits. Dietary peptides are important molecules extracted from dietary proteins. Cicer arietinum peptides can be produced by enzymatic methods using pepsin, trypsin, chymotrypsin, alkaline protease, flavor enzyme and papain alone or in combination. However, the sequences of many peptides in the hydrolysate of chickpea protein remain unknown. It has biological function including antioxidant activity, cholesterol-lowering and angiotensin-1-converting enzyme inhibition, anti-carcinogenic, antibacterial and anti-inflammatory effects. And Cicer arietinum hydrolysate can be added to the diet as a functional component to prevent diabetes (Chandrasekaran et al., 2020).

Cultivation: A single colony containing the XCP2-Cicer arietinum plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the XCP2-Cicer arietinum coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 11. Result of gel electrophoresis of XCP2-Cicer arietinum

2. Add a Composite Part, BBa_25ULT0GX(pET28a-papain); BBa_25ZPQ33J(pET28a-XCP2-Abrus precatorius); BBa_25TUUN1F(pET28a-XCP2-Actinidia eriantha); BBa_25FRHJRN(pET28a-XCP2-Cajanus cajan); BBa_251N76W5(pET28a-XCP2-Cicer arietinum)

2.1 BBa_25ULT0GX(pET28a-papain)

Composition: pET-28a(+) backbone; papain gene fragment.

Apparatus used: pET-28a(+) plasmid, papain gene fragment, restriction endonucleases (NdeI, HindIII), Homologous recombination enzyme.

Figure 12. Plasmid map of pET28a-papain

Engineering Principle: The pET-28a-papain construct is designed to express the Carica papaya papain protease in E. coli under the control of the T7 promoter, enabling high-level, inducible protein expression. The system utilizes the lac operon-based regulation, where IPTG induction activates T7 RNA polymerase, initiating transcription of the papain coding sequence. A His-tag is fused to the protein to facilitate purification via affinity chromatography. This design enables efficient production and downstream application of recombinant papain for functional assays such as protein degradation or skin exfoliation.

The papain gene fragment (678 bp) was amplified using PCR, and both the gene and the pET-28a(+) vector were digested with NdeI and HindIII restriction enzymes to generate compatible sticky ends. Figure 13 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of Papain is approximately 678 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 13. Agarose gel electrophoresis results of PCR for papain

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E. coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E. coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 14A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 14B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 14C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 14. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-papain

Cultivation, Purification and SDS-PAGE:

Protein expression was induced with IPTG (1 mM) in E. coli BL21(DE3), followed by cell lysis using a sonicator under cold conditions to preserve enzyme activity. The recombinant papain protein was purified using nickel affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high (buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 15, distinct protein bands were observed in all lysate samples. A ~24 kDa band was detected in the papain-expressing strain, consistent with the expected molecular weight of papain. These results confirmed that XCP2-papain recombinant enzymes were successfully expressed in E. coli.

Figure 15. SDS-PAGE results of pET28a-Papain

2.2 BBa_25ZPQ33J(pET28a-XCP2-Abrus precatorius)

Composition: pET-28a(+) backbone; XCP2-Abrus gene fragment.

Apparatus used: pET-28a(+) plasmid, XCP2-Abrus fragment, restriction endonucleases (NdeI, HindIII), Homologous recombination enzyme.

Figure 16. Plasmid map of pET28a-XCP2-Abrus

Engineering Principle: The pET-28a-XCP2-Abrus precatorius construct is engineered to express the XCP2 cysteine protease derived from Abrus precatorius in E.coli. The gene is placed downstream of the T7 promoter, enabling tightly controlled and high-yield expression upon IPTG induction via the lac operon system. The vector includes an N-terminal His-tag for simplified purification using nickel affinity chromatography. This design allows for efficient production of functional XCP2 protease to evaluate its keratin-degrading and exfoliation potential in vitro.

The XCP2-Abrus gene (1083 bp), a cysteine protease from Abrus precatorius, was amplified by PCR. Both the PCR product and the pET-28a(+) vector were digested with NdeI and HindIII to generate compatible ends. Figure 17 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of XCP2-Abrus is approximately 1083 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 17. Agarose gel electrophoresis results of PCR for XCP2-Abrus

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E.coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E.coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 18A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 18B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 18C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 18. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Abrus

Cultivation, Purification and SDS-PAGE: Protein expression was induced using 1 mM IPTG. Cells were lysed by sonication at low temperature. The recombinant XCP2-Abrus protein was purified using nickel affinity chromatography. The washing steps included buffer A (low imidazole) and buffer B (high imidazole), followed by SDS-PAGE analysis of elution fractions.Verified clones were transformed into E. coli BL21(DE3) for protein expression. As shown in Figure 18, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected in the Abrus-expressing strain, consistent with the expected molecular weight of XCP2-Abrus. These results confirmed that recombinant enzymes were successfully expressed in E. coli.

Figure 19. SDS-PAGE results of pET28a-XCP2-Abrus

2.3 BBa_25TUUN1F(pET28a-XCP2-Actinidia eriantha)

Composition: pET-28a(+) backbone; XCP2-like gene from Actinidia eriantha.

Apparatus used: pET-28a(+) plasmid, XCP2-Actinidia insert, restriction endonucleases (NdeI, HindIII), and Homologous recombination enzyme.

Figure 20. Plasmid map of pET28a-XCP2-Actinidia

Engineering Principle: The pET-28a-XCP2-Actinidia eriantha construct is designed to express the XCP2 cysteine protease derived from Actinidia eriantha (hardy kiwifruit) in E. coli under the control of the T7 promoter. The system employs IPTG-inducible lac operon regulation, ensuring tightly controlled and high-level protein expression. A 6×His-tag is fused to the N-terminus to enable convenient purification via nickel affinity chromatography. This recombinant expression system facilitates functional studies of XCP2’s proteolytic activity and its potential applications in keratin degradation and exfoliation.

The XCP2-like gene from Actinidia eriantha (1089 bp) was PCR-amplified and digested along with the pET-28a(+) vector using NdeI and HindIII. After purification, the digested fragments were ligated using Homologous recombination enzyme. Figure 21 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of XCP2-Actinidia is approximately 1089 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 21. Agarose gel electrophoresis results of PCR for XCP2-Actinidia

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E.coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E.coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 22A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 22B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 22C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 22. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Actinidia

Cultivation, Purification and SDS-PAGE: The verified plasmid was transformed into E.coli BL21(DE3) for protein expression. Following IPTG induction, the cells were harvested and lysed with sonication at low temperature. The recombinant protein was purified using Ni-NTA affinity chromatography, including stepwise washes with imidazole buffers. As shown in Figure 23, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected in the Actinidia-expressing strain, consistent with the expected molecular weight of XCP2-Actinidia. These results confirmed that recombinant enzymes were successfully expressed in E.coli.

Figure 23. SDS-PAGE result of pET28a-XCP2-Actinidia

2.4 BBa_25FRHJRN(pET28a-XCP2-Cajanus cajan)

Composition: pET-28a(+) backbone; XCP2 gene from Cajanus cajan.

Apparatus used during construction: pET-28a(+) plasmid, PCR-amplified XCP2-Cajanus cajan, restriction endonucleases (NdeI, HindIII), Homologous recombination enzyme.

Figure 24. Plasmid map of pET28a-XCP2-Cajanus

Engineering Principle: The pET-28a-XCP2-Cajanus cajan construct is engineered to express the XCP2 cysteine protease derived from Cajanus cajan (pigeon pea) in E. coli under the control of the T7 promoter. The system utilizes the lac operon for IPTG-inducible expression, enabling tight regulation and high yield of the recombinant protein. An N-terminal His-tag is incorporated to facilitate purification through nickel affinity chromatography. This construct allows for efficient production and activity analysis of XCP2, with the goal of evaluating its keratin-degrading and exfoliation potential for skincare-related applications.

The XCP2-Cajanus gene (1083bp) was obtained by PCR amplification from a codon-optimized synthetic template. Both the insert and pET-28a(+) backbone were digested with NdeI and HindIII to create sticky ends. The digested fragments were ligated using Homologous recombination enzyme to generate the plasmid pET28a-XCP2-Cajanus. Figure 25 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of XCP2-Cajanus is approximately 1083 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 25. Agarose gel electrophoresis results of PCR for XCP2-Cajanus

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E.coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E.coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 26A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 26B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 26C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 26. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Cajanus

Cultivation, Purification and SDS-PAGE:

The confirmed plasmid was transformed into E.coli BL21(DE3) for protein expression. IPTG was added to induce expression, and the culture was harvested after 20 hours. Cell lysis was performed using a sonicator under low-temperature conditions to prevent protease denaturation. Purification was carried out using nickel affinity chromatography. The column was washed with buffer A (low imidazole) and eluted with buffer B (high imidazole). Collected fractions including lysate, flow-through, and elution were analyzed via SDS-PAGE. As shown in Figure 27, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected in the Cajanus-expressing strain, consistent with the expected molecular weight of XCP2-Cajanus. These results confirmed that recombinant enzymes were successfully expressed in E.coli.

Figure 27. SDS-PAGE result of pET28a-XCP2-Cajanus

2.5 BBa_251N76W5(pET28a-XCP2-Cicer arietinum)

Composition: pET28a(+) backbone; XCP2-like gene from Cicer arietinum.

Apparatus used during construction: pET28a(+) backbone, XCP2-Cicer insert, restriction endonucleases (NdeI, HindIII), Homologous recombination enzyme.

Figure 28. Plasmid map of pET28a-XCP2-Cicer

Engineering Principle: The pET-28a-XCP2-Cicer arietinum construct is designed to express the cysteine protease XCP2 from Cicer arietinum (chickpea) in E.coli. The coding sequence is placed under the control of the T7 promoter, allowing IPTG-inducible expression via the lac operon. An N-terminal His-tag is included to facilitate protein purification using nickel affinity chromatography. This design enables efficient recombinant production of XCP2 for functional assays, with the goal of evaluating its keratin-degrading ability and potential use in skin exfoliation or biological material recycling.

The XCP2-like Cicer gene (1095bp) was PCR-amplified from a synthesized, codon-optimized template. The gene fragment and pET28a(+) plasmid were digested with NdeI and HindIII to generate sticky ends. Using Homologous recombination enzyme, we ligated the insert into the linearized vector to form the recombinant plasmid pET28a-XCP2-Cicer. Figure 29 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of XCP2-Cicer is approximately 1095bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 29. Agarose gel electrophoresis results of PCR for XCP2-Cicer

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E.coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E.coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 30A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 30B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 30C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 30. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Cicer

Cultivation, Purification and SDS-PAGE: The verified plasmid was transformed into E.coli BL21(DE3). IPTG was added to induce expression of the XCP2-Cicer protein. The cells were lysed using a sonicator under cold conditions to preserve enzyme activity. Purification was conducted using nickel affinity chromatography, with sequential washes using low-imidazole buffer A and high-imidazole buffer B. As shown in Figure 31, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected in the Cajanus-expressing strain, consistent with the expected molecular weight of XCP2-Cicer. These results confirmed that recombinant enzymes were successfully expressed in E.coli.

Figure 31. SDS-PAGE result of pET28a-XCP2-Cicer

3. Functional Test:

3.1. Papain-mediated Casein Clot Dissolution Assay

To evaluate the initial proteolytic potential of the recombinant enzyme papain, we conducted a qualitative assay based on its ability to degrade acid-precipitated casein from milk.

As illustrated in Figure 32, untreated milk containing 6% (w/v) casein appeared uniformly dispersed (A). The addition of dilute hydrochloric acid caused the formation of a dense white precipitate, indicating the aggregation of denatured casein proteins (B). Following incubation with papain at 37 °C for 1 hour, the precipitate visibly diminished and the solution became notably clearer (C), indicating enzymatic breakdown of the protein network.

These results confirm that papain can hydrolyze casein aggregates and exhibits functional caseinolytic activity. While this assay qualitatively supports its protease function, it does not allow for comparison between different enzyme efficiencies.

Figure 32. Result of casein clot dissolution assay

3.2. FN1 Matrix Dissolution Assay

To investigate whether the recombinant proteases are capable of degrading biologically relevant substrates, fibronectin-1 (FN1) was chosen as the model protein for enzymatic digestion.

As depicted in Figure 33, the FN1 control sample (without enzyme treatment) showed a single, sharp band at approximately 47 kDa, indicating that the protein remained intact in the absence of enzymatic activity. In contrast, samples treated with each of the five enzymes—Papain, Abrus, Actinidia, Cajanus, and Cicer—exhibited a clear decrease in the intensity of the full-length FN1 band. Additionally, prominent new bands emerged near 24 kDa, reflecting the formation of low-molecular-weight fragments resulting from proteolytic cleavage.

The consistent generation of these ~24 kDa bands across all enzyme groups suggests that, despite originating from different species, the recombinant proteases may share similar substrate recognition or cleavage patterns. These findings demonstrate that all five enzymes are active against FN1 and may be effective tools in applications related to extracellular matrix degradation or remodeling.

Figure 33. Result of FN1 matrix dissolution assay

3.3. ELISA-based Enzyme Activity Assay

To measure and compare the proteolytic activity of different crude enzyme lysates, we carried out an ELISA-based quantification assay. Prior to analysis, total protein concentrations were determined using NanoDrop, and each lysate was diluted to various working concentrations. A standard curve was constructed from serial dilutions of the kit’s reference standard, generating a linear model that allowed us to estimate relative enzymatic activities from OD₄₅₀ values.

To quantitatively evaluate the enzymatic activity of the crude lysates, an ELISA assay was performed. Protein concentrations were first normalized using NanoDrop, and all lysates were diluted to different concentrations.

Figure 34. The standard table of papain concentration

A standard curve was generated based on serial dilutions of the ELISA kit standard, yielding a linear regression model for activity estimation.

Figure 35. The standard curve of papain concentration

We used ELISA kits to individually test and analyze the activity of several enzymes. Based on the above standard curves, we calculated the activity of each enzyme and performed significance analysis using Graphpad to calculate the P-value. As shown in Figure 36, the enzyme activity was Papain > Act > Abr > Caj > Cic.

Figure 36. Single enzyme activity detection data

Single-enzyme activity results showed that Papain had the highest protease activity among all five enzymes, consistent with the qualitative results from the casein assay. No statistically significant differences were observed between Cajanus and Cicer.

Figure 37. Graphs of single-enzyme activity comparison

We used an ELISA kit to analyze the activity of the dual-enzyme combinations and verify whether there was a synergistic effect. Based on the standard curve, we calculated the activity of the dual-enzyme combinations and performed a significance analysis using Graphpad to calculate the P-value. As shown in Figure 38, the optimal combination was Caj+Cic.

Figure 38. Dual-enzyme activity detection data

In the dual-enzyme combinations, the mixture of Cajanus and Cicer displayed the highest activity. Other combinations, such as papain with Cajanus or Cicer, and Abrus with Actinidia, showed moderate activity but no significant enhancement compared to single enzymes.

Figure 39. Graphs of dual-enzyme combination comparison

We used an ELISA kit to analyze the activity of the Three-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated the activity of the Three-enzyme combination and performed a significance analysis using Graphpad to calculate the P value. The optimal combinations were Papain + Abrus + Actinidia and Abrus + Actinidia + Cajanus.

Figure 40. Three-enzyme activity detection data

Three-enzyme combinations revealed that the groups Papain + Abrus + Actinidia and Abrus + Actinidia + Cajanus produced the strongest activity levels, although their differences were not statistically significant. These combinations outperformed most others in the same group.

Figure 41. Graphs of Three-enzyme combinations comparison

We used an ELISA kit to analyze the activity of the four-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated the activity of the four-enzyme combination and performed a significance analysis using Graphpad to calculate the P value. The optimal combination was Abrus + Actinidia + Cajanus + Cicer.

Figure 42. Four-enzyme activity detection data

In the four-enzyme combinations, the mixture of Abrus + Actinidia + Cajanus + Cicer showed the highest value, indicating the strongest activity among all tested combinations of four enzymes.

Figure 43. Graphs of four-enzyme combinations comparison

We used an ELISA kit to analyze the activity of the five-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated the activity of the five-enzyme combination and performed a significance analysis using Graphpad to calculate the P value.Subsequently, we compared the combinations with the strongest enzyme activity under different combinations. The results showed that the dual enzyme combination had the strongest effect.

Figure 44. Different combinations activity detection data

As shown in Figure 45, the dual combination of Cajanus + Cicer displayed the highest overall activity, even exceeding that of the full five-enzyme mixture. This suggests that specific pairings may exhibit synergistic effects stronger than broader mixtures.

Figure 45. Graphs of optimal combinations comparison

Among the five individual enzymes tested, Papain demonstrated the strongest activity, in line with its rapid casein-clearing effect observed earlier. Cajanus and Cicer showed similar performance, and no statistically significant difference was detected between them.

Interestingly, the Cajanus + Cicer combination outperformed even the full five-enzyme mix, highlighting that targeted pairing of compatible enzymes can deliver higher activity than complex multi-enzyme blends. This observation underscores the value of rational enzyme pairing in optimizing proteolytic systems.

Other Contributions

We have established a reusable natural protease structure-function prediction workflow that covers the entire process from sequence screening to function prediction. First, we use the NCBI BLAST tool to screen for promising papain isoenzyme sequences; then, we utilise AlphaFold for three-dimensional structure prediction of the candidate enzymes, combined with molecular docking tools such as AutoDock to analyse the spatial conformation of their substrate-binding sites and active centres; finally, based on structural features, affinity, and stability, we select target enzymes with application potential. This workflow is not only applicable to the initial development of proteases for skincare applications but also to the structural screening and optimisation of functional proteins for cleaning, antibacterial, and other functions. It offers broad transferability and reference value, providing systematic support for other iGEM teams in the direction of functional enzyme screening.

References

1. Trevisol, T. C., Henriques, R. O., Souza, A. J. A., Cesca, K., & Furigo Jr, A. (2023). Starch-and carboxymethyl cellulose-based films as active beauty masks with papain incorporation. International Journal of Biological Macromolecules, 231, 123258.
2. Qian, H., Wang, L., Li, Y., Wang, B., Li, C., Fang, L., & Tang, L. (2022). The traditional uses, phytochemistry and pharmacology of Abrus precatorius L.: a comprehensive review. Journal of ethnopharmacology, 296, 115463.
3. Okoro, E. E., Maharjan, R., Jabeen, A., Ahmad, M. S., Azhar, M., Shehla, N., ... & Choudhary, M. I. (2021). Isoflavanquinones from Abrus precatorius roots with their antiproliferative and anti-inflammatory effects. Phytochemistry, 187, 112743.
4. Xiang, Z., Chen, Y., & Qiu, J. (2022). An integrated chemical analysis and network pharmacology approach to identify quality markers of Actinidia eriantha Benth radix on gastric cancer. Phytochemical Analysis, 33(6), 851-868.
5. Wang, S., Gao, X., Sun, Q., Zhu, Y., Qin, L., & Zhu, B. (2022). The phytochemical properties, pharmacological effects and traditional uses of Actinidia eriantha Benth.: A review. Frontiers in Pharmacology, 13, 959900.
6. Begum, N., Khan, Q. U., Liu, L. G., Li, W., Liu, D., & Haq, I. U. (2023). Nutritional composition, health benefits and bio-active compounds of chickpea (Cicer arietinum L.). Frontiers in nutrition, 10, 1218468.
7. Acevedo Martinez, K. A., Yang, M. M., & Gonzalez de Mejia, E. (2021). Technological properties of chickpea (Cicer arietinum): Production of snacks and health benefits related to type‐2 diabetes. Comprehensive reviews in food science and food safety, 20(4), 3762-3787.
8. Chandrasekaran, S., Luna-Vital, D., & De Mejia, E. G. (2020). Identification and comparison of peptides from chickpea protein hydrolysates using either bromelain or gastrointestinal enzymes and their relationship with markers of type 2 diabetes and bitterness. Nutrients, 12(12), 3843.