Expression and Purification of a Subtilin spaS RiPP Precursor in E. coli

ABSTRACT

Antibiotic resistance is a critical global health challenge causing over two million infections and 23,000 deaths annually in the United States. The rise of resistant pathogens underscores the need for new treatment options since many antibiotics that once cured infections are now ineffective. Natural products particularly ribosomally synthesized and post-translationally modified peptides (RiPPs) offer promising leads to these highly needed treatments. Subtilin a lathibiotic RiPP disrupts gram positive bacterial membranes but its precursor protein spaS has not been studied thoroughly in Escherichia coli. E. coli is the universal model organism since it is fast growing, inexpensive and efficient in labs.  In this study the subtilin precursor spaS was heterologously expressed in E. coli BL21 using the chitin-binding domain (CBD) purification system. After a transformation, induction, purification and SDS-Page analysis a faint but distinct protein band was detected near the predicted molecular weight of 10 kDa. This confirmed the expression and purification of spasS at a low yield. These results show that spaS can be produced in E. coli establishing a foundation for further research. Continuing the study, testing 5 spaS mutants will help design new RiPP- derived antibiotics. This study aims to advance efforts to engineer stable and effective antimicrobial compounds, contributing to the development of promising strategies against antibiotic resistant infections.

INTRODUCTION.

In the U.S more than 2 million people get infections that are resistant to antibiotics and yearly 23,000 people die as a result [1]. This global health threat is pushing the need for new antibiotics to be discovered and studied. This project focuses on producing and purifying the spaS precursor of subtilin a potential antibiotic in E. coli. Due to antibiotic resistance being a major health issue, studying ribosomally synthesized and post-translationally modified peptides (RiPPs), like subtilin can help in the designing process of new treatments.

Antibiotic resistance.

Antibiotic resistance (AMR) occurs when bacteria change in order to resist antibiotics that are used to effectively treat them. They evolve mechanisms that protect them from the effects of antibiotics, such as changing drug targets, producing enzymes that destroy antibiotics, or pumping drugs out of their cells [2]. This is a critical global health challenge because it limits the effectiveness of existing medicines and increases the risk of untreatable infections [3]. In 2021 it was reported that 5.1% of people from ages 12 and up were misusing prescriptions. This includes people who take prescriptions in higher doses than prescribed, use medications not intended for them, or take drugs for non-medical reasons [4]. Though many discoveries of new antibiotics were made AMR continues to rise and affect the effectiveness of antibiotics. Finding new antibiotics is essential to counter this problem and decrease the number of deaths and infections.

Natural products.

Natural products are molecules made by living organisms, including bacteria, fungi, animals and plants [5]. Around 50% of the medicines used today originate from natural sources, including widely used antibiotics such as penicillin and vancomycin [6]. Penicillin was discovered by Alexander Fleming, a bacteriologist in 1928. Fleming observed that colonies of Staphylococcus bacteria didn’t grow near a mold on an agar plate, indicating the mold was secreting an antibacterial substance. After identifying the mold as Penicillium, he isolated the active agent, naming it penicillin. Penicillin kills bacteria by preventing them from making strong cell walls, it blocks proteins that help form cross-links between parts of the wall, which are needed to keep the wall stable. Without these cross-links, the wall becomes weak, and the bacteria burst. Since human cells don’t have cell walls, penicillin doesn’t harm them, which is why it’s a foundational principal in antibiotic therapy [7]. Vancomycin was discovered and isolated in the 1950s from soil samples collected in jungles of Borneo from a fungus named Streptomyces Orientalis. The isolated compound, produced from broth fermentation, showed strong bactericidal against staphylococci. Initial studies showed that staphylococci failed to develop resistance to vancomycin even after repeated exposure through serial passages in media containing it [8]. Vancomycin works by stopping bacteria from building their cell wall. It binds to the end of the peptidoglycan precursors the key building blocks of the wall and prevents them from being added to the growing structure [9]. Without a strong cell wall the bacteria can’t survive and eventually die. This makes vancomycin useful against gram-positive bacteria. Natural products have evolved to act against microbes and are a rich source of potential new drugs.

RiPPS (Ribosomally synthesized and post-translationally modified Peptides).

RiPPs are a class of natural products that begin as simple peptides synthesized by ribosomes. After translation, specialized enzymes carry out post-translational modifications (PTMs) that chemically alter the peptide [10]. Examples of modifications, include adding rings, cross-links, or unusual amino acids, which give them unique 3D structures. The complex structures allow RiPPs to tightly bind to bacterial enzymes or cell membranes increasing their antibacterial potency, which is the reason why RiPPs are a promising candidate for developing new antibiotics. The biosynthesis of a RiPP molecule begins with a biosynthetic gene cluster (BGC) that encodes several functional genes, including those for the precursor peptide, modification enzymes, protease, export and regulation. The precursor gene is first transcribed and translated into a precursor protein composing of a leader, core, and follower region. The leader peptide and the recognition sequences guide the modification enzymes to the core region, where PTMs create the bioactive structure. After modification, the leader (and follower) peptides are cleaved off by a protease, producing the mature RiPP, which is then exported out of the cell (see Figure 1) [11].

Subtilin.

Subtilin is a well-known lantibiotic that demonstrates how structural modifications can enhance antimicrobial potency [13]. It is synthesized as a linear precursor peptide that undergoes extensive PTMs to form several thioether cross-links between cysteine residues and dehydrated amino acids such as dehydroalanine (Dha) and dehydrobutyrine (Dhb). These reactions create lanthionine and methyllanthionine rings that give subtilin its unique ring structure, which are visible in Figure 2. The rings allow subtilin to be more stable, durable and help it attach tightly to bacterial membranes [14]. Subtilin disrupts bacterial cell wall synthesis by binding to lipid II, an essential precursor in peptidoglycan formation, leading to cell death [11]. The combination of structural stability, precise molecular targeting and potent antibacterial activity makes subilin a strong example on how post-translationally modified peptides serve as natural antibiotics.

Spa S protein and mutants.

SpaS is the unmodified ribosomally produced precursor peptide in the subtilin BGC. It contains a leader sequence that helps guide the modification process and a core sequence that becomes the active RiPP after enzymes like SpaB act on it. Without SpaB and other enzymes the SpaS precursor would remain inactive and wouldn’t fold into a functional antibiotic. SpaS mutants are versions of the SpaS protein that have intentional changes (mutations) introduced into their DNA sequence. By expressing and purifying these mutants’ researchers can study PTMs effect on SpaS’s folding and stability. This helps identify which sections of the protein are important for its function. This would help guide the design of the improved antibiotic.

Study Analysis.

Antibiotic resistance is a growing global problem that increases the need for developing new and effective antibiotics. Subtilin is a natural antibiotic, and current research is focused on determining whether its starting protein SpaS and its mutants can be successfully produced and purified in E. coli, as understanding this process would help scientists design and engineer new antibiotics based on subtilin’s structure and activity. The objective of this project is to heterologously express and purify the subtilin precursor SpaS in E. coli using a CBD purification system and verify expression by SDS-PAGE gel. It can be hypothesized that the subtilin precursor protein SpaS and its mutants can be successfully expressed in E. coli using the CBD system; if expression doesn’t occur, this would suggest that SpaS requires additional auxiliary biosynthetic enzymes for production.

MATERIALS AND METHODS.

Transformation into BL21 bacteria.

The New England Biolabs High Efficiency Transformation protocol (C2566) was performed without any modifications to introduce the plasmid DNA into T7 Express E. coli cells. Competent cells were first mixed with plasmid DNA and kept on ice to stabilize the membranes and let the DNA attach. The mixture was then heat-shocked at 42°C for 30 seconds to increase membrane permeability so the plasmid could enter. Right after, the cells were returned to ice to help the membranes reseal. Media was added to give the cells nutrients and allow them to recover while expressing the antibiotic resistance gene. The cells were then plated on selective agar plates so only those containing the plasmid would form colonies. T7 Express E. coli cells were used because they’re engineered for high-level protein expression from a T7 promoter. This step is critical because it’s the controlled way to get the foreign DNA inside the bacteria and turn them into tiny “factories” that can make the protein.

Growing cells.

The New England Biolabs Protocol for Expression Using T7 Express (C2566) was performed without any adjustments. Carbenicillin was used instead of ampicillin because it is more chemically stable, especially at 37 °C. This protocol was used to carry out protein expression using E. coli T7 Express, a strain optimized for high-level expression from T7 promoters and reduced protein degradation. The expression plasmid containing the target gene was first transformed into competent cells to introduce the gene into the host. Cells were plated on selective agar to isolate colonies that successfully took up the plasmid. A single colony was picked to start a starter culture, ensuring a uniform population for expression. The culture was grown to mid-log phase (OD₆₀₀ ≈ 0.6), when the cells are most metabolically active. Protein expression was induced by adding IPTG, which activates T7 RNA polymerase and drives strong transcription of the target gene. After induction, the cultures were incubated for several hours to allow protein accumulation. Finally, the cells were separated from the growth medium by centrifugation so the pellet could be used for the purification steps.

 Protein Purification.

The New England Biolabs IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system was used to purify proteins without leaving any extra tags. It uses a self-cleaving protein element called an intein, fused to a chitin-binding domain (CBD). The gene of interest is cloned into the pTXB1 vector so it is expressed as a fusion with the intein-CBD tag. This vector was used because it is specifically designed for tag-free purification of recombinant proteins in this system. After expressing the fusion protein in E. coli, the cells are lysed and the lysate is applied to a chitin resin column. The CBD binds to the chitin, allowing other proteins to be washed away. A reducing agent like DTT is then added to trigger intein self-cleavage, releasing the target protein in its native, untagged form while the intein-CBD tag stays on the resin. Figure 2 shows the process in a simple, easy-to-follow way. Each step is designed to maximize purity and yield without needing external proteases or extra purification tags, making this system ideal for obtaining functional, native proteins.

SDS-PAGE gel.

The Novex™ Tris-Glycine Mini Gels (WedgeWell™ format) protocol was performed to ensure accurate and reliable protein separation. First, the protein samples were mixed with SDS sample buffer and heated to denature the proteins. This coats them with a uniform negative charge so they separate based only on size. The samples were then loaded into the pre-cast WedgeWell™ gel wells, which are designed for even and consistent loading. The gel was run using Tris-Glycine SDS running buffer, which maintains a stable pH and ionic environment while keeping the proteins denatured during electrophoresis. As the current is applied, proteins migrate through the polyacrylamide gel matrix, with smaller proteins moving faster and farther than larger ones. After separation, the gel is stained—commonly with Coomassie Blue—to visualize the protein bands. Finally, the stained gel is imaged for analysis to assess protein size, expression levels, or sample purity.

Protein staining.

The precast gel was extracted from the cassette by prying it open with a gel knife. The gel was carefully placed in a tray, and the prepared Coomassie dye stain was added. The tray was briefly microwaved for 1 minute and then gently rocked for 1 hour to ensure even dye distribution. After staining, the gel was destained overnight using Coomassie destain solution. This staining step was important to visualize the protein bands within the gel, allowing for evaluation of protein presence, size, and approximate abundance following electrophoresis.

RESULTS.

Protein expression and purification was evaluated using an SDS-PAGE gel (Figure 3).  The gel contains a molecular weight ladder, a crude uninduced and induced extract, a flow through, a wash and two elution lanes. The ladder lane displays the molecular weight markers used for estimating the size of the proteins in the other lanes. The uninduced crude extract shows the baseline protein composition of the cells before induction. It contains only naturally expressed E. coli proteins and doesn’t show a visible target band. In the induced crude extract, there is a new slightly darker band that appears around 10 kDa, indicating successful induction of the target protein. The crude lysate contains all the proteins released after cell disruption, which includes soluble and insoluble proteins, cell debris and the expressed target protein.  The smears observed in the lane are likely results from the large mixture of proteins varying in sizes and the partial denaturation causes by the microwave heating. Microwave heating was used for denaturation, which can lead to protein aggregation, uneven binding, and improper folding. The flow-through lane shows proteins that did not bind to the purification column, which mainly includes non-target proteins. There are faint smearing bands that may indicate that some target protein was last during this step due to incomplete biding. The wash lane contains proteins that were loosely attached to the resin, and the faint bands suggest that the wash effectively removed contaminates while retaining most of the target protein on the column. The elution lanes display a clear band around 10 kDa, which corresponds to the expected molecular wight of the target protein. These bands confirm that the target protein was successfully eluted form the column.

DISCUSSION.

To facilitate the denaturation step before loading samples onto the SDS-PAGE gel, the samples were briefly heated using a microwave instead of a conventional heat block. Although this saved time, microwaving can cause parts of the samples to overheat while others stay cool, leading to incomplete or inconsistent protein unfolding. This likely affected the clarity and consistency of the protein bands seen on the gel. Even with these limitations, the detection of a low–molecular-weight band at the expected size confirms that the precursor peptide was expressed and purified, establishing a foundation for further optimization. Although yields were lower than expected, the results show that SpaS can be heterologously expressed in E. coli. Since E. coli does not naturally produce this protein, successful expression in a laboratory setting provides a safe and controllable environment for studying the peptide. These findings are significant in the broader effort to address antibiotic resistance. RiPPs include many natural antimicrobials and verifying that the SpaS precursor can be expressed is an essential step toward characterizing its modification enzymes and testing designed variants. Accessing these variants will help researchers understand how structural features influence stability and interactions with bacterial targets, guiding the development of improved RiPP-derived antibiotics.

CONCLUSION.

This study successfully demonstrated the heterologous expression and purification of the subtilin precursor peptide SpaS in E. coli. The appearance of a faint but distinct band at the predicted molecular weight confirmed that SpaS was produced and isolated, though at lower-than-expected yields with limited gel resolution. These findings establish a foundation for improving culture conditions, purification strategies, and analytical methods in future work. Moving forward, refining experimental approaches such as optimizing growth parameters, enhancing purification steps, and improving gel imaging will be essential for increasing SpaS yield and clarity. Scaling up culture volumes will also support greater protein recovery. A critical next step is the expression and purification of the six remaining SpaS mutants under consistent conditions to allow direct comparison of solubility, stability, and expression levels. Evaluating how these mutations influence folding and behavior in E. coli will provide valuable insights into SpaS processing and support a more reliable platform for studying post-translational modifications. Ultimately, these efforts will advance the engineering of RiPP-derived antimicrobial compounds and contribute to the development of new strategies for addressing rising antibiotic resistance.

ACKNOWLEDGMENTS.

I’d like to give a thank you to my PI Dr. Allison Walker, my mentor Srujan Vadlamudi and the grad students in the lab Claiborne Tydings and Emilee Patterson for allowing me to work along their side and guidance. Thank you to REHSS for allowing me to participate in this program and expanding my science experience beyond a high school lab.

REFERENCES.

1. 8 Things Everyone Should Know About Antibiotic Resistance – NFID, https://www.nfid.org/. https://www.nfid.org/8-things-everyone-should-know-about-antibiotic-resistance/.
2. W. C. Reygaert, An overview of the antimicrobial resistance mechanisms of bacteria. AIMSMICRO 4, 482–501 (2018).
3. Antimicrobial resistance. https://www.who.int/news-room/fact-sheets/‌detail/antimicrobial-resistance.
4. N. I. on D. Abuse, What is the scope of prescription drug misuse in the United States? | National Institute on Drug Abuse (NIDA) (–). https://nida.nih.gov/publications/research-reports/misuse-prescription-drugs/what-scope-prescription-drug-misuse.
5. F. Les, M. et al., Natural Product Chemistry and Biological Research. IJMS 25, 3774 (2024).
6. S. Mushtaq, et al., Natural products as reservoirs of novel therapeutic agents. EXCLI Journal, 17, 420-451 (2018).
7. D. W. et al., “Penicillin” in StatPearls (StatPearls Publishing, Treasure Island (FL), 2025; http://www.ncbi.nlm.nih.gov/books/NBK554560/).
8. E. Rubinstein, et al., Vancomycin Revisited – 60 Years Later. Front. Public Health 2 (2014).
9. S. Patel, et al., “Vancomycin” in StatPearls (StatPearls Publishing, Treasure Island (FL), 2025; http://www.ncbi.nlm.nih.gov/books/‌NBK459263/).
10. A. L. Vagstad, Engineering ribosomally synthesized and posttranslationally modified peptides as new antibiotics. Current Opinion in Biotechnology 80, 102891 (2023).
11. K. J. et al., Ribosomally synthesized and post-translationally modified peptide natural product discovery in the genomic era. Current Opinion in Chemical Biology 38, 36–44 (2017).
12. S. Zhu, et al., Computational advances in biosynthetic gene cluster discovery and prediction. Biotechnology Advances 79, 108532 (2025).
13. J. Parisot, et al., Molecular Mechanism of Target Recognition by Subtilin, a Class I Lanthionine Antibiotic. Antimicrob Agents Chemother 52, 612–618 (2008).
14. Y. Goto, B. et al., Discovery of Unique Lanthionine Synthetases Reveals New Mechanistic and Evolutionary Insights. PLoS Biol 8, e1000339 (2010).
15. A. Chakicherla, et al., Role of the Leader and Structural Regions of Prelantibiotic Peptides as Assessed by Expressing Nisin-Subtilin Chimeras in Bacillus subtilis 168, and Characterization of their Physical, Chemical, and Antimicrobial Properties. Journal of Biological Chemistry 270, 23533–23539 (1995).

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Posted by buchanle on Friday, May 15, 2026 in May 2026.

Tags: Antibiotic Resistance, Expression, Protein Purification, RiPPs, Subtilin