Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production.

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.

Protein purification via consecutive histidine-polyphosphate interaction.

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.

Cryo-EM structure of cytochrome bo3 quinol oxidase assembled in peptidiscs reveals an "open" conformation for potential ubiquinone-8 release.

Cytochrome bo3 quinol oxidase belongs to the heme­copper-oxidoreductase (HCO) superfamily, which is part of the respiratory chain and essential for cell survival. While the reaction mechanism of cyt bo3 has been studied extensively over the last decades, specific details about its substrate binding and product release have remained unelucidated due to the lack of structural information. Here, we report a 2.8 Å cryo-electron microscopy structure of cyt bo3 from Escherichia coli assembled in peptidiscs. Our structural model shows a conformation for amino acids 1-41 of subunit I different from all previously published structures while the remaining parts of this enzyme are similar. Our new conformation shows a "U-shape" assembly in contrast to the transmembrane helix, named "TM0", in other reported structural models. However, TM0 blocks ubiquinone-8 (reaction product) release, suggesting that other cyt bo3 conformations should exist. Our structural model presents experimental evidence for an "open" conformation to facilitate substrate/product exchange. This work helps further understand the reaction cycle of this oxidase, which could be a benefit for potential drug/antibiotic design for health science.

A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

Screening for Metal-Chelating Activity in Potato Protein Hydrolysates Using Surface Plasmon Resonance and Peptidomics.

Metal-catalyzed lipid oxidation is a major factor in food waste, as it reduces shelf life. Addressing this issue, our study investigates the potential of hydrolysates derived from potato protein, a by-product of potato starch production, as metal-chelating antioxidants. Through sequential enzymatic hydrolysis using alcalase or trypsin combined with Flavourzyme, we produced various hydrolysates, which were then fractionated using ultrafiltration. Using a combination of peptidomics and bioinformatics, we predicted the presence of metal-chelating and free radical-scavenging peptides across all hydrolysate fractions, with a trend indicating a higher content of antioxidant peptides in lower molecular weight fractions. To validate these predictions, we utilized surface plasmon resonance (SPR) and a 9-day emulsion storage experiment. While SPR demonstrated potential in identifying antioxidant activity, it faced challenges in differentiating between hydrolysate fractions due to significant standard errors. In the storage experiment, all hydrolysates showed lipid oxidation inhibition, though not as effectively as ethylenediaminetetraacetic acid (EDTA). Remarkably, one fraction (AF13) was not significantly different (p < 0.05) from EDTA in suppressing hexanal formation. These results highlight SPR and peptidomics/bioinformatics as promising yet limited methods for antioxidant screening. Importantly, this study reveals the potential of potato protein hydrolysates as antioxidants in food products, warranting further research.

Prokaryotic Expression and Affinity Purification of DDX3 Protein.

BACKGROUND: DDX3 is a protein with RNA helicase activity that is involved in a variety of biological processes, and it is an important protein target for the development of broad-spectrum antiviral drugs, multiple cancers and chronic inflammation. OBJECTIVES: The objective of this study is to establish a simple and efficient method to express and purify DDX3 protein in E. coli, and the recombinant DDX3 should maintain helicase activity for further tailor-made screening and biochemical function validation. METHODS: DDX3 cDNA was simultaneously cloned into pET28a-TEV and pNIC28-Bsa4 vectors and transfected into E. coli BL21 (DE3) to compare one suitable prokaryotic expression system. The 6×His-tag was fused to the C-terminus of DDX3 to form a His-tagging DDX3 fusion protein for subsequent purification. Protein dissolution buffer and purification washing conditions were optimized. The His-tagged DDX3 protein would bind with the Ni-NTA agarose by chelation and collected by affinity purification. The 6×His-tag fused with N-terminal DDX3 was eliminated from DDX3 by TEV digestion. A fine purification of DDX3 was performed by gel filtration chromatography. RESULTS: The recombinant plasmid pNIC28-DDX3, which contained a 6×His-tag and one TEV cleavage site at the N terminal of DDX3 sequence, was constructed for DDX3 prokaryotic expression and affinity purification based on considering the good solubility of the recombinant His-tagging DDX3, especially under 0.5 mM IPTG incubation at 18°C for 18 h to obtain more soluble DDX3 protein. Finally, the exogenous recombinant DDX3 protein was obtained with more than 95% purity by affinity purification on the Ni-NTA column and removal of miscellaneous through gel filtration chromatography. The finely-purified DDX3 still retained its ATPase activity. CONCLUSION: A prokaryotic expression pNIC28-DDX3 system is constructed for efficient expression and affinity purification of bioactive DDX3 protein in E. coli BL21(DE3), which provides an important high-throughput screening and validation of drugs targeting DDX3.

Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M.

Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.

Poly-Histidine-Tagged Protein Purification Using Immobilized Metal Affinity Chromatography (IMAC).

His-tagging is the most widespread and versatile strategy used to purify recombinant proteins for biochemical and structural studies. Recombinant DNA methods are first used to engineer the addition of a short tract of poly-histidine tag (His-tag) to the N-terminus or C-terminus of a target protein. The His-tag is then exploited to enable purification of the "tagged" protein by immobilized metal affinity chromatography (IMAC). In this chapter, we describe efficient procedures for the isolation of highly purified His-tagged target proteins from an Escherichia coli host using IMAC in a bind-wash-elute strategy that can be performed under both native and denaturing conditions.

Immunogenicity Characterization of the Recombinant gI Protein Fragment from Pseudorabies Virus and an Evaluation of Its Diagnostic Use in Pigs.

Serological testing is an important method for the diagnosis of pseudorabies virus (PRV) infection. We aimed to investigate the envelope glycoprotein I (gI) of PRV, a strong immunogen, and its potential as an efficient and low-cost diagnostic reagent. In this study, the DNA of the PRV SC strain was used as the template, and the recombinant fragment of gI (633 bp) was amplified via PCR using synthetic primers, and was then ligated into the pET-30a expression vector. The constructs were transferred into Escherichia coli (E. coli) for prokaryotic expression, and the antigenicity of the expression products was identified by Western blot analysis with pig positive serum against PRV. The recombinant protein was purified by a Ni column, and BALB/c mice were immunized with purified gI protein to obtain anti-gI-positive serum. After PK-15 cells had been infected by PRV for 48 h, the immunogenicity of purified gI protein was identified with a fluorescence immunoassay using anti-gI mouse serum. The recombinant plasmid (pET-30a-gI) was expressed, and the native gI protein was obtained after denaturation by urea and renaturation by dialysis. A small-scale ELISA test containing 1.0 µg/mL of purified gI protein was designed to evaluate pig serum (80 samples), and the results of the ELISA test were compared to those of competitive ELISA (cELISA) tests using IDEXX Kits, which resulted in 97.5% consistency. The results suggested that the truncated gI protein may be a potential diagnostic reagent.

Simple purification and characterization of soluble and homogenous ABC-F translation factors from Enterococcus faecium.

The family of ATP-binding cassette F proteins (ABC-F) is mainly made up of cytosolic proteins involved in regulating protein synthesis, and they are often part of a mechanism that confers resistance to ribosome-targeting antibiotics. The existing literature has emphasized the difficulty of purifying these recombinant proteins because of their very low solubility and stability. Here, we describe a rapid and efficient three-step purification procedure that allows for the production of untagged ABC-F proteins from Enterococcus faecium in the heterologous host Escherichia coli. After four purified ABC-F proteins were produced using this protocol, their biological activities were validated by in vitro experiment. In conclusion, our study provides an invaluable tool for obtaining large amounts of untagged and soluble ABC-F proteins that can then be used for in vitro experiments.

Screening and identification of potential MERS-CoV papain-like protease (PLpro) inhibitors; Steady-state kinetic and Molecular dynamic studies.

MERS-CoV belongs to the coronavirus group. Recent years have seen a rash of coronavirus epidemics. In June 2012, MERS-CoV was discovered in the Kingdom of Saudi Arabia, with 2,591 MERSA cases confirmed by lab tests by the end of August 2022 and 894 deaths at a case-fatality ratio (CFR) of 34.5% documented worldwide. Saudi Arabia reported the majority of these cases, with 2,184 cases and 813 deaths (CFR: 37.2%), necessitating a thorough understanding of the molecular machinery of MERS-CoV. To develop antiviral medicines, illustrative investigation of the protein in coronavirus subunits are required to increase our understanding of the subject. In this study, recombinant expression and purification of MERS-CoV (PLpro), a primary goal for the development of 22 new inhibitors, were completed using a high throughput screening methodology that employed fragment-based libraries in conjunction with structure-based virtual screening. Compounds 2, 7, and 20, showed significant biological activity. Moreover, a docking analysis revealed that the three compounds had favorable binding mood and binding free energy. Molecular dynamic simulation demonstrated the stability of compound 2 (2-((Benzimidazol-2-yl) thio)-1-arylethan-1-ones) the strongest inhibitory activity against the PLpro enzyme. In addition, disubstitutions at the meta and para locations are the only substitutions that may boost the inhibitory action against PLpro. Compound 2 was chosen as a MERS-CoV PLpro inhibitor after passing absorption, distribution, metabolism, and excretion studies; however, further investigations are required.

Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens.

A catalytic antibody has multiple functions compared with a monoclonal antibody because it possesses unique features to digest antigens enzymatically. Therefore, many catalytic antibodies, including their subunits, have been produced since 1989. The catalytic activities often depend on the preparation methods and conditions. In order to elicit the high catalytic activity of the antibodies, the most preferable methods and conditions, which can be generally applicable, must be explored. Based on this view, systematic experiments using two catalytic antibody light chains, #7TR and H34, were performed by varying the purification methods, pH, and chemical reagents. The experimental results obtained by peptidase activity tests and kinetic analysis, revealed that the light chain's high catalytic activity was observed when it was prepared under a basic condition. These data imply that a small structural modulation of the catalytic antibody occurs during the purification process to increase the catalytic activity while the antigen recognition ability is kept constant. The presence of NaCl enhanced the catalytic activity. When the catalytic light chain was prepared with these preferable conditions, #7TR and H34 hugely enhanced the degradation ability of Amyloid-beta and PD-1 peptide, respectively.

Bioinformatics Predicted Linear Epitopes of the Major Coat Protein of the Beet Yellows Virus for Detection of the Virus in the Cell Extract of the Infected Plant.

Beet yellows virus, which belongs to the genus Closterovirus, family Closteroviridae and has a significant negative economic impact, has proven to be challenging to detect and diagnose. To obtain antibodies against BYV, we propose an easier bioinformatics approach than the isolation and purification of the wild virus as an antigen. We used the SWISS-MODEL Workspace (Biozentrum Basel) protein 3D prediction program to discover epitopes of major coat protein p22 lying on the surface of the BYV capsid. Sequences coding these epitopes were cloned into plasmid pQE-40 (Qiagen) in frame with mouse dihydrofolate reductase gene. Fused epitopes were expressed in Escherichia coli and isolated by the Ni-NTA affinity chromatography. Murine antibodies were raised against each epitope and in a combination of both and characterized by dot-ELISA and indirect ELISA. We successively used these antibodies for diagnosis of virus disease in systemically infected Tetragonia tetragonioides. We believe the approach described above can be used for diagnostics of difficult-to-obtain and hazardous-to-health viral infections.

Studies of functional properties of espin 1: Its interaction to actin filaments.

Actin is a multifunctional biomolecule that forms not only basic structural bodies such as filopodia and lamellipodia, but also large microvilli-like organelles like stereocilia. Actin consists of four sub-domains (S1, S2, S3, and S4), and the "target-binding groove" formed between S1 and S3 is the major binding site for various actin binding proteins. Actin filament dynamics are regulated by numerous actin binding proteins with different mechanisms of actin binding, assembly, and disassembly such as actin severing, branching, and bundling. Ectoplasmic specialization protein 1 (espin 1) is an actin binding and bundling protein that is specifically implicated in the elongation and stabilization of stereocilia as a binding partner with myosin III. However, little is known about the molecular structure, actin bundling, and stabilizing mechanism of espin 1; hence, we investigated the interaction between actin and espin 1 through structural data. In this study, we first purified human espin 1 in an E. coli system following a new detergent-free approach and then demonstrated the 2D structure of full-length espin 1 using transmission electron microscopy along with Nickel nitrilotriacetic acid nanogold labeling and 2D averaging using SPIDER. Furthermore, we also determined the espin 1 binding domain of actin using a co-sedimentation assay along with gelsolin and myosin S1. These findings are not only beneficial for understanding the actin binding and bundling mechanism of espin 1, but also shed light on its elongation, stabilization, and tip-localization mechanisms with myosin III. This study thus provides a basis for understanding the molecular structure of espin 1 and can contribute to various hearing-related diseases, such as hearing loss and vestibular dysfunction.

Ligand Immobilization Methods for Affinity Chromatography.

There is a plethora of chromatographic supports available in different shapes, sizes, functionalities, and chemical compositions, that could be employed for the numerous applications such as purification, scavenging, and target quantification. Therefore, it becomes critical to understand the chemical nature of these materials to select optimum conditions for ligand immobilization. In this chapter, we have explained some commonly employed ligand immobilization techniques to graft the ligand on the resin surfaces. For a quick reference, we have also included some functionalities of the resins that are commercially available.

Establishment of purification method for prokaryotic expression of Serpin gene for Dermatophagoides farinae.

This study aimed to develop an effective method for the expression and purification of the Dermatophagoides farinae serpin protein and to establish an experimental foundation for elucidating its role in the temperature stress response. The total RNA of D. farinae was extracted, and specific primers were designed for serpin amplification. Serpin was joined with pET32a vector and transformed into BL21 (DE3) cells. Expression of recombinant proteins was induced. Proteins were extracted by enzymatic lysis or enzymatic lysis combined with ultrasonication. Recombinant proteins were purified by Ni-NTA method. SDS-PAGE was conducted to evaluate protein expression, extraction, and purification efficiency. Agarose gel electrophoresis and sequencing analysis showed that the amplified serpin open reading frame was 1284 bp, encoding a hydrophilic and stable protein with a relative molecular weight of 48.30 kD. SDS-PAGE demonstrated that there was a specific band at 55-70 kD, which was consistent with the predicted size of the recombinant pET32a-Serpin protein. Enzymatic lysis combined with 30% ultrasonic power promoted the release of soluble protein more effectively than enzymatic lysis alone. 16 °C for 4 h was optimal for inducing expression. The optimal imidazole concentrations for washing non-His-tagged protein and eluting His-tagged protein were determined to be 20 mM and 200 mM, respectively. In this study, A prokaryotic expression and purification system for the D. farinae serpin protein was successfully established, providing a technical reference for functional gene research in mites at the protein level.

High level expression and purification of recombinant 3ABC non-structural protein of foot-and-mouth disease virus using SUMO fusion system.

The detection of antibody to non-structural protein (NSP) of Foot-and-mouth disease virus (FMDV) is the reliable diagnostic method for differentiating infected from vaccinated animals (DIVA). For this purpose, the detection of antibodies to non-structural 3ABC protein is suitable for identification of virus activity in the animals exposed to FMDV infection. However, large-scale production of recombinant 3ABC protein is challenging due to the formation of inclusion bodies in Escherichia coli and low yield due to protein aggregation during in vitro refolding. In this study, 3ABC gene was fused with SUMO (small ubiquitin-like modifiers) fusion system which significantly enhanced expression of recombinant 3ABC protein in E. coli. The solubility of the recombinant 6xHis-SUMO 3ABC fusion protein was improved by mild detergent treatment and purified through Ni-NTA chromatography under non-denaturing conditions which yielded 9 mg protein obtained from 1-L bacterial fermentation culture. The diagnostic potential of recombinant 3ABC protein was also tested by ELISA that provided reliable diagnostic performance (DSn = 92%, DSp = 94%) upon comparison with commercially available kit. The thermal stability of fusion protein was also tested which presented reliable performance at different temperatures. In conclusion, we presented SUMO fusion for the enhanced expression in E. coli and purification of active recombinant 3ABC protein using non-denaturing conditions without refolding steps. This protein can be used as a suitable diagnostic antigen to detect antibodies following FMDV infection.

Development of the Antileishmanial Vaccine.

Search for an efficacious antileishmanial vaccine has led to clinical trials of numerous vaccine candidates in the past few decades. As no promising candidate has emerged from these studies, novel vaccine modalities and vaccine assessment techniques are still emerging for antileishmanial vaccine development. Briefly, this chapter discusses: (a) history and timeline of antileishmanial vaccine development; (b) techniques utilized for developing whole-parasite and subunit-based antileishmanial vaccine formulations, and (c) immunogenicity and post-challenge protective efficacy assessment of vaccine candidates.

The bone-degrading enzyme machinery: From multi-component understanding to the treatment of residues from the meat industry.

Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm Osedax mucofloris, and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.6%) encoding such enzymes were retrieved. The taxonomic assignation and the median identity of 72.2% to homologous proteins reflect microbial and functional novelty associated to a specialized bone-degrading marine community. Binning suggests that only one generalist hosting all ten targeted activities, working in synergy with multiple specialists hosting a few or individual activities. Collagenases were the most abundant enzyme class, representing 48% of the total hits. A total of 47 diverse enzymes, representing 8 hydrolytic activities, were produced in Escherichia coli, whereof 13 were soluble and active. The biochemical analyses revealed a wide range of optimal pH (4.0-7.0), optimal temperature (5-65 °C), and of accepted substrates, specific to each microbial enzyme. This versatility may contribute to a high environmental plasticity of bone-degrading marine consortia that can be confronted to diverse habitats and bone materials. Through bone-meal degradation tests, we further demonstrated that some of these enzymes, particularly those from Flavobacteriaceae and Marinifilaceae, may be an asset for development of new value chains in the biorefinery industry.

Advances in engineered Bacillus subtilis biofilms and spores, and their applications in bioremediation, biocatalysis, and biomaterials.

Bacillus subtilis is a commonly used commercial specie with broad applications in the fields of bioengineering and biotechnology. B. subtilis is capable of producing both biofilms and spores. Biofilms are matrix-encased multicellular communities that comprise various components including exopolysaccharides, proteins, extracellular DNA, and poly-γ-glutamic acid. These biofilms resist environmental conditions such as oxidative stress and hence have applications in bioremediation technologies. Furthermore, biofilms and spores can be engineered through biotechnological techniques for environmentally-friendly and safe production of bio-products such as enzymes. The ability to withstand with harsh conditions and producing spores makes Bacillus a suitable candidate for surface display technology. In recent years, the spores of such specie are widely used as it is generally regarded as safe to use. Advances in synthetic biology have enabled the reprogramming of biofilms to improve their functions and enhance the production of value-added products. Globally, there is increased interest in the production of engineered biosensors, biocatalysts, and biomaterials. The elastic modulus and gel properties of B. subtilis biofilms have been utilized to develop living materials. This review outlines the formation of B. subtilis biofilms and spores. Biotechnological engineering processes and their increasing application in bioremediation and biocatalysis, as well as the future directions of B. subtilis biofilm engineering, are discussed. Furthermore, the ability of B. subtilis biofilms and spores to fabricate functional living materials with self-regenerating, self-regulating and environmentally responsive characteristics has been summarized. This review aims to resume advances in biological engineering of B. subtilis biofilms and spores and their applications.