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1.
Int J Mol Sci ; 24(2)2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36674796

RESUMO

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.


Assuntos
Inteínas , Staphylococcus aureus , Humanos , Inteínas/genética , Ligantes , Termodinâmica , Imunoglobulina G , Calorimetria/métodos , Ligação Proteica
2.
Nat Commun ; 13(1): 7672, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36539402

RESUMO

Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.


Assuntos
RNA Polimerase II , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Nucleares/metabolismo , Regulação da Expressão Gênica , Inteínas/genética , Transcrição Genética
3.
Chem Commun (Camb) ; 58(95): 13282-13285, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36373598

RESUMO

A split intein-based method has been developed to detect peptide:N-glycanase (PNGase) activity in live cells. PNGase cleaves the linkage between N,N'-diacetylchitobiose and the Asn side-chain of N-intein peptides and the products react rapidly with C-intein by protein trans-splicing to generate an active luciferase.


Assuntos
Inteínas , Luminescência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Processamento de Proteína , Peptídeos
4.
STAR Protoc ; 3(4): 101807, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36386891

RESUMO

Neural network studies require efficient genetic tools to analyze individual neural circuit functions in vivo. Thus, we developed an advanced circuit-selective gene manipulating tool utilizing anterograde and retrograde adeno-associated viruses (AAVs) encoding split-intein-mediated split-Cre. This strategy can be applied to visualize a specific neural circuit as well as manipulate multiple genes in the circuit neurons. Here, we describe the production and purification of the AAVs, viral injection to the mouse brain, and imaging analysis for a specific neural circuit. For complete details on the use and execution of this protocol, please refer to Kim et al. (2022).


Assuntos
Inteínas , Processamento de Proteína , Animais , Camundongos , Integrases/genética , Dependovirus/genética , Encéfalo/diagnóstico por imagem
5.
Curr Protoc ; 2(10): e571, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36222539

RESUMO

Heterologous expression in Escherichia coli is a commonly used method to produce ribosomally synthesized peptides for further study. This generally requires expression of the target protein with an affinity fusion tag, followed by isolation of the fusion protein from a cellular lysate by affinity purification, and finally by removal of the fusion tag and purification of the desired peptide. Sometimes, however, fusion proteins may be degraded during recombinant expression in E. coli. We recently reported an expression system that sandwiches the target peptide between an N-terminal small ubiquitin-like modifier (SUMO) protein and a C-terminal intein. This SUMO-peptide-intein (SPI) fusion protein protects the central peptide from degradation and can lead to improved peptide yield after purification. In this report, we detail the cloning, expression, and isolation procedures for the SPI fusion system, with comments on conditions that can be optimized for different peptides to obtain maximal yield for each construct. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cloning to construct SPI gene Basic Protocol 2: Expression of SPI fusion proteins in E. coli BL21(DE3) Support Protocol: Optimization of expression and induction conditions Basic Protocol 3: Isolation and purification of SPI fusion proteins with a chitin column Alternate Protocol: Isolation and purification of SPI fusion proteins without chitin.


Assuntos
Escherichia coli , Inteínas , Quitina/metabolismo , Escherichia coli/genética , Inteínas/genética , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Ubiquitinas/metabolismo
6.
J Am Chem Soc ; 144(41): 19196-19203, 2022 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-36194550

RESUMO

Split intein-mediated protein trans-splicing (PTS) is widely applied in chemical biology and biotechnology to carry out traceless and specific protein ligation. However, the external residues immediately flanking the intein (exteins) can reduce the splicing rate, thereby limiting certain applications of PTS. Splicing by a recently developed intein with atypical split architecture ("Cat") exhibits a stark dependence on the sequence of its N-terminal extein residues. Here, we further developed Cat using error-prone polymerase chain reaction (PCR) and a cell-based selection assay to produce Cat*, which exhibits greatly enhanced PTS activity in the presence of unfavorable N-extein residues. We then applied solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to explore how the dynamics of a conserved B-block histidine residue (His78) contribute to this extein dependence. The enhanced extein tolerance of Cat* reported here should expand the applicability of atypically split inteins, and the mechanism highlights common principles that contribute to extein dependence.


Assuntos
Exteínas , Inteínas , Histidina/metabolismo , Processamento de Proteína , Proteínas/metabolismo
7.
ACS Synth Biol ; 11(10): 3296-3304, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36150110

RESUMO

Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.


Assuntos
Ginsenosídeos , Inteínas , Inteínas/genética , Processamento de Proteína , Proteínas/metabolismo
8.
ACS Infect Dis ; 8(9): 1851-1868, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-35948057

RESUMO

Drug resistance is a significant concern in the treatment of diseases, including cryptococcosis caused by Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga). Alternative drug targets are necessary to overcome drug resistance before it attains a critical stage. Splicing of inteins from pro-protein precursors is crucial for activities of essential proteins hosting intein elements in many organisms, including human pathogens such as Cne and Cga. Through a high-throughput screening, we identified calcimycin (CMN) as a potent Prp8 intein splicing inhibitor with a minimum inhibitory concentration (MIC) of 1.5 µg/mL against the wild-type Cne-H99 (Cne-WT or Cne). In contrast, CMN inhibited the intein-less mutant strain (Cne-Mut) with a 16-fold higher MIC. Interestingly, Aspergillus fumigatus and a few Candida species were resistant to CMN. Further studies indicated that CMN reduced virulence factors such as urease activity, melanin production, and biofilm formation in Cne. CMN also inhibited Cne intracellular infection in macrophages. In a target-specific split nanoluciferase assay, the IC50 of CMN was 4.6 µg/mL. Binding of CMN to recombinant Prp8 intein was demonstrated by thermal shift assay and microscale thermophoresis. Treating Cne cells with CMN reduced intein splicing. CMN was fungistatic and showed a synergistic effect with the known antifungal drug amphotericin B. Finally, CMN treatment at 20 mg/kg body weight led to 60% reduction in lung fungal load in a cryptococcal pulmonary infection mouse model. Overall, CMN represents a potent antifungal with a novel mechanism of action to treat Cne and possibly Cga infections.


Assuntos
Criptococose , Cryptococcus neoformans , Animais , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Calcimicina/metabolismo , Calcimicina/farmacologia , Criptococose/tratamento farmacológico , Proteínas Fúngicas/química , Humanos , Inteínas , Camundongos , Alinhamento de Sequência
9.
Sheng Wu Gong Cheng Xue Bao ; 38(6): 2365-2376, 2022 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-35786486

RESUMO

DLP4 (defensin-like peptide 4) is a novel insect defensin, which has strong antibacterial activity against Gram-positive bacteria and is not susceptible to develop drug resistance. In this study, an elastin-like polypeptide (ELP) and an intein fusion system were used for production and purification of DLP4, which combined the characteristics of the phase transition of ELP and the C-cleavage of the intein. A recombinant expression plasmid pET-ELP-I-DLP4 was constructed and transformed into Escherichia coli. Subsequently, DLP4 was purified by simple centrifugation, alternation of pH and temperature. However, the C-cleavage of the intein occurred unexpectedly during the process of expression and purification. To solve this problem, the intein was split into N-intein (I0N) and C-intein (I0C), and fused with ELP or DLP4 to construct two recombinant expression plasmids pET-ELP-I0N and pET-ELP-I0C-DLP4, respectively. These two plasmids were transformed into E. coli separately. The mixture of the two cultures of E. coli strains restored the C-cleavage activity of the intein. This operation yielded DLP4 of about 1.49 mg/L. Antibacterial test confirmed that the purified DLP4 exhibited expected activity. Thus, this approach can be used as an effective way for DLP4 expression and purification in the prokaryotic system.


Assuntos
Elastina , Inteínas , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Peptídeos Antimicrobianos , Defensinas/metabolismo , Elastina/química , Elastina/genética , Elastina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Inteínas/genética , Peptídeos/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia
10.
J Pharmacol Toxicol Methods ; 116: 107174, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35779849

RESUMO

Angiotensin-converting enzyme I (ACE) is a key part of the renin-angiotensin system. Its main function is to regulate blood pressure and the balance of salts in the body. Somatic ACE has two domains, N-C-, each of which has a catalytic site that exhibits 60%sequence identity. The N-domain has a specific action in the hydrolysis of beta-amyloid bodies and angiotensin (1-7), which activates the MAS receptor and triggers anti-thrombotic and anti-inflammatory actions. Our goal was to obtain the catalytic site Ala361 to Gly468 of the N domain region, csACEN, without needing purification by chromatography. We employed a method that uses an Elastin-like Polypeptide (ELP) and Intein sequences linked to the peptide of interest. The more differential for obtaining the pure peptide was the cultivation temperatures in the synthesis of ELPcsACEN at 37 °C, with a significant increase in expression. In the purification by ELP precipitation, we recorded the highest efficiency in the concentrations of 0.57 M and 0.8 M of ammonium sulfate buffer. Intein autocleavage study allows removal of the ELP sequence at acidic pH, with the buffers MES and Tris-HCl The present study defined the best conditions for obtaining pure csACEN that the literature has not yet described for peptides. Obtaining pure csACEN aims at future studies for therapeutic use in hypertension, Alzheimer's, and oncology.


Assuntos
Elastina , Inteínas , Angiotensinas , Domínio Catalítico , Elastina/química , Elastina/metabolismo , Peptídeos/química
11.
ACS Synth Biol ; 11(7): 2513-2517, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35767601

RESUMO

Virus-assisted delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. Among the CRISPR/Cas systems, CRISPR/Cas9 is most widely used; however, to pack the relatively large size of the CRISPR/Cas9 system into viral vectors with confined packaging capacity is challenging. To address this technical challenge, we developed a strategy based on split inteins that splits the required CRISPR/Cas9 components across a dual-vector system. The CRISPR/Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by a successful demonstration of split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-nCas9 can induce efficient base editing in plant cells. We identified several split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.


Assuntos
Sistemas CRISPR-Cas , Inteínas , Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma de Planta , Inteínas/genética , Plantas/genética
12.
Nat Commun ; 13(1): 3605, 2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35739125

RESUMO

The cell-type-specific recording and manipulation is instrumental to disentangle causal neural mechanisms in physiology and behavior and increasingly requires intersectional control; however, current approaches are largely limited by the number of intersectional features, incompatibility of common effectors and insufficient gene expression. Here, we utilized the protein-splicing technique mediated by intervening sequences (intein) and devised an intein-based intersectional synthesis of transactivator (IBIST) to selectively control gene expression of common effectors in multiple-feature defined cell types in mice. We validated the specificity and sufficiency of IBIST to control fluorophores, optogenetic opsins and Ca2+ indicators in various intersectional conditions. The IBIST-based Ca2+ imaging showed that the IBIST can intersect five features and that hippocampal neurons tune differently to distinct emotional stimuli depending on the pattern of projection targets. Collectively, the IBIST multiplexes the capability to intersect cell-type features and controls common effectors to effectively regulate gene expression, monitor and manipulate neural activities.


Assuntos
Inteínas , Optogenética , Animais , Inteínas/genética , Camundongos , Neurônios , Processamento de Proteína , Transativadores/genética
13.
Biosensors (Basel) ; 12(5)2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35624584

RESUMO

Cell-based sensors provide a flexible platform for screening biologically active targets and for monitoring their interactions in live cells. Their applicability extends across a vast array of biological research and clinical applications. Particularly, cell-based sensors are becoming a potent tool in drug discovery and cell-signaling studies by allowing function-based screening of targets in biologically relevant environments and enabling the in vivo visualization of cellular signals in real-time with an outstanding spatiotemporal resolution. In this review, we aim to provide a clear view of current cell-based sensor technologies, their limitations, and how the recent improvements were using intein-mediated protein engineering. We first discuss the characteristics of cell-based sensors and present several representative examples with a focus on their design strategies, which differentiate cell-based sensors from in vitro analytical biosensors. We then describe the application of intein-mediated protein engineering technology for cell-based sensor fabrication. Finally, we explain the characteristics of intein-mediated reactions and present examples of how the intein-mediated reactions are used to improve existing methods and develop new approaches in sensor cell fabrication to address the limitations of current technologies.


Assuntos
Técnicas Biossensoriais , Inteínas , Técnicas Biossensoriais/métodos , Engenharia de Proteínas , Transdução de Sinais
14.
EMBO Mol Med ; 14(6): e15199, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35491676

RESUMO

Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralise F8 activity. Taking advantage of split-intein-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV-intein vectors whose co-administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti-F8 antibodies unlike animals treated with the single oversized AAV-F8 vector under clinical development. Therefore, liver gene therapy with AAV-F8-N6 intein should be considered as a potential therapeutic strategy for HemA.


Assuntos
Hemofilia A , Inteínas , Animais , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Hemofilia A/genética , Hemofilia A/terapia , Inteínas/genética , Fígado , Camundongos , Trans-Splicing
15.
Protein Sci ; 31(5): e4316, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35481634

RESUMO

Recombinant peptide production in Escherichia coli is often accomplished through cloning and expression of a fusion protein. The fusion protein partner generally has two requirements: (a) it contains an affinity tag to assist with purification and (b) it can be cleaved off to leave only the desired peptide sequence behind. Common soluble fusion partners include small ubiquitin-like modifier protein (SUMO), maltose-binding protein (MBP), glutathione S-transferase (GST), or intein proteins. However, heterologously expressed peptides can suffer from proteolytic degradation or instability. This degradation can pose a major issue for applications requiring a large amount of purified peptide, such as NMR structural assignments or biochemical assays. Improving peptide yield by testing various expression and isolation conditions requires a significant amount of effort and may not lead to improved results. Here, we cloned and expressed four different peptides as SUMO fusion proteins. These peptides (lactococcin A, leucocin A, faerocin MK, neopetrosiamide A) were truncated during expression and isolation as SUMO fusions, resulting in low yields of purified peptide. To prevent this degradation and improve yield, we designed a new expression system to create a "sandwiched" fusion protein of the form: His6 -SUMO-peptide-intein (SPI). These sandwiched peptides were more stable and protected against degradation, resulting in improved yields (up to 17-fold) under a set of standard expression and isolation procedures. This SPI expression system uses only two commercially available vectors and standard protein purification techniques, and therefore may offer an economical and facile route to improve yields for peptides that undergo degradation.


Assuntos
Inteínas , Peptídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
16.
Biotechnol Prog ; 38(5): e3266, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35488391

RESUMO

This research focuses on the construction of an affinity purification system based on Cfa DnaE split intein. Cfa DnaE intein is an artificially constructed intein with the advantages of a fast cleavage reaction and good stability. In a previous study, a purification system that uses Cfa intein as a tag was constructed, the separation of the target protein and the tag during the purification process was completed, and the purity of the purified target protein reached 98.21%. Guided by molecular docking results, we identified flexible regions in the split intein and inserted several glycines into the protein to decrease the stability of the Cfa IC , thereby improving the regenerability of the IN media. Inserting 6 glycines between amino acids 14 and 15 of IC improved the regeneration rate of IC -GFP on the column to approximately 96%.


Assuntos
DNA Polimerase III , Inteínas , Cromatografia de Afinidade/métodos , DNA Polimerase III/metabolismo , Glicina , Inteínas/genética , Simulação de Acoplamento Molecular
17.
Chembiochem ; 23(12): e202200115, 2022 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-35420232

RESUMO

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.


Assuntos
Antineoplásicos , Peptídeos Penetradores de Células , Inteínas , Processamento de Proteína , Proteínas Recombinantes
18.
J Magn Reson ; 338: 107195, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35398651

RESUMO

Protein trans-splicing catalyzed by split inteins has been used for segmental isotopic labeling of proteins for alleviating the complexity of NMR signals. Whereas inteins spontaneously trigger protein splicing upon protein folding, inteins from extremely halophilic organisms require a high salinity condition to induce protein splicing. We designed and created a salt-inducible intein from the widely used DnaE intein from Nostoc punctiforme by introducing 29 mutations, which required a lower salt concentration than naturally occurring halo-obligate inteins. We determined the NMR solution structure of the engineered salt-inducible DnaE intein in 2 M NaCl, showing the essentially identical three-dimensional structure to the original one, albeit it unfolds without salts. The NMR structure of a halo-obligate intein under high salinity suggests that the stabilization of the active folded conformation is not a mere result of various intramolecular interactions but the subtle energy balance from the complex interactions, including the solvation energy, which involve waters, ions, co-solutes, and protein polypeptide chains.


Assuntos
Inteínas , Nostoc , DNA Polimerase III/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Inteínas/genética , Espectroscopia de Ressonância Magnética , Nostoc/química , Nostoc/genética , Nostoc/metabolismo , Processamento de Proteína
19.
Carbohydr Polym ; 288: 119398, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35450651

RESUMO

Here a versatile fusion tag composed of His-tag, intein, and elastin-like polypeptide (ELP) tag was prepared for the first time to be fused with levansucrase SacB to construct a recombinant His-ELP-intein-SacB (HEIS) protein to realize nonchromatographic purification of SacB. The efficient biomimetic mineralization of CaHPO4 and HEIS-based hybrid-hydrangea (CaHPO4-HEIS-HH) with good reusability, excellent storage stability and 254.3% improved relative levan yield was prepared with the biomimetic mineralization method. Additionally, the CaHPO4-HEIS-HH showed outstanding operation activity when catalyzing sucrose in solution and up to 75% sucrose conversion rate in fruit juices. The mechanism of biomimetic mineralization was analyzed to show that the HEIS protein might serve as a "binder" to assemble the nanoflakes during biomimetic mineralization. The CaHPO4-HEIS-HH was applicable for efficient production of the levan-type prebiotic polysaccharides, and this approach should be highly valuable for nonchromatographic purification and convenient preparation of various encapsulated enzymes for more efficient catalysis.


Assuntos
Elastina , Inteínas , Biomimética , Elastina/química , Elastina/metabolismo , Frutanos , Peptídeos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Sacarose
20.
J Biotechnol ; 347: 49-55, 2022 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-35240202

RESUMO

Antibiotic resistance is a major public health threat to both humans and animals. There is an urgent need for antimicrobial agents with novel modes of action. Antimicrobial peptides (AMPs) with broad-spectrum antimicrobial activity become the ideal alternative to traditional antibiotics. Here, the ELP-intein self-cleavage system was used to produce antimicrobial peptide arasin-likeSp in Escherichia coli. The tagged target protein (ELP-intein-arasin-likeSp) was mainly expressed in soluble, separated from cell lysates by the inverse transition cycling (ITC), and the arasin-likeSp was further purified by the self-cleavage of intein and the second round of ITC. The final yield of arasin-likeSp was about 3.56 mg/L. Purified arasin-likeSp exhibited significant antibacterial activities against the Gram-positive Bacillus subtilis and Gram-negative Vibrio harveyi bacteria. FE-SEM and PI staining analysis revealed that the arasin-likeSp treatment altered the morphology and membrane permeability of Bacillus subtilis and Vibrio harveyi. Collectively, these data suggest that arasin-likeSp is a candidate AMP for effective inhibition of Vibrio harveyi, a significant bacterial pathogen infecting marine fish and invertebrates. The ELP-intein self-cleavage system described here is a low-cost, simple and potential method for producing antimicrobial peptides, which lays foundations for the large-scale production of antimicrobial peptides in the future.


Assuntos
Escherichia coli , Inteínas , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia
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