Production of Lysinibacillus sphaericus Mosquitocidal Protein Mtx2 from Bacillus subtilis as a Secretory Protein

2021 ◽  
Vol 28 ◽  
Author(s):  
Chutchanun Trakulnaleamsai ◽  
Boonhiang Promdonkoy ◽  
Sumarin Soonsanga
Keyword(s):  
Bacillus Subtilis ◽  
Larvicidal Activity ◽  
Mosquito Control ◽  
Expression System ◽  
Secretory Protein ◽  
Signal Peptides ◽  
Secreted Protein ◽  
Lysinibacillus Sphaericus ◽  
Putative Signal Peptide ◽  
Set Up

Background: Mtx2 is a mosquitocidal toxin produced during the vegetative growth of Lysinibacillus sphaericus. The protein shows synergism with other toxins against mosquito larvae; hence it could be used in mosquito control formulations. The protein expression system is needed for Mtx2 development as a biocontrol agent. Objective: The objective of the study was to set up a Bacillus subtilis system to produce Mtx2 as a secreted protein since the protein contains a putative signal peptide. Methods: Initially, four different promoters (P43, Pspac, PxylA, and PyxiE) were compared for their strength using GFP as a reporter in B. subtilis. Subsequently, six different signal peptides (SacB, Epr, AmyE, AprE, LipA, and Vip3A)were tested in conjunction with the selected promoter and mtx2 to evaluate levels of Mtx2 secreted by B. subtilis WB800, an extracellular protease-deficient strain. Results: The promoter PyxiE showed the highest GFP intensity and was selected for further study. Mtx2 was successfully produced as a secreted protein from signal peptides LipA and AmyE, and exhibited larvicidal activity against Aedesaegypti. Conclusion: B. subtilis was successfully developed as a host for the production of secreted Mtx2 and the protein retained its larvicidal activity. Although the Mtx2 production level still needs improvement, the constructed plasmids could be used to produce other soluble proteins.

Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system

2013 ◽  
Vol 8 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Yasuhiko Soejima ◽  
Jae Lee ◽  
Yudai Nagata ◽  
Hiroaki Mon ◽  
Kazuhiro Iiyama ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Signal Peptide ◽  
Protein Production ◽  
Expression System ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Secretion Pathway ◽  
Key Factor ◽  
Secretion Efficiency

AbstractThe baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

scholarly journals Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 523
Author(s):  
Maria Helena Neves Lobo Silva-Filha ◽  
Tatiany Patricia Romão ◽  
Tatiana Maria Teodoro Rezende ◽  
Karine da Silva Carvalho ◽  
Heverly Suzany Gouveia de Menezes ◽  
...  
Keyword(s):  
Mode Of Action ◽  
Mosquito Control ◽  
Binary Toxin ◽  
Lysinibacillus Sphaericus ◽  
Control Programs ◽  
Resistance Selection ◽  
Single Receptor ◽  
Combined Use ◽  
Environmentally Safe ◽  
Medical Importance

Larvicides based on the bacteria Bacillus thuringiensis svar. israelensis (Bti) and Lysinibacillus sphaericus are effective and environmentally safe compounds for the control of dipteran insects of medical importance. They produce crystals that display specific and potent insecticidal activity against larvae. Bti crystals are composed of multiple protoxins: three from the three-domain Cry type family, which bind to different cell receptors in the midgut, and one cytolytic (Cyt1Aa) protoxin that can insert itself into the cell membrane and act as surrogate receptor of the Cry toxins. Together, those toxins display a complex mode of action that shows a low risk of resistance selection. L. sphaericus crystals contain one major binary toxin that display an outstanding persistence in field conditions, which is superior to Bti. However, the action of the Bin toxin based on its interaction with a single receptor is vulnerable for resistance selection in insects. In this review we present the most recent data on the mode of action and synergism of these toxins, resistance issues, and examples of their use worldwide. Data reported in recent years improved our understanding of the mechanism of action of these toxins, showed that their combined use can enhance their activity and counteract resistance, and reinforced their relevance for mosquito control programs in the future years.

scholarly journals Generating Electricity Using Microbial Fuel Cell Powered by Benthic Mud Collected From Two Locations in Akure, Nigeria

2017 ◽  
Vol 13 (18) ◽  
pp. 242
Author(s):  
Adegunloye D. V ◽  
Olotu T. M
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Fuel Cell ◽  
Microbial Fuel Cell ◽  
Lactobacillus Plantarum ◽  
Salt Bridge ◽  
Peak Voltage ◽  
River Bed ◽  
Bacteria And Fungi ◽  
Set Up

Generating electricity using microbial fuel cell powered by benthic mud collected from two locations in Akure was carried out. The locations were Riverbed of FUTA and Apatapiti area of Akure. This was achieved by building anode and cathode containers connected together by a salt bridge and an external circuit was made to transfer the electrons from the anode to the cathode. Bacteria and fungi were isolated from the benthic mud for eight days using standard microbiological techniques. Lactobacillus plantarum, Escherichia coli, Bacillus subtilis, Enterobacter aerogenes, Trichoderma sp, Mucor sp and Alterania sp; Lactobacillus plantarum, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Myrothecium sp and Geotrichum candidum were bacteria and fungi isolated from the benthic mud of Apatapiti area and Riverbed of Futa, Akure respectively. This was used for the generation of electricity using unsterilized mud sample and the control setup was sterilized mud from same source. The set-up was monitored every 24hrous to determine the voltage and current generated. The pH, concentration and temperature were measured. The temperature remains constant throughout the experiment. The set-up were operated at a normal temperature of 27oC and 29oC for Riverbed of FUTA and Apatapiti area of Akure respectively. The peak voltage was between 182.5V and 192.5V and current produced from the main set-up was between 0.3A to 0.53A for Futa river bed while for Apatapiti area of Akure the peak voltage and current were 192.5V and 0.3A respectively. Higher microbial population, current and voltage were observed to be generated in River bed of Futa than Apatapiti area. The difference in the voltage and current and the control set-up shows that anaerobic microorganisms are capable of producing electricity from microbial fuel cell under appropriate conditions.

scholarly journals Antimicrobial genes from Allium sativum and Pinellia ternata revealed by a Bacillus subtilis expression system

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Xi Kong ◽  
Mei Yang ◽  
Hafiz Muhammad Khalid Abbas ◽  
Jia Wu ◽  
Mengge Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Allium Sativum ◽  
Expression System ◽  
Pinellia Ternata ◽  
Bacillus Subtilis Expression

scholarly journals Translational Control of Secretory Proteins in Health and Disease

2020 ◽  
Vol 21 (7) ◽  
pp. 2538 ◽  
Author(s):  
Andrey L. Karamyshev ◽  
Elena B. Tikhonova ◽  
Zemfira N. Karamysheva
Keyword(s):  
Translational Control ◽  
Molecular Mechanisms ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Human Diseases ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Protein Biogenesis ◽  
Health And Disease

Secretory proteins are synthesized in a form of precursors with additional sequences at their N-terminal ends called signal peptides. The signal peptides are recognized co-translationally by signal recognition particle (SRP). This interaction leads to targeting to the endoplasmic reticulum (ER) membrane and translocation of the nascent chains into the ER lumen. It was demonstrated recently that in addition to a targeting function, SRP has a novel role in protection of secretory protein mRNAs from degradation. It was also found that the quality of secretory proteins is controlled by the recently discovered Regulation of Aberrant Protein Production (RAPP) pathway. RAPP monitors interactions of polypeptide nascent chains during their synthesis on the ribosomes and specifically degrades their mRNAs if these interactions are abolished due to mutations in the nascent chains or defects in the targeting factor. It was demonstrated that pathological RAPP activation is one of the molecular mechanisms of human diseases associated with defects in the secretory proteins. In this review, we discuss recent progress in understanding of translational control of secretory protein biogenesis on the ribosome and pathological consequences of its dysregulation in human diseases.

Sign in / Sign up

Export Citation Format

Share Document