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Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP

Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP

Current antibiotics have limited action mode, which makes it difficult for the antibiotics dealing with the emergence of bacteria resisting the existing antibiotics. As a need for new bacteriolytic agents alternative to the antibiotics, AMPs have long been considered substitutes for the antibiotics.
Cecropin B was expressed in a fusion form to six-histidine and SUMO tags in Escherichia coli. Six-histidine tag attached to SUMO was for purification of SUMO-cecropin B fusion proteins and removal of the SUMO tag from cecropin B. Chimeric gene was constructed into pKSEC1 vector that was designed to be functional in both Escherichia coli and chloroplast.
To maximize translation of the fusion protein, sequences were codon-optimized. Four different constructs were tested for the level of expression and solubility, and the construct with a linker, 6xHisSUMO3xGly-cecropin B, showed the highest expression.
In addition, cleavage of the SUMO tag by SUMOase in the three fusion constructs which have no linker sequence (3xGly, three glycines) was not as efficient as the construct with the linker between SUMO and cecropin B. The cleaved cecropin B showed bacteriolytic activity against Bacillus subtilis at a concentration of 0.0625 μg/μL, while cecropin B fused to SUMO had no activity at a higher concentration, 0.125 μg/μL.
As an expression system for AMPs in prokaryotic hosts, the use of tag proteins and appropriate codon-optimization strategy can be employed and further genetic modification of the fusion construct should help the complete removal of the tag proteins from the AMP in the final step of purification.

A new prokaryotic expression vector for the expression of antimicrobial peptide abaecin using SUMO fusion tag.

Despite the growing demand for antimicrobial peptides (AMPs) for clinical use as an alternative approach against antibiotic-resistant bacteria, the manufacture of AMPs relies on expensive, small-scale chemical methods. The small ubiquitin-related modifier (SUMO) tag is industrially practical for increasing the yield of recombinant proteins by increasing solubility and preventing degradation in expression systems.
A new vector system, pKSEC1, was designed to produce AMPs, which can work in prokaryotic systems such as Escherichia coli and plant chloroplasts. 6xHis was tagged to SUMO for purification of SUMO-fused AMPs. Abaecin, a 34-aa-long antimicrobial peptide from honeybees, was expressed in a fusion form to 6xHis-SUMO in a new vector system to evaluate the prokaryotic expression platform of the antimicrobial peptides.
The fusion sequences were codon-optimized in three different combinations and expressed in E. coli. The combination of the native SUMO sequence with codon-optimized abaecin showed the highest expression level among the three combinations, and most of the expressed fusion proteins were detected in soluble fractions.
Cleavage of the SUMO tag by sumoase produced a 29-aa-long abaecin derivative with a C-terminal deletion. However, this abaecin derivative still retained the binding sequence for its target protein, DnaK. Antibacterial activity of the 29-aa long abaecin was tested against Bacillus subtilis alone or in combination with cecropin B.
The combined treatment of the abaecin derivative and cecropin B showed bacteriolytic activity 2 to 3 times greater than that of abaecin alone.Using a SUMO-tag with an appropriate codon-optimization strategy could be an approach for the production of antimicrobial peptides in E.coli without affecting the viability of the host cell.

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities.
Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability.
Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease.
In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake.
Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.
Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP

The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis.

Heterologous gene expression is well established for various prokaryotic model systems. However, low yield, incorrect folding and instability still impede the production of soluble, bioactive proteins. To improve protein production with the Gram-positive host Bacillus subtilis, a secretory expression system was designed that enhances translocation, folding and stability of heterologous proteins, and simplifies purification.
Based on the theta-replication plasmid pHT01, a B. subtilis secretory expression vector was constructed that encodes a fusion protein consisting of a signal peptide and a StrepII-tag linked to a SUMO-tag serving as a folding catalyst. The gene of a protein of interest can be translationally fused to the SUMO cassette and an additional 6xHis-tag encoding region.
In order to maximize secretory expression of the construct by fitting the signal peptide to the StrepII-SUMO part of the fusion protein, a B. subtilis signal-peptide library was screened with the Escherichia coli alkaline phosphatase PhoA as a reporter.The YoaW signal peptide-encoding region (SPyoaW) was identified with highest secretory expression capacity in context with the StrepII-SUMO-tag fusion in a B. subtilis eightfold extracellular protease deletion strain.
PhoA activity and fusion protein production was elevated by a factor of approximately five when compared to an α-amylase (AmyQ) signal peptide construct. Replacement of PhoA with a single-chain variable fragment antibody specific for GFP or the B. amyloliquefaciens RNase barnase, respectively, resulted in a similar enhancement of secretory expression, demonstrating universality of the YoaW signal peptide-StrepII-SUMO encoding cassette for secretory expression in B. subtilis.
Optimisation of codon usage and culture conditions further increased GFP-specific scFv fusion-protein production, and a simple affinity purification strategy from culture supernatant with removal of the StrepII-SUMO-tag by SenP-processing yielded 4 mg of pure, soluble and active GFP-specific scFv from 1 l of culture under standard laboratory conditions.

Recombinant Human NEDD8 (N-6His, SUMO tag)

CG48-1mg 1mg
EUR 456
Description: Lyophilized from a 0.2 μm filtered solution of 20mM PB,150mM NaCl,5% Trehalose,pH7.4.

Recombinant Human NEDD8 (N-6His, SUMO tag)

CG48-500ug 500ug
EUR 303
Description: Lyophilized from a 0.2 μm filtered solution of 20mM PB,150mM NaCl,5% Trehalose,pH7.4.

Recombinant Human NEDD8 (N-6His, SUMO tag)

CG48-50ug 50ug
EUR 95
Description: Lyophilized from a 0.2 μm filtered solution of 20mM PB,150mM NaCl,5% Trehalose,pH7.4.

Tag-100 tag Antibody

abx019172-100ug 100 ug
EUR 356
  • Shipped within 5-10 working days.

Mono Anti-HA tag (hemeagglutinin-tag; HA-tag)

HA13-M 100 ug
EUR 482

Recombinant Human Ubiquitin-Conjugating Enzyme E2 K/UBE2K (N-6His, SUMO tag)

CG45-10ug 10ug
EUR 80
Description: Supplied as a 0.2 μm filtered solution of 20mM PB,150mM NaCl,pH7.4.

Recombinant Human Ubiquitin-Conjugating Enzyme E2 K/UBE2K (N-6His, SUMO tag)

CG45-1mg 1mg
EUR 1014
Description: Supplied as a 0.2 μm filtered solution of 20mM PB,150mM NaCl,pH7.4.

Recombinant Human Ubiquitin-Conjugating Enzyme E2 K/UBE2K (N-6His, SUMO tag)

CG45-500ug 500ug
EUR 659
Description: Supplied as a 0.2 μm filtered solution of 20mM PB,150mM NaCl,pH7.4.

Recombinant Human Ubiquitin-Conjugating Enzyme E2 K/UBE2K (N-6His, SUMO tag)

CG45-50ug 50ug
EUR 141
Description: Supplied as a 0.2 μm filtered solution of 20mM PB,150mM NaCl,pH7.4.

Histidine Tag (His-Tag) Antibody

abx412016-01mg 0.1 mg
EUR 495
  • Shipped within 1 week.

Histidine Tag (His-Tag) Antibody

abx413027-1mg 1 mg
EUR 815
  • Shipped within 1 week.

Histidine Tag (His-Tag) Antibody

abx413031-2mg 2 mg
EUR 1483
  • Shipped within 1 week.

Histidine Tag (His-Tag) Antibody

abx413032-01mg 0.1 mg
EUR 411
  • Shipped within 1 week.

HA-Tag Polyclonal Tag Antibody

A26004 50 µg Ask for price
Description: reagents widely cited

T7 tag (T7-tag, fusion tag) control/blocking peptide

T711-P 100 ug
EUR 164
The new expression system employing a YoaW signal peptide-StrepII-SUMO fusion will simplify secretory protein production and purification with B. subtilis. It can obviate the need for time consuming individual signal-peptide fitting to maximize yield for many different heterologous proteins of interest.

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