UNLABELLED The antimicrobial peptide NZ17074, which is derived from arenicin-3 isolated from Arenicola marina, displayed high activity against a broad range of pathogenic bacteria and fungi. However, NZ17074 has not been produced using fermentation technology. The aim of this work was to study the expression of difficult-to-express NZ17074 in Pichia pastoris by fusing with SUMO3. The DNA fragments of NZ17074 and SUMO3 were fused into SUMO3-NZ17074 using overlap PCR and cloned into the pPICZαA vector to construct the pPICZ-SUMO3-NZ17074 expression vector. The rSUMO3-NZ17074 fusion protein, purified by Ni(2) (+) -chelating affinity chromatography, was cleaved by 50% formic acid at 50°C for 28 h to release recombinant NZ17074 (rNZ17074). After purification with second affinity column, 4·1 mg rNZ17074 peptide with the purity over 90% was obtained from per litre fermentation culture. The rNZ17074 peptide exhibited the significant inhibition activity against Gram-negative bacteria: its minimal inhibitory concentrations (MICs) against Escherichia coli, Salmonella enteritidis and Pseudomonas aeruginosa were 2-4, 2 and 8-16 μg ml(-1) , respectively, which indicated that SUMO3 is a good fusion partner for the expression of the toxic peptide. SIGNIFICANCE AND IMPACT OF THE STUDY Recombinant active NZ17074 was produced with Pichia pastoris by using high-density fermentation technology for the first time. Our findings demonstrated the usefulness of SUMO-fusion technology as an effective expression strategy for synthesizing peptides in yeast. This SUMO3 expression system with a lower cost would likely be widely used for the production of other cytotoxic proteins including antimicrobial peptides.

https://sfamjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/lam.12246

Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner.

Provided are small molecule inhibitors of ubiquitin specific protease 1 (USP1) activity and methods for their use in treating and characterizing cancers. The small molecule USP1 inhibitors of the invention are particularly useful in the treatment of cancers that are resistant to DNA cross-linking agents.

Small molecule inhibitors of USP1 deubiquitinating enzyme activity

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system

The dermcidin-derived peptide DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. Thus, it offers a promising alternative to conventional antibiotics. Furthermore, it plays a role in wound healing, atopic dermatitis and acne vulgaris, indicating applications in cosmetic industries. Recently, dermcidin has been identified as a tumor marker improving cancer prognosis. Hence, large quantities of purified DCD-1L peptide are required to meet the needs of basic research and clinical trials. In the current study, we demonstrate SUMO-based heterologous DCD-1L production in Escherichia coli, followed by affinity chromatography purification. The SUMO tag is cleaved with SUMO-specific protease following purification, leaving free DCD-1L peptide without any additional amino acids. The mass of the peptide was further confirmed by MALDI-TOF-TOF analysis. Furthermore, the cleaved DCD-1L showed antimicrobial activity against the E. coli DH5 alpha test strain. The production and purification of DCD-1L using SUMO tag compare advantageously to other protocols previously described. Thus, the SUMO tag system enables large scale recombinant production of the antimicrobial peptide DCD-1L, which constitutes pharmaceutical and therapeutic potential as an alternative antibiotic.

/pdf/sumo-based-expression-and-purification-of-dermcidin-derived-e3f9hqnncs.pdf

SUMO-based expression and purification of dermcidin-derived DCD-1L, a human antimicrobial peptide, in Escherichia coli

Nuclear transport receptors (NTRs) mediate the translocation of cargos between the cytoplasm and the nucleus. NTRs are classified either as importins or exportins according to the directionality of the cargo transport. To identify new interaction partners of so far poorly characterized NTRs from S. cerevisiae, we developed a novel protein purification approach based on an engineered SUMO-specific protease (SUMOvera protease) and its specific SUMO substrate (SUMOvera). SUMOvera can be used as a stable fusion tag in virtually all eukaryotic systems since it is not recognized by SUMO-specific proteases from yeast, plant, human, amphibian, insect and protozoa. In addition, the SUMOvera protease can be over-expressed in S. cerevisiae without causing toxicity unlike yeast and B. distachyon SUMO-specific proteases. The SUMOvera system has also an orthogonal specificity to the SUMOstar/SUMOstar protease pair to allow the purification of protein complexes with a defined stoichiometry in eukaryotic hosts. 
Using the SUMOvera system, we discovered that yeast Lph2 and Pdr6 mediate the nuclear export and import of different cargos. Specifically, we showed that Lph2 exports and binds to the translation initiation factor eIF4A in a Ran-GTP-dependent manner, and that Pdr6 recognizes Ubc9 as an import substrate as well as eEF2 and eIF5A as export cargos. Lph2 and Pdr6 have been only described as importins; however, our data indicate that they act as bidirectional NTRs that shuttle distinct sets of cargos in opposite directions through the nuclear envelope.
Overall, we describe two novel bidirectional NTRs in S. cerevisiae in addition to Msn5. The reported findings suggest that bidirectionality in NTRs might be more common than previously assumed, and that there might be other bidirectional NTRs in different organisms that still need to be identified.

Novel export and import pathways in S. cerevisiae identified by an engineered SUMO system

The preparation of large amounts of recombinant protein is a major bottleneck for many areas of scientific research, including structural proteomics and therapeutic drug discovery. Recently the Small Ubiquitin-like Modifier (SUMO)-based fusion tag technology has gained increasing use for the expression of recombinant proteins. In E. coli recombinant proteins expressed as SUMO fusions have demonstrated enhanced stability and solubility, leading to greatly increased yields (5 to 20-fold) over constructs lacking this tag. Furthermore, efficient removal of the SUMO tag by SUMO protease allows the generation of a native N-terminus of the target protein. The use of SUMO-fusion technology in E. coli, termed SUMOpro, as well as the use of SUMOstar, an adaptation of SUMO for use in eukaryotic hosts, will be reviewed. This review will also provide a comparison between SUMOpro technology and other fusion tags commonly used to enhance protein production and facilitate purification.

The use of SUMO as a fusion system for protein expression and purification

Methods for detection of the activity of proteolytic enzymes, particularly isopeptidases, are disclosed.

Di- and poly-ubiquitin deubiquitinase substrates and uses thereof

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The use of SUMO as a fusion system for protein expression and purification

Di- and poly-ubiquitin deubiquitinase substrates and uses thereof