enterokinase

Single-cell analyses have recently become important to understand cell heterogeneity, the mechanism of cell function, and diseases. In contrast to single-cell analyses that target nucleic acids, single-cell protein analyses still pose challenges. We have proposed a general concept of integration and extended this concept to the 10-1000 nm scale with femtoliter-picoliter volumes which are smaller than the volume of a single cell exploring ultimate analytical performances (e.g. single-cell target proteomics)...

The plant-based expression systems are now accredited as bioreactors for the high production of various biopharmaceuticals. However, low levels of agglomeration and the absence of effective procedures for purification of recombinant proteins have remained two essential obstacles in molecular farming. In this research, we have studied the production of human interferon gamma (hIFN-γ) in tobacco and analyzed the effects of elastin-like polypeptide (ELP) tag and subcellular localization on its accumulation. We report a remarkable enhancement of accumulation of the fusion proteins versus the corresponding unfused hIFN-γ proteins...

BACKGROUND: The heterologous production of antimicrobial peptides in bacterial models can produce insoluble proteins due to the lack of proper folding. Fusion proteins have been used to increase the expression and solubility of these types of proteins with varying degrees of success. OBJECTIVES: Here, we demonstrate the use of the small metal-binding proteins CusF3H+ (9.9kDa) and SmbP (9.9kDa) as fusion partners for the soluble expression of the bioactive antimicrobial peptide VpDef(6...

A dynamic mucosal layer shields the epithelial cells lining the body cavities and is made up of high molecular weight, heavily glycosylated, multidomain proteins called mucins. Mucins, broadly grouped into transmembrane and secreted mucins, are the first responders to any mechanical or chemical insult to the epithelia and help maintain tissue homeostasis. However, their intrinsic properties to protect and repair the epithelia are exploited during oncogenic processes, where mucins are metamorphosed to aid the tumor cells in their malignant journey...

AIMS: To develop an oral delivery system of glucagon-like peptide 1 (GLP-1)(28-36) for treating Type-2 Diabetes, B.S-GLP-1(28-36), a recombinant Bacillus subtilis spores transformed with a plasmid vector encoding five consecutive GLP-1 (28-36) nonapeptides with an enterokinase site was constructed. METHODS AND RESULTS: GLP-1(28-36) nonapeptide was successfully expressed on the surface of B. subtilis spores and validated by western blot and immunofluorescence. The therapeutic effect of oral administration of B...

The antimicrobial peptide PMAP-36 is a cationic peptide derived from porcine myeloid. The N-terminally paired lysine of PMAP-36 was substituted with tryptophan, and the C-terminal hydrophobic tail was deleted, thereby obtaining the antimicrobial peptide PRW4. PRW4 is a α-helical antimicrobial peptide with broad-spectrum antimicrobial activity. In this study, PRW4 was fused to the 6× His-Trx, and the fusion protein was successfully expressed in Pichia pastoris GS115 from the vector pPICZαA. The maximal induction of recombinant protein occurred in the presence of 1% methanol after 96 h at pH 6...

Emerging evidence indicates that mitochondria contribute to drug resistance in cancer, but how to selectively target the mitochondria of cancer cells remains less explored. Here, we show perimitochondrial enzymatic self-assembly for selectively targeting the mitochondria of liver cancer cells. Nanoparticles of a peptide-lipid conjugate, being a substrate of enterokinase (ENTK), encapsulate chloramphenicol (CLRP), a clinically used antibiotic that is deactivated by glucuronidases in cytosol but not in mitochondria...

Human acidic fibroblast growth factor (haFGF) is a multifunctional protein involved in regulating a wide range of cellular processes. As a potent therapeutic agent, it is highly desirable to produce recombinant haFGF (r-haFGF) at low cost. However, the complex structure and formation of aggregation confines its high-level soluble expression and functional form. Herein, to produce r-haFGF efficiently in E. coli, we devised a novel soluble expression and cost-effective purification approach based on fusion with Scl2-M (a novel modified collagen-like protein) for the first time...

Energetic challenges match intestinal size to dietary intake but less is known about how the intestine responds to specific macronutrient challenges. We examined how intestinal size responds to insufficient dietary protein at the microscopic level. Villi, enterocytes, and surface area were measured across the length of the small intestine in non-reproductive and lactating Mus musculus fed isocaloric control or protein-deficient diets. Lactating mice on the protein-deficient diet exhibited a 24% increase in villus height and a 30% increase in enterocyte width in the proximal small intestine and an overall similar increase in surface area; on the control diet changes in villus height were localized in the mid region...

Human basic fibroblast growth factor (hbFGF) is involved in a wide range of biological activities that affect the growth, differentiation, and migration. Due to its wound healing effects and therapy, hbFGF has the potential as therapeutic agent. Therefore, large-scale production of biologically active recombinant hbFGF with low cost is highly desirable. However, the complex structure of hbFGF hinders its high-level expression as the soluble and functional form. In the present study, an efficient, cost-effective, and scalable method for producing recombinant hbFGF was developed...

Heat shock protein 90 (HSP90) plays essential roles in the normal physiology and comprises four distinct domains, including NH2 -terminal (N), charged linker region (LR), middle (M), and COOH-terminal (C) domains, all of which regulate HSP90 biological functions. We reported herein detailed protocols to produce recombinant full-length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET-32b(+) expression vector...

Hispidalin is a novel antimicrobial peptide isolated from the seeds of Benincasa hispida and is reported to have broad antimicrobial activity against various bacterial and fungal pathogens. To produce significant amounts of Hispidalin, a recombinant Hispidalin with an N-terminal 6 × His tag and an enterokinase sequence, for the first time, was successfully expressed in Escherichia coli or Pichia pastoris cell factory. Results showed that the E. coli-derived recombinant Hispidalin did not show any antimicrobial activity against all the tested strains, whereas the P...

RATIONALE: LysargiNase is a novel characterized metalloprotease that can cleave N-terminal of lysine or arginine residue. The peptides generated by LysargiNase were just mirrors to those by trypsin. This characters of LysargiNase provide a powerful tool for mass spectrometry-based proteomics research. High activity and stable LysargiNase produced with easy and low-cost method could great benefit proteomics research. Here, we reported the soluble recombinant expression, purification and acetyl modification of LysargiNase in Escherichia coli...

Enterokinase (EK) is one of the most popular enzymes for the in vitro cleavage of fusion proteins due to its high degree of specificity for the amino-acid sequence (Asp)4 -Lys. Enzyme reusability is desirable for reducing operating costs and facilitating the industrial application of EK. In this work, we report the controlled, site-specific and covalent cross-linking of an engineered EKLC on amine-modified magnetic nanoparticles (NH2 -MNPs) via microbial transglutaminase-catalyzed bioconjugation for the development of the oriented-immobilized enzyme, namely, EKLC @NH2 -MNP biocatalyst...

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction...

BACKGROUND: Studying proteins and enzymes involved in important biological processes in the Aedes aegypti mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in Escherichia coli prove to be effective in producing milligram quantities of the expressed product...

Fungus defensin is a kind of important natural peptide resource, such as plectasin from the soil fungus Pseudoplectania nigrella with potential application in the antimicrobial peptide lead drug discovery. Here, a fungus defensin named Bldesin with Kv1.3 channel and serine protease inhibitory activities was first explored. By GST-Bldesin fusion expression and enterokinase cleaving strategy, recombinant Bldesin was obtained successfully. Antimicrobial assays showed that Bldesin had potent activity against Gram-positive Staphylococcus aureus, but had no effect on Gram-negative Escherichia coli...

The emergence of antimicrobial resistance among pathogenic microorganisms has been led to an urgent need for antibiotic alternatives. Antimicrobial peptides (AMPs) have been introduced as promising therapeutic agents because of their remarkable potentials. A new modified cathelicidin-BF peptide (Cath-A) with 34 amino acid sequences, represents the potential antimicrobial effects against methicillin-resistant Staphylococcus aureus (MRSA) with slight hemolytic and cytotoxic activities on eukaryotic cells. In this study, the effects of Cath-A on Acinetobacter baumannii , and Pseudomonas aeruginosa isolated from medical instruments were studied...

Background: Biosynthetic teriparatide (1-34) (TPD) is a N-terminally truncated version of human parathyroid hormone (hPTH). The recombinant form of this polypeptide has been expressed in Escherichia coli (E. coli) and approved as the first anabolic treatment of osteoporosis in the EU and the USA. Feasibility of expression and secretion of a tag- fused form of TPD into Bacillus subtilis (B. subtilis) was examined due to several advantages of B. subtilis over E. coli in production of recombinant proteins with pharmacological activities...

Soluble HIV-1 envelope glycoprotein (Env) trimers are under active investigation as vaccine candidates in relevant pre-clinical models. Like SOSIPs, the cleavage-independent native flexibly linked (NFL) trimers are faithful mimics of the Env spike. Here, we analyzed multiple new designs to explore alternative modifications, informing tertiary interactions, while maintaining NFL trimer homogeneity and integrity. Accordingly, we performed a proline (P) substitution screen in the gp41 heptad repeat 1 region, identifying other trimer-enhancing Ps, including L555P...