AI Article Synopsis
Article Abstract
The human hemofiltrate peptide HF6478, a putative serine proteinase inhibitor, which is part of the precursor protein LEKTI, was cloned, overexpressed, and purified. HF6478 contains two disulfide bridges with 1-4, 2-3 connectivity, sharing partial homology to Kazal-type domains and other serine proteinase inhibitors. It was expressed as thioredoxin (Trx) fusion protein, and disulfide formation occurred in the oxidative cytoplasm of Escherichia coli Origami (DE3) strain which carries a trxB(-)/gor522(-) double mutation. The soluble fusion protein was purified using metal-chelating affinity chromatography. Cleavage of the Trx fusion protein with factor Xa and subsequent purification yielded the final product in amounts sufficient for structural studies. Characterization of recombinant HF6478 was done by amino acid sequencing, mass spectrometry, capillary zone electrophoresis, and CD spectroscopy. Taking the blood filtrate peptide HF6478 as example, we present a strategy which should facilitate the expression of different extracellular proteins in the E. coli cytoplasm.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1006/prep.2001.1415 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Impact of Recent Translational and Therapeutic Developments on Clinical Course of BCR::ABL1-Positive and -Negative Myeloproliferative Neoplasms.
Hematol Oncol
January 2025
University of California Irvine, Irvine, California, USA.
Despite the study of BCR::ABL1-positive and -negative myeloproliferative neoplasms (MPNs) providing seminal insights into cancer biology, tumor evolution and precision oncology over the past half century, significant challenges remain. MPNs are clonal hematopoietic stem cell-derived neoplasms with heterogenous clinical phenotypes and a clonal architecture which impacts the often-complex underlying genetics and microenvironment. The major driving molecular abnormalities have been well characterized, but debate on their role as disease-initiating molecular lesions continues.
View Article and Find Full Text PDFThe role of MEGF10 in myoblast fusion and hypertrophic response to overload of skeletal muscle.
J Muscle Res Cell Motil
January 2025
School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
Biallelic mutations in multiple EGF domain protein 10 (MEGF10) gene cause EMARDD (early myopathy, areflexia, respiratory distress and dysphagia) in humans, a severe recessive myopathy, associated with reduced numbers of PAX7 positive satellite cells. To better understand the role of MEGF10 in satellite cells, we overexpressed human MEGF10 in mouse H-2k-tsA58 myoblasts and found that it inhibited fusion. Addition of purified extracellular domains of human MEGF10, with (ECD) or without (EGF) the N-terminal EMI domain to H-2k-tsA58 myoblasts, showed that the ECD was more effective at reducing myoblast adhesion and fusion by day 7 of differentiation, yet promoted adhesion of myoblasts to non-adhesive surfaces, highlighting the importance of the EMI domain in these behaviours.
View Article and Find Full Text PDFRecombinant Expression of a New Antimicrobial Peptide Composed of hBD-3 and hBD-4 in Escherichia coli and Investigation of Its Activity Against Multidrug-Resistant Bacteria.
Probiotics Antimicrob Proteins
January 2025
State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, No. 20 Dongda Street, Beijing, 100071, Fengtai District, China.
Human β-defensin (HBD) has been recognized as a promising antimicrobial agent due to its broad-spectrum antimicrobial activity against various pathogens. In our previous work, we engineered a chimeric human β-defensin, designated H4, by fusing human β-defensin 3 and human β-defensin 4, resulting in enhanced antimicrobial activity and salt stability. However, the high cost of chemical synthesis due to the relatively large number of amino acids in H4 has limited its applications.
View Article and Find Full Text PDFMolecular architecture of human LYCHOS involved in lysosomal cholesterol signaling.
Nat Struct Mol Biol
January 2025
Key Laboratory of RNA Innovation, Science, and Engineering; Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.
Lysosomal membrane protein LYCHOS (lysosomal cholesterol signaling) translates cholesterol abundance to mammalian target of rapamycin activation. Here we report the 2.11-Å structure of human LYCHOS, revealing a unique fusion architecture comprising a G-protein-coupled receptor (GPCR)-like domain and a transporter domain that mediates homodimer assembly.
View Article and Find Full Text PDFEfficient production of recombinant hybrid mussel proteins with improved adhesion.
Int J Biol Macromol
January 2025
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, China; College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China. Electronic address:
Mussel foot proteins (mfps) play important roles in surface interaction and underwater adhesion. However, limited production and the lack of adhesion of recombinant mfps have restricted their widespread use. Here, we present a general strategy for enhancing both the expression and function of mfps by connecting multiple protein fragments.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!