To establish a standardized technical framework for large‐scale production of recombinant collagen, we have employed Pichia pastoris GS115 as a host expression system and optimized the fermentation and purification processes for a transdermal peptide–type III collagen fusion protein (transdermal peptide–hCOL3A) at the 15‐L bioreactor scale. A recombinant vector encoding the fusion gene was constructed through codon optimization and transformed into the GS115 strain. The seed culture was prepared via shake‐flask cultivation in BMGY medium. High‐cell‐density fed‐batch fermentation was conducted in a 15‐L fermenter with an inoculum size ranging from 3% to 10%, achieving a final recombinant protein yield of 1.9 g/L. In the downstream purification, optimization of membrane‐based concentration parameters and the incorporation of strong cation‐exchange chromatography using an SP column enhanced the recovery rate of the target protein to 68%. Gel‐filtration analysis demonstrated that transdermal peptide–hCOL3A exists as a trimer, and a CCK‐8 assay revealed that it promotes proliferation of HaCaT cells in vitro , indicating biological functionality. This work successfully establishes a robust and scalable collagen production process suitable for pilot‐scale manufacturing and provides a solid technical foundation for future industrial‐scale translation. © 2025 Wiley Periodicals LLC.
High-throughput screening (HTS) of proteins is used in a wide range of applications across the biology, biotechnology, and medicine disciplines. These include yield optimization, drug or biomarker discovery, and protein engineering, among others. Factors that need to be considered in designing high-throughput protein expression and screening methods (be that for expression, activity, stability, or binding assays), include the required yield, reproducibility, solubility, stability, purity, and activity of the protein. Thus, larger culture volumes and time-consuming manual protein extraction and purification steps are normally required to produce enough protein of appropriate purity. This limits the type of assay and number of protein variants that can be simultaneously tested in an experiment. Here, we describe a HTS protocol that allows the overnight expression, export, and assay of recombinant proteins from Escherichia coli cells in the same microplate well. The protocol uses a recently described Vesicle Nucleating peptide (VNp) technology that promotes high yield vesicular export of functional proteins from E. coli into the culture medium. The resulting protein is of sufficient purity and yield that it can be used directly in plate-based enzymatic assays without additional purification. This simple single-plate protocol allows itself to a wide range of high-throughput research and development screening applications, ranging from streamlining protein production and identification of activity enhancing mutations, to ligand screening for basic research, biotechnological and drug discovery applications. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expression, export, and isolation of vesicular-packaged recombinant protein Support Protocol 1: 96-well plate cold-shock transformation Support Protocol 2: In-plate affinity-tag protein purification Support Protocol 3: Example in-plate enzymatic assay.
Recombinant monoclonal antibodies (mAbs) are widely used across therapeutic areas, and their glycosylation plays a critical role in product quality, manufacturing consistency, and biosimilarity assessment. A middle-down nuclear magnetic resonance (NMR) method has been developed to profile mAb glycan distribution while preserving the covalent bond between glycan and mAb domains, e.g., the fragment crystallizable (Fc) domain. The workflow uses the immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS) to generate Fc fragments that are purified, denatured, and dissolved in urea prior to high-resolution two-dimensional 1H-13C heteronuclear single quantum coherence (2D 1H-13C HSQC) NMR spectrum collection. The resulting anomeric peak distribution reveals major and minor glycan species, including the trimannosyl core, high-mannose variants, and branch-specific galactosylation. Compared with traditional glycan mapping, which requires enzymatic cleavage on glycan and liquid chromatography separation, middle-down NMR provides a non-invasive analysis that preserves glycan integrity and enables comprehensive, semi-quantitative monosaccharide profiling. The method requires 3 to 4 days with ∼4 to 5 hr of hands-on time and can be readily implemented in regulated environments for development and quality control. Basic biochemistry and 2D NMR skills are enough to efficiently apply this protocol in a streamlined workflow. Published 2025. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: mAb-Fc sample preparation Basic Protocol 2: 2D NMR of HSQC peak profile.
Chromatin remodeling is a process of regulating gene expression levels by altering chromatin structure, facilitated by chromatin remodeling factors. ISWI (Imitation SWItch), a type of chromatin remodeling factor, plays a crucial role in epigenetic regulatory processes, such as cell differentiation and development. It primarily achieves remodeling effects by promoting the direct sliding of nucleosomes and their relocation on DNA. To study the function and structural characteristics of ISWI, this basic protocol describes a simple and rapid procedure for the large-scale expression and purification of soluble CtISWI77-1038 and CtATPase77-722 proteins in Escherichia coli (E. coli). The procedure provides high yields (∼20 mg) and is followed by in vitro measurement of ATP hydrolysis activity. © 2025 Wiley Periodicals LLC. Basic Protocol: Expression, purification, and ATP hydrolysis activity measurement of CtISWI and CtATPase.
Neutrophil extracellular traps (NETs) are pivotal in the immune response, trapping and neutralizing pathogens. The process of NET formation, or NETosis, is a critical innate immune response mechanism implicated in diverse physiological and pathological processes, including infections, chronic inflammation, and cancer. Accurate imaging of NETosis is essential for unraveling its mechanisms and therapeutic implications, although limitations of specificity and flexibility remain. In this article, we present modified nanobodies targeting Ly6C/G and histones for imaging of NETosis. Single-domain antibodies (VHHs), also known as nanobodies, targeting Ly6C/G specifically bind to neutrophils, and anti-histone nanobodies selectively target NET-associated histones, enabling NETosis monitoring. These nanobodies are engineered to optimize fluorescent probe labeling and can enable high-resolution visualization of NETosis through whole-body fluorescence tomography and/or intravital microscopy. Our approach offers an alternative for expanding the existing NETosis imaging toolkit. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Cloning, cytoplasmic expression, and purification of proteins Basic Protocol 2: Fluorophore binding assay Support Protocol 1: Analysis by gel staining and immunoblotting Support Protocol 2: In-gel tryptic digestion and sample preparation for LC-MS/MS analysis Support Protocol 3: SpyCatcher003/SpyTag003 binding assay.
