Protein expression and purification最新文献

Several pharmaceuticals produced by transgenic chickens, Kanuma and Epovet, are already in commercial use, which indicates the potential cost competitiveness of this approach as an "animal bioreactor." Since the establishment of reliable cultivation and genetic modification methods for chicken primordial germ cells, the robustness of this system has increased significantly. Moreover, even proteins that might otherwise be deleterious if expressed systemically may be produced when their expression is restricted to the oviduct through the use of tissue-specific promoters. In this review, we discuss several critical issues that must be addressed in order to advance chicken bioreactor systems to the next stage. First, glycosylation profiles should be optimized according to the intended application. Although chickens generally exhibit mammalian-like glycosylation patterns, egg white proteins are known to carry relatively short glycans that lack terminal galactose and sialic acid residues. Therefore, technologies enabling the introduction of appropriate glycan structures need to be developed. Second, the screening process for establishing transgenic lines should be minimized to reduce overall production costs. Recent advances in site-specific genome modification technology are promising in this regard, provided that patent disputes can be resolved. The integration of these emerging strategies into transgenic chicken bioreactor platforms could further enhance their competitiveness as systems for recombinant protein production.

The Escherichia coli (E. coli) expression system is widely used for recombinant protein production; however, protein insolubility remains a major challenge. Although fusion tags can enhance solubility, they often complicate purification due to the need for tag removal. Here, we demonstrate that tandem fusion of Trigger Factor (TF) and SUMO markedly improves the solubility and expression of feline junctional adhesion molecule A (fJAMA) and Leptotrichia wadei CRISPR-associated protein 13a (LwCas13a). Furthermore, we developed a novel self-cleaving tandem tag by fusing SUMO protease Ulp1 to SUMO, enabling autonomous tag removal during expression and purification. This integrated design allows one-step purification of target proteins, thereby streamlining the workflow and reducing both time and cost.

Increasing evidence highlights the gut microbiota as a source of diagnostic biomarkers and a key modulator of anti-tumor immunity and cancer immunotherapy responses. Pilin-like proteins from the uncultured TANB77 clade have shown potential to enhance immune checkpoint blockade therapy, yet protocols for their recombinant production have not been established. Here, we present the first optimized method for the expression and purification of two TANB77-derived pilin-like proteins, 935 and 938, using the Escherichia coli (E. coli) BL21(DE3)/pET system. The workflow integrates optimized expression conditions, two-step purification by Ni-NTA affinity and size-exclusion chromatography (SEC), and endotoxin removal for downstream applications. We reveal significant differences in the expression profiles and biophysical properties of the two proteins: protein 935 was expressed at high levels (∼2.5 mg/100 mL) and formed stable oligomers. In contrast, protein 938 exhibited >30-fold lower yields (∼0.08 mg/100 mL) and significant fragmentation, indicating intrinsic instability within this expression system. This study provides the first systematic framework for producing TANB77 pilin-like proteins, facilitating future exploration of their biochemical properties and therapeutic potential in cancer immunotherapy.

Solid-state NMR (ssNMR) provides unique advantages for resolving the structure and interactions of membrane proteins within native-mimicking lipid membranes. However, the challenge is preparing milligrams of stable, homogeneous proteoliposomes samples to achieve high-resolution data. Mechanosensitive channels of small conductance (MscS) are important members of the bacterial mechanosensitive channel family, functioning as emergency release valves to protect cells from osmotic lysis. In this study, AlphaFold was employed to predict the structures of MscS proteins from four bacterial species, shedding light on further experimental work. To obtain homogeneous MscS protein in high yield, several key parameters were systematically optimized, including the expression host strain, induction temperature, and optical density at induction. A "dual-media" approach was also applied. Pa-MscS demonstrated stable high-level expression, yielding about 30 mg/L using "dual-media" method. Gelfiltration chromatography and chemical crosslinking confirmed that Pa-MscS assembles homogeneously into a homoheptamer. Pa-MscS was successfully reconstituted into proteoliposomes, by optimizing the lipid composition of the liposomes, detergent removal conditions, and the phospholipid-to-protein ratio. Finally, 2D ¹⁵N-¹³Cα (NCA) spectroscopy of the Pa-MscS proteoliposomes revealed well-resolved spectra by ssNMR, enabling high-resolution structural studies. This integrative approach provides a robust framework for obtaining high-yield and homogeneous MscS sample for ssNMR-based functional and mechanistic studies, laying the groundwork for future investigations into MscS and therapeutic development.

Depth filtration (DF) is widely utilized as a clarification step in pharmaceutical manufacturing, concurrently removing process-related impurities such as host cell proteins (HCPs) and DNA. This study demonstrates DF's capability in eliminating polysorbates (PS)-degrading impurities (esterases and metals) that may compromise the long-term stability of biopharmaceuticals. By using mass spectrometry-based methods, screening experiments with various commercial depth filters revealed significant brand-dependent variations in impurity removal efficiency. Our findings provide valuable reference data for projects requiring HCP removal, particularly those targeting challenging esterase or metals contaminants.

The human basic fibroblast growth factor 2 (hbFGF2) is a highly conserved protein that is crucial for cell proliferation, differentiation, and migration. Commercial hbFGF2 is expensive and contributes significantly to the cost of cultivated meat (CM) media, affecting the scalability of the production process. This study aims to reduce media costs by achieving a high yield of purified active hbFGF2 from Escherichia coli T7 Express. A codon-optimized hbFGF2 variant, hbFGF2-G3, was cloned into the pET-28a (+) plasmid and expressed in E. coli T7 Express. Cultivation parameters, including temperature and induction time, were optimized in both shake flasks and batch bioreactors. Optimized conditions yielded a titer of 1.3 g/L in shake flasks at 20 °C and an impressive 2.95 g/L in a batch bioreactor, quantified by densitometry using an in-house purified hbFGF2-G3 as the standard. The protein was subsequently purified using His-trap affinity and size exclusion chromatography. The functional activity of the purified hbFGF2-G3 was validated through FGF/FGFR activation assays in HepG2 cell lines and cell proliferation studies in Ph9F-1x fish cell lines. Our findings demonstrate a cost-effective method for producing active hbFGF2-G3, which has significant implications for the economic viability of CM production.