Ferritin is considered as an ideal delivery system due to its precise targeting, reversible self-assembly, high biocompatibility, and easy modification. this study aims to express, purify, and identify three fusion ferritin proteins, and explore their tumor targeting. Three fusion ferritin genes were synthesized and cloned into prokaryotic expression vectors, and the recombinant proteins were purified by affinity chromatography with nickel columns. The fusion ferritin proteins were identified by native polyacrylamide gel electrophoresis (native-PAGE), Western blotting, and circular dichroism. Fluorescein 5-isothiocyanate (FITC) was used to react with fusion ferritin, and confocal laser scanning microscopy was employed to evaluate the tumor targeting of fusion ferritin. The reaction system of sulfo-cyanine7 (Cy7-SE) with fusion ferritin was injected into the tail vein of melanoma mice for in vivo tumor imaging to explore the tumor targeting of fusion ferritin. The results showed that soluble fusion ferritin proteins of about 21 kDa were expressed under the induction by isopropylthio-β-d-galactoside (IPTG), and the recombinant proteins with high purity were obtained. Western blotting showed that the recombinant proteins could be recognized by the corresponding antibodies. The target proteins were identified as multimers with α helixes by native-PAGE and circular dichroism. In vitro and in vivo tumor uptake experiments demonstrated that fusion ferritin was taken up by tumor cells and tumor tissue. This study successfully expressed, purified, and identified fusion ferritin, and verified its tumor uptake in vitro and in vivo, which laid a foundation for the application of ferritin in biomedicine.
Animals ; Mice ; Recombinant Fusion Proteins/isolation & purification* ; Ferritins/metabolism* ; Escherichia coli/metabolism* ; Melanoma, Experimental/metabolism* ; Humans

Reactive oxygen species (ROS) are major contributors to radiation therapy-induced side effects in cancer patients. A fusion antioxidant enzyme comprising glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and a transmembrane peptide has been shown to effectively mitigate ROS-induced damage. To enhance its targeting capability, the fusion protein was further modified by incorporating a matrix metalloproteinase-2/9 substrate peptide (X) and the transmembrane peptide R9, yielding the antioxidant enzyme GST-SOD1-X-R9 (GS1XR). This modification reduced its transmembrane ability in tumor cells, thereby selectively protecting normal cells from oxidative stress. However, the use of non-human GST poses potential immunogenicity risks. In this study, we employed seamless cloning technology to construct an expression vector containing the human GST gene to replace the non-human GST gene, and then expressed and purified novel fusion antioxidant enzymes GS1R and GS1XR. The protective effects of newly constructed GS1R and GS1XR against hydrogen peroxide (H₂O₂)-induced oxidative damage in L-02 cells were then evaluated using GS1 as a control. Enzymatic activity assays revealed that the specific activity of GST in GS1XR remained unchanged compared to the unmodified protein, while SOD activity was enhanced. Exposure to 200 μmol/L H₂O₂ transiently activated the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway; however, this activation diminished after 24 h, reducing cell viability to 48.4%. Both GS1R and GS1XR effectively scavenged intracellular ROS, directly counteracting oxidative stress and promoting Nrf2 nuclear translocation, thereby activating antioxidant pathways and restoring cell viability to normal levels. The two enzymes showed comparable efficacy. In contrast, GS1, lacking transmembrane capability, was restricted to scavenging extracellular ROS and provided only limited protection. In conclusion, both novel fusion antioxidant enzymes demonstrated significant potential in safeguarding normal cells from ROS-mediated oxidative damage. The findings provide a foundation for further investigation in related field.
Humans ; Oxidative Stress/drug effects* ; Hydrogen Peroxide ; Antioxidants/metabolism* ; Glutathione Transferase/metabolism* ; Recombinant Fusion Proteins/pharmacology* ; Superoxide Dismutase-1 ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/biosynthesis*

In this study, we constructed a series of recombinant Fc variants of immunoglobulin G4 (IgG4), screened the fragment crystallizable (Fc) variants with significantly prolonged serum half-lives, and analyzed the relationship between mutation site and half-life, aiming to provide a theoretical basis for the development of IgG4 antibodies and Fc fusion protein-based drugs. Nine gene sites were selected for mutation, and different mutation sites were combined. The variant expression plasmids pET24b-Fc were constructed by molecular cloning and point mutation. The plasmids were transformed into Escherichia coli BL21(DE3) for the expression of different recombinant proteins of Fc. Fc2 and Fc3 variants had slightly lower recombinant protein yields, and the expression of other variants was not affected. The toxicity of different Fc variants was determined by cell counting kit-8 (CCK-8) and calcein acetoxymethyl ester/ propidium iodide (calcein AM/PI) in vitro and enzyme-linked immuno sorbent assay (ELISA) in vivo. The results showed that the recombinant Fc variants had good biocompatibility and safety. Finally, the Fc variants were labeled with fluorescent markers, and the effects of different mutations on their serum half-lives were investigated by in vivo experiments. The Fc5 variant with prolonged serum half-life was successfully screened out, which provided a theoretical and practical basis for the optimal design of IgG4 subtype antibody and Fc fusion protein drugs.
Immunoglobulin G/blood* ; Immunoglobulin Fc Fragments/biosynthesis* ; Half-Life ; Animals ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Recombinant Proteins/biosynthesis* ; Mice

The spike (S) protein plays a crucial role in the entry of SARS-CoV-2 into host cells. The S protein contains two subunits, S1 and S2. The receptor-binding domain (RBD) of the S1 subunit binds to the receptor angiotensin-converting enzyme 2 (ACE2) to enter the host cells. Therefore, degrading S1 is one of the feasible strategies to inhibit SARS-CoV-2 infection. The purpose of this study is to develop a degradation tool targeting S1. First, we constructed a HEK 293 cell line stably expressing S1 by using a three-plasmid lentivirus system. The overexpression of the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) in this cell line promoted the ubiquitination of S1 and accelerated its proteasomal degradation. Further research showed the polyubiquitination of S1 catalyzed by MUL1 mainly occurred via the addition of K48-linked chains. Moreover, the specific peptide LCB1, which targets and recognizes S1, was combined with MUL1 to create the chimeric E3 ubiquitin ligase LCB1-MUL1. In comparison to MUL1, this chimeric enzyme demonstrated improved catalytic efficiency, resulting in a reduction of S1's half-life from 12 h to 9 h. In summary, this study elucidated the mechanism by which MUL1 promotes the ubiquitination modification of S1 and facilitates its degradation through the proteasome, and preliminarily validated the effectiveness of targeted degradation of S1 by chimeric enzyme LCB1-MUL1.
Ubiquitin-Protein Ligases/genetics* ; Humans ; HEK293 Cells ; Ubiquitination ; Spike Glycoprotein, Coronavirus/genetics* ; SARS-CoV-2/metabolism* ; Recombinant Fusion Proteins/metabolism* ; Proteasome Endopeptidase Complex/genetics* ; COVID-19/metabolism* ; Angiotensin-Converting Enzyme 2/genetics*

Human epidermal growth factor (hEGF) can be applied in the treatment of surgical trauma (burns, scalds), tissue repair, skin moisturizing, beauty, skincare, etc. However, the low expression and high cost limit the application of hEGF. In order to improve the expression level of hEGF and reduce the production cost, considering the high expression of polyhedrin, this study fused a partial sequence of polyhedrin with hEGF and expressed the fused sequence by using a silkworm baculovirus expression vector system. In view of the small molecular weight of hEGF, we connected hEGF genes in series and optimized the codons to construct multiple fusion expression vectors by fusing different partial sequences of polyhedrin at the N-terminus. The results showed that through the above strategy, the protein expression level of hEGF was significantly increased. The expression vector containing three concatenated hEGF genes with optimized codons and fused with the sequence encoding 25 or 35 residues at the N-terminus of polyhedrin showed the highest expression level.
Humans ; Epidermal Growth Factor/biosynthesis* ; Genetic Vectors/genetics* ; Recombinant Fusion Proteins/biosynthesis* ; Animals ; Bombyx/metabolism* ; Occlusion Body Matrix Proteins/genetics* ; Nucleopolyhedroviruses/genetics* ; Amino Acid Sequence

The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.
Codon/genetics* ; Protein Sorting Signals/genetics* ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Interferon-alpha/metabolism* ; Immunoglobulin Heavy Chains/immunology* ; Cell Line ; Immunoglobulin Variable Region/immunology*

Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration (IC₅₀) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.
Metallothionein/metabolism* ; Elastin/chemistry* ; Recombinant Fusion Proteins/pharmacology* ; Mice ; Animals ; Peptides/metabolism* ; Escherichia coli/metabolism* ; NIH 3T3 Cells

Human fibroblast growth factor 21 (hFGF21) has become a candidate drug for regulating blood glucose and lipid metabolism. The poor stability and short half-life of hFGF21 resulted in low target tissue availability, which hampers its clinical application. In this study, the hFGF21 was fused with a recombinant human serum albumin (HSA), and the resulted fusion protein rHSA-hFGF21 was expressed in Pichia pastoris. After codon optimization, the recombinant gene fragment rHSA-hFGF21 was inserted into two different vectors (pPIC9k and pPICZαA) and transformed into three different strains (X33, GS115 and SMD1168), respectively. We investigated the rHSA-hFGF21 expression levels in three different strains and screened an engineered strain X33-pPIC9K-rHSA-hFGF21 with the highest expression level. To improve the production efficiency of rHSA-hFGF21, we optimized the shake flask fermentation conditions, such as the OD value, methanol concentration and induction time. After purification by hollow fiber membrane separation, Blue affinity chromatography and Q ion exchange chromatography, the purity of the rHSA-hFGF21 protein obtained was 98.18%. Compared to hFGF21, the biostabilities of rHSA-hFGF21, including their resistance to temperature and trypsinization were significantly enhanced, and its plasma half-life was extended by about 27.6 times. Moreover, the fusion protein rHSA-hFGF21 at medium and high concentration showed a better ability to promote glucose uptake after 24 h of stimulation in vitro. In vivo animal studies showed that rHSA-hFGF21 exhibited a better long-term hypoglycemic effect than hFGF21 in type 2 diabetic mice. Our results demonstrated a small-scale production of rHSA-hFGF21, which is important for large-scale production and clinical application in the future.
Animals ; Blood Glucose/metabolism* ; Diabetes Mellitus, Experimental ; Fibroblast Growth Factors ; Humans ; Hypoglycemic Agents/metabolism* ; Methanol/metabolism* ; Mice ; Pichia/metabolism* ; Recombinant Fusion Proteins ; Recombinant Proteins/metabolism* ; Saccharomycetales ; Serum Albumin/metabolism* ; Serum Albumin, Human/metabolism*

Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
Ammonium Sulfate ; Antioxidants ; Cell-Penetrating Peptides/pharmacology* ; Endopeptidases ; Glutathione Transferase/metabolism* ; Humans ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Recombinant Fusion Proteins ; Recombinant Proteins ; Superoxide Dismutase/metabolism* ; Superoxide Dismutase-1

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.
Animals ; CTLA-4 Antigen ; genetics ; Cell Proliferation ; HEK293 Cells ; Humans ; Lung Neoplasms ; metabolism ; Programmed Cell Death 1 Receptor ; genetics ; Protein Binding ; Protein Domains ; genetics ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism