The ubiquitin specific protease 26 (USP26) gene is located at Xq26.2 and present as a single exon on the X chromosome encoding for a protein of 913 amino acids. It belongs to a large family of deubiquitinating enzymes, and is exclusively expressed in the testis. There are conflicting reports on whether mutations in USP26 are associated with male infertility. This article updates the researches on the USP26 gene, its complicated relationship with male spermatogenesis dysfunction, the role of its mutation in male infertility, its geographical or ethnic distribution, and its evolution.
Cysteine Endopeptidases ; genetics ; Humans ; Infertility, Male ; genetics ; Male ; Spermatogenesis ; genetics

The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.
COVID-19 ; Codon/genetics* ; Cysteine Endopeptidases/genetics* ; Escherichia coli/genetics* ; Humans ; Peptide Hydrolases ; SARS-CoV-2 ; Viral Nonstructural Proteins/genetics*

OBJECTIVEThis study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.

METHODSEukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.

RESULTSIt proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.

CONCLUSIONThe eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.

Animals ; Bacterial Proteins ; genetics ; COS Cells ; Cercopithecus aethiops ; Cysteine Endopeptidases ; genetics ; Genetic Vectors ; Plasmids ; Porphyromonas gingivalis ; genetics ; Transfection

OBJECTIVETo study the correlation of nucleotide polymorphism in the ubiquitin-specific protease 26 (Usp26) gene with idiopathic male infertility and its action mechanism in spermatogenesis.

METHODSBased on the WHO criteria (4th ed.), we selected 41 patients with idiopathic infertility from 150 infertile males, and enlisted 50 normal fertile men as controls. We examined the selected patients for mutations using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, and determined how and where the mutations occurred by gene sequencing.

RESULTSLow sperm concentration and poor sperm motility were found in the 41 men with idiopathic infertility. Nine (22.0%) of them exhibited changes in the Usp26 gene (P = 0.01), including compound mutations of 364insACA and 460G > A in 8 (19.5%, P = 0.01) and 1 044T > A substitution in 1 (2.4%, P > 0.05). The above three variations led to changes in the coding amino acids. No other changes were found in the remaining patients and normal fertile controls.

CONCLUSIONThe nucleotide polymorphisms of the Usp26 gene might be closely related with idiopathic male infertility, and exert negative effect on the testis function.

Case-Control Studies ; Cysteine Endopeptidases ; genetics ; Humans ; Infertility, Male ; genetics ; pathology ; Male ; Polymorphism, Single Nucleotide ; Semen Analysis

We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.
Bacterial Proteins ; biosynthesis ; genetics ; Catalytic Domain ; Cysteine Endopeptidases ; biosynthesis ; genetics ; Endopeptidases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Luminescent Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; SUMO-1 Protein ; biosynthesis ; genetics

OBJECTIVE: Our previous study revealed many genes differentially expressed in human laryngeal squamous cell carcinoma tissues (LSCC) and related adjacent normal tissues. We verificated the differentiated expressions of target genes, possibly related to LSCC. And these results can be foundations of further study of these target genes function. METHOD: SENP1, CD109, Laminin alpha 2, Laminin alpha 3 were selected according to the cDNA microarray results. The expression of these genes mRNA and protein were detected by RT-PCR and Western blot in 12 cases of LSCC and related adjacent normal tissues. RESULT: The mRNA expression of SENP1, CD109 and Laminin alpha 3 were significantly higher while Laminin alpha 2 were significantly lower in LSCC tissues than in corresponding adjacent normal tissues by Semiquantitative RT-PCR. Western blot analysis revealed SENP1, CD109 protein expression were significantly higher in LSCC tissues than in corresponding adjacent normal tissues. CONCLUSION SENP1, CD109, Laminin alpha 2 and Laminin alpha 3 may correlated with tumorigenesis and development of LSCC and can provide beneficial clue for study pathogenesis of LSCC in molecular level.
Aged ; Aged, 80 and over ; Antigens, CD ; genetics ; Carcinoma, Squamous Cell ; genetics ; Cysteine Endopeptidases ; Endopeptidases ; genetics ; GPI-Linked Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Laminin ; genetics ; Laryngeal Neoplasms ; genetics ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; Oligonucleotide Array Sequence Analysis

HC-pro gene of Watermelon Mosaic virus was obtained by RT-PCR was 1371bp in length. It was cloned into pPI(9K, then the eucaryotic recombinant expression plasmid pPIC9K-WHC was constructed. After being linearized with restriction endonuclease Sal I , the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The high copy transformants with Mut+ /His+ phenotype were selected by RT-PCR and screening on G418, MD and MM medium. Induced by methanol for 5 days, the culture supernatant was analyzed by SDS-PAGE, the results showed that a specific protein with a molecular weight of about 66 kD was expressed. Western blot analysis proved that the expression protein could specifically bind to HC-Pro polyclonal antibody. Far western blot analysis proved that the expression protein could bind to coat protein, given support to "bridge" hypothesis that HC-Pro help aphid transmission of non-persistent viruses.
Citrullus ; virology ; Cysteine Endopeptidases ; biosynthesis ; genetics ; Mosaic Viruses ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Viral Proteins ; biosynthesis ; genetics

The aim of the study is to obtain an efficient expression of recombinant ubiquitin-like specific protease 1 (Ulp1) by gene engineering. We cloned the Ulp1p, active fragment (403 aa-621 aa) of Ulp1, from Saccharomyces cerevisia, and subcloned into pGEX/Rosetta (DE3) to form an expression plasmid, pGEX-Ulp1p-His6. In order to enhance the solubility of GST-Ulp1p-His6, we purified the fusion protein GST-Ulp1p-His6 by either glutathione S-transferase agarose or Ni-NTA resin chromatography, the purity was up to 98%. We utilized the protein to cleave the SUMO fusions, and the specific activity of GST-Ulp1p-His6 was 1.375 x 10(4) U/mg. This study showed that the recombinant protein GST-Ulp1p-His6 displayed high specificity and activity.
Cloning, Molecular ; Cysteine Endopeptidases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; biosynthesis ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; enzymology ; Solubility

According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD86:6His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.
Cloning, Molecular ; Cystatins ; biosynthesis ; genetics ; Cysteine Endopeptidases ; metabolism ; Cysteine Proteinase Inhibitors ; genetics ; Escherichia coli ; genetics ; metabolism ; Genes, Plant ; genetics ; Mutation ; Oligonucleotides ; chemical synthesis ; genetics ; Oryza ; genetics ; Papain ; antagonists & inhibitors ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism

The recombinant plasmid carrying the gene encoding 3C protease of Enterovirus 71 (EV71) was constructed, the recombinant protein was then expressed and purified, the functional activity was also measured. Firstly, the 3C protease gene was inserted into pET28a vector, the constructed recombinant plasmid was transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The expressed protein was purified by affinity chromatography (Ni-NTA) and the N-terminus His-tag was cleaved by enterokinase from 3C protease. The activity of 3C protease was evaluated with fluorescent peptide substrates. It was verified by restriction analysis and sequencing that recombinant plasmid pET28a-3C was constructed correctly and functionally expressed in E. coli BL21 (DE3) resulting in the production of recombinant 3C protease with a size of 22kD. Both His-tag and non-His-tag (cleaved by enterokinase) 3C protease exhibited similar enzyme activity to 3B-3C fluorescent peptide with Km, Vmax and Kcat values of 22 microM, 434nM. Min(-1) and 0.0669 Min(-1), respectively. The optimial pH and temperature were 7.0 and 30-37 degrees C, respectively. The acquirement of recombinant purified 3C protease with high activity has paved the way of further studies on anti-viral inhibitors, structural protein assembly, vaccine development and detection methods of EV71.
Cloning, Molecular ; Cysteine Endopeptidases ; chemistry ; genetics ; metabolism ; Enterovirus A, Human ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Kinetics ; Viral Proteins ; chemistry ; genetics ; metabolism