Limulus Factor C, a serine protease zymogen from the amoebocytes of the limulus, has high affinity for endotoxin. When Factor C is activated by endotoxin, it hydrolyses artificial tripeptide substrate and measurable products are released, so it can be used as an alternative reagent for endotoxin analysis. Factor C gene of Tachypleus tridentatus was obtained through RT-PCR and the recombinant protein was expressed by Bac-to-Bac/BmNPV baculovirus expression system in silkworm larvae. The activity of Factor C was detected with diluted serum of silkworm larvae, and the sensitivity of endotoxin detected was 0.2 EU/mL when the serum was diluted at 1:500. The silkworm larvae expressed limulus Factor C could be used to develop a new low-cost endotoxin test reagent.
Animals ; Arthropod Proteins ; biosynthesis ; Baculoviridae ; Bombyx ; metabolism ; Enzyme Precursors ; biosynthesis ; Genetic Vectors ; Larva ; metabolism ; Recombinant Proteins ; biosynthesis ; Serine Endopeptidases ; biosynthesis

To express bovine chymosin in yeast, we amplified the prochymosin gene from the plasmid pMD18T-Prochy by PCR, and then cloned the gene into the expression vector pPICZaA, resulting in pPICZaA-Prochy. Pichia pastoris GS115 was used as host cells. Integration of the prochymosin cDNA into the Pichia pastoris genome was confirmed by PCR and sequencing analysis. Chymosin was expressed in Pichia pastoris successfully, and a strong band at about 37 kD was shown by SDS-PAGE. Activity tests showed that the chymosin activity of the culture supernatant was 12.2 SU/mL. This is the first report of successful expression of chymosin in Pichia pastoris. The recombinant Pichia pastoris strain obtained in this study could be further used to produce recombinant chymosin for cheese making.
Animals ; Cattle ; Chymosin ; biosynthesis ; genetics ; Cloning, Molecular ; Enzyme Precursors ; genetics ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Effect of CTAB addition on the accumulation of microbial transglutaminase (MTG) with Streptomyces hygroscopicus was investigated. The results showed that the addition of CTAB enhanced MTG accumulation, and the optimal addition time and concentration of CTAB were 32 h and 1%. The maximum MTG activity in the culture broth was 5.04 u/mL and increased by 21.8% compared with the control. With the addition of CTAB, pro-MTG was activated to become MTG. CTAB could enhance the production of pro-MTG by relieving the product inhibition, and the accumulation of MTG was improved.
Bacterial Proteins ; metabolism ; Cetrimonium Compounds ; pharmacology ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Enzyme Activation ; drug effects ; Enzyme Precursors ; metabolism ; Industrial Microbiology ; methods ; Streptomyces ; drug effects ; enzymology ; metabolism ; Surface-Active Agents ; pharmacology ; Time Factors ; Transglutaminases ; metabolism

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chyl with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.
Animals ; Cattle ; Chymosin ; biosynthesis ; genetics ; Enzyme Precursors ; biosynthesis ; genetics ; Gene Expression Regulation, Fungal ; genetics ; Genetic Vectors ; genetics ; Kluyveromyces ; genetics ; growth & development ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics

Platelet is activated through signal transduction, that mainly includes phospholipase-beta (PLCbeta) pathway, protein tyrosine kinases (PTK) pathway, phosphatidylinositol3-kinase (PI3-K) pathway, mitogen-activated protein kinases (MAPK) pathway, cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway and phospholipase A2 (PLA2) pathway. This article focuses on the relationship between signal transduction and platelet activation.
Calcium ; metabolism ; Enzyme Precursors ; metabolism ; Guanosine Triphosphate ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; Phosphatidylinositol 3-Kinases ; physiology ; Platelet Activation ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Syk Kinase ; von Willebrand Factor ; physiology

Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.
Codon ; Enzyme Precursors ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; Lipase ; biosynthesis ; genetics ; metabolism ; Pichia ; enzymology ; genetics ; Protein Engineering ; methods ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics ; Rhizopus ; enzymology ; genetics ; Substrate Specificity

BACKGROUNDTo clone and express the ss1 recombinant gene containing S gene and preS1 (10-50 AA) gene in P. pastoris expression system.

METHODSThe fusion gene ss1 containing the S (1-222 AA) gene and preS1 (10-50 AA) gene was constructed with PCR method. The fusion ss1 gene was cloned into the expression vector of pPIC3.5k. The linear vector DNA was transformed into the host cell of GS115 with electroporation method. After screening with G418, the product was induced to express with methanol and its antigenicity was analyzed.

RESULTSThe molecular weight of expressed ss1 protein was about 30,000 dalton. The product was reactive to anti-HBs and anti-preS1 mAb.

CONCLUSIONThe fusion gene was efficiently expressed in P. pastoris expression system.The expressed products have the antigenicity of both S and preS1 protein.

Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Pichia ; genetics ; Plasmids ; genetics ; Protein Precursors ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transformation, Genetic

OBJECTIVETo evaluate the effect of intrajejunal nutrition on uptake of amino acid and enzyme-protein synthesis in pancreatic acinar cell and subcellular fractionation and zymogen granules in dogs with acute pancreatitis.

METHODSFifteen dogs were induced acute pancreatitis by retrograde injection of 5% sodium-taurocholate into the pancreatic duct. Radioactive tracing and electron microscope were used to evaluate the change of amino acid uptake, enzyme-protein synthesis in acinar cell, subcellular fractionation, the quantitative analysis of mean zymogen granule number and mean zymogen granule area after injection L-(3)H-phenylalanine 30, 60, 120 1nd 180 min on the 7(th) day.

RESULTSThe radioactivity of L-(3)H phenylalanine uptake by pancreatic acinar cells and incorporations of L-(3)H phenylalanine into newly synthesized enzyme-protein peaked at 60 min. In enteral nutrition (EN) group it was higher that that in parenteral nutrition (PN) group (P < 0.05), and then gradually declined. The radioactivity peaked at 60 min in zymogen granule, lysosomal-mitochondria and microsomal subcellular fractionation. The latter two decreased, bat there was no significant difference (P > 0.05). The change of the mean number and mean area of zymogen granules were not significant different between the EN group and PN group (P > 0.05).

CONCLUSIONEN or PN do not stimulate pancreatic acinar uptake amino acid and enzyme-protein synthesis in acinar cell and subcellular fractionation.

Acute Disease ; Amino Acids ; metabolism ; Animals ; Disease Models, Animal ; Dogs ; Enteral Nutrition ; Enzyme Precursors ; biosynthesis ; Female ; Male ; Pancreas, Exocrine ; metabolism ; Pancreatitis ; pathology ; physiopathology ; therapy ; Parenteral Nutrition ; Random Allocation ; Treatment Outcome

The presence of matrix metalloproteinase-2 (MMP-2) in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP-2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner (ID), middle (MD), and outer dentin (OD) regions and demineralized. MMP-2 was extracted with 0.33 mol x L(-1) EDTA/2 mol xL(-1) guanidine-HCl, pH 7.4, and MMP-2 concentration was estimated with enzyme-linked immunoabsorbant assay (ELISA). Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP-2 per mg dentin protein in the dentin regions were significantly different (P = 0.043): 0.9 ng (ID), 0.4 ng (MD), and 2.2 ng (OD), respectively. The pattern of MMP-2 concentration was OD > ID > MD. Western blotting analysis detected -.66 and -72 kDa immunopositive proteins corresponding to pro- and mature MMP-2, respectively, in the ID and MD, and a -66 kDa protein in the OD. Gelatinolytic activity consistent with MMP-2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD > ID > OD. The concentration of MMP-2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP-2, potentially indicating region specific protein interactions.
Blotting, Western ; Dentin ; enzymology ; Electrophoresis, Polyacrylamide Gel ; Enzyme Precursors ; analysis ; Female ; Humans ; Male ; Matrix Metalloproteinase 2 ; analysis ; metabolism ; Molar, Third ; enzymology ; Tissue Distribution ; Tooth Crown ; enzymology ; Tooth Demineralization ; enzymology

OBJECTIVETo analyze the effects of interaction between vascular endothelial cells and monocytes on the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2), as well as the regulation of pravastatin.

METHODSA co-cultured system of monocytes and endothelial cells was established through addition of THP-1 to human umbilical vein endothelial cells (HUVECs) in various rates. After 24 hours, the changes in activity and expression of MMP-2 and TIMP-2 in the co-culture system were studied by zymography and reverse zymography. The 1:1 co-culture system was selected and one control group (no pravastatin added) and experimental groups (with concentration of pravastatin being 0.1, 0.5 and 1.0 micromol/ml respectively) were studied. All groups were cultured for another 24 hours and analyzed in the same way.

RESULTSCompared to the single cultured HUVECs, the activity of proMMP-2 in the co-cultured system increased by 2.09, 2.46 and 2.07 folds respectively (number = 8, P < 0.01). There was also activated MMP-2 secretion in the co-culture system. The secretion of proMMP-2 and active MMP-2 in the 1:1 co-cultured system was most obvious. After pravastatin treatment, the activity of proMMP-2 and MMP-2, decreased significantly (number = 8, P < 0.01). MMP-2 secretion was completely suppressed after 1.0 micromol/ml pravastatin treatment. Reverse zymography revealed that, compared to the single culture HUVECs or THP-1, the secretion of TIMP-2 decreased in the co-cultured system, regardless of the ratio of mixture. However, pravastatin had no obvious effect on TIMP-2.

CONCLUSIONSInteraction between vascular endothelial cells and monocytes may contribute to the secretion and activation of MMP-2 and suppress secretion of TIMP-2. Pravastatin may inhibit the secretion and activation of MMP-2.

Anticholesteremic Agents ; pharmacology ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Endothelial Cells ; cytology ; metabolism ; Enzyme Precursors ; metabolism ; Gelatinases ; metabolism ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Metalloendopeptidases ; metabolism ; Monocytes ; cytology ; metabolism ; Pravastatin ; pharmacology ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Umbilical Veins ; cytology