Affinity purification of a cholesterol oxidase expressed in Escherichia coli

A cholesterol oxidase (COD) gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), an affinity protocol was developed for the preparation, and industrial application of this method was of great potential. Riboflavin was chosen as the affinity ligand, and it was coupled with Sepharose 4B through some spacers. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture the target protein. The purified cholesterol oxidase was 99.5% pure analyzed on HPLC Vydac C4 column, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 9.8% of crude extracted proteins; the recovery of typical cholesterol oxidase activity was 90.1%, higher than that of other reported traditional protocols. Reducing SDS-PAGE analysis showed that the enzyme was a single polypeptide with the mass of ～50 kDa. The desorption constant K(d) and the theoretical maximum absorption Q(max) on the affinity medium were 1.0 μg/g medium and 74.5 mg/g medium in absorption analysis. K(m) and V(max) of cholesterol oxidase activity for the purified enzyme were 25.5 μM and 16.4 μmol/(min mg), respectively.     

Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis

Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG‐75, DEAE‐Sephadex A‐50, and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). VLCII as a thrombin‐like enzyme was able to hydrolyze Nα‐CBZ L‐arginine‐p‐nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10‐phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration‐dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway.     

Isolation and characterization of an arginine ester hydrolase from Bothrops jararacussu venom which induces contractions of the isolated rat uterus.

The isolation and partial characterization of a serine protease with arginine ester hydrolase activity from Bothrops jararacussu snake venom are described. The purification procedure consisted of a gel filtration of the crude venom on Sephadex G-75 followed by an ion-exchange chromatography of the active fraction on DEAE-cellulose and a rechromatography on Bio-Rex 70 resin. The esterase fraction (DI-III), M(r) = 25,000 by SDS-PAGE, showed proteolytic activity on fibrinogen and casein. After 2 hr incubation, the A alpha and B beta chains of fibrinogen were intensely hydrolysed, while the gamma chain kept apparently intact, even after 20 hr of incubation. In spite of that, DI-III did not clot fibrinogen. DI-III induced edema in the rat paw. Although unable to release bradykinin, it induced contractions of the isolated rat uterus. DI-III did not catalyse the hydrolysis of bradykinin. Its arginine ester hydrolase activity was completely inhibited by diisopropyl fluorophosphate after 1 hr incubation, but not by phenylmethylsulfonyl fluoride under the same conditions.     

Isolation, purification, and separation of the complex preparation of extracellular proteinases with fibrinolytic and anticoagulant properties from Aspergillus ochraceus 513]

The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus 513 was isolated, purified, and separated by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650 M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodon snake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated.     

Structural and functional characterization of a prothrombin activator from the venom of Bothrops neuwiedi

A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a Km of 38 microM and a Vmax of 120 mumol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl2. These results indicate that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrombin activator from B. neuwiedi are similar to those reported for the venom activator from Echis carinatus.     

A trypsin and chymotrypsin inhibitor from Caesalpinia bonduc seeds: isolation, partial characterization and insecticidal properties.

Evolution of proteinase inhibitor diversity in leguminous plants of tropical rainforests is under immense pressure from the regular upregulation of proteolytic machinery of their pests. The present study illustrates the isolation and bioinsecticidal potency of a serine proteinase inhibitor from the seeds of Caesalpinia bonduc (CbTI), inhabiting Great Nicobar Island, India. Following initial fractionation by ammonium sulfate precipitation, CbTI was purified to homogeneity by ion exchange, gel filtration and trypsin affinity chromatography. SDS-PAGE of gel filtrated CbTI showed a couple of proteins CbTI-1 ( approximately 16kDa) and CbTI-2 (20kDa) under non-reducing conditions, which subsequent to trypsin affinity chromatography yielded only CbTI-2. Both Native PAGE as well as iso-electric focusing showed 2 iso-inhibitors of CbTI-2 (pI values of 5.35 and 4.6). CbTI exhibited tolerance to extremes of temperatures (0-60 degrees C) and pH (1-12). A 1:1 stoichiometric ratio was noted during CbTI-2-trypsin complex formation, which was absent on binding with chymotrypsin. Further, SDS-PAGE analysis also showed that CbTI-1 has affinity only towards chymotrypsin, whereas both trypsin and chymotrypsin formed complexes with CbTI-2. Dixon plot analysis of CbTI-2 yielded inhibition constants (K(i)) of 2.75 x 10(-10)M and 0.95 x 10(-10)M against trypsin and chymotrypsin activity respectively. Preliminary investigations on the toxicological nature of CbTI revealed it to be a promising bioinsecticidal candidate.     

Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.     

Isolation,Purification, and Resolution of the Extracellular Proteinase Complex of Aspergillus ochraceus513 with Fibrinolytic and Anticoagulant Activities

The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus513 was isolated, purified, and resolved by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodonsnake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated.     

Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus)

A novel serine proteinase inhibitor has been purified to homogeneity from the skeletal muscle of white croaker (Argyrosomus argentatus). The purification was carried out by ammonium sulfate fractionation, DEAE-Sephacel, heating treatment followed by column chromatographies on SP-Sepharose, Sephadex G-150 and gel-filtration high performance liquid chromatography. The molecular mass of the inhibitor was 55 kDa as estimated by SDS-PAGE and gel filtration. It specifically inhibited a myofibril-bound serine proteinase (MBSP) isolated from the skeletal muscle of lizard fish (Saurida wanieso). No inhibition, however, was detected toward other serine proteinases such as bovine trypsin, bovine chymotrypsin and a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle. Interestingly, the sequences of tryptic digested peptide fragments of MBSPI revealed high identity to that of porcine phosphoglucose isomerase (PGI) (76%) and other PGIs. Furthermore, purified MBSPI exhibits PGI activity, suggesting the inhibitor is a protein closely related to PGI. When rabbit muscle PGI was investigated, it also specifically suppressed the activity of MBSP. It thus strongly suggests that MBSPI is actually PGI and conversely, PGI is a specific inhibitor toward myofibril-bound serine proteinase(s).     

Purification and characterization of an intracellular heat-stable proteinase (pernilase) from the marine hyperthermophilic archaeon Aeropyrum pernix K1

A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5–10) with an optimum pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90°C. The enzyme was strongly inhibited by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85 min at 100°C and 12 min at 110°C. Received September 24, 1997 / Accepted May 20, 1998     

Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom.

1. An arginine ester hydrolase was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. Molecular weight was determined to be ca 29 kDa, with an isoelectric point of 4.4. 5. The enzyme was stable to heat treatment (95 degrees C, 10 min) and to pH changes over the range 2-11. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), beta-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.     

Purification of a novel myofibril-bound serine proteinase inhibitor (MBSPI) from the skeletal muscle of lizard fish

A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish (Saurida wanieso). Purification was carried out by ammonium sulfate fractionation, followed by column chromatographies on DEAE-Sephacel, SP-Sepharose and Sephadex G-150. MBSPI was purified 7.7-fold starting from the DEAE-Sephacel fraction, with a yield of 0.2%. It is a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle.     

Purification and properties of arginine esterases from Bitis arietans (puff adder) venom

An arginine esterase (FT1) was purified from B. arietans venom by gel-filtration and ion-exchange chromatography. The purified enzyme contains 21.6% of carbohydrate, 240 amino acids including 12 half-cystine residues and has a mol. wt of approximately 43,000. The purified enzyme has a high esterolytic activity towards N-alpha-benzoyl-L-arginine ethyl ester but shows no proteolytic activity against Azocoll and no clotting activity with fibrinogen. The N-terminal sequence of the arginine esterase from B. arietans venom shares a significant degree of sequence homology with the arginine esterase of B. nasicornis, the thrombin-like enzyme of C. adamanteus and the kallikrein-like enzymes of C. atrox venoms. It would appear that the arginine esterase from B. arietans venom exists in various multiple forms of the enzyme.     

Purification and characterization of aspartic proteinase from sunflower seeds

Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained noncovalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.     

Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase

Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.     

Extracellular proteinase fromEnterococcus faecalis subsp.liquefaciens

An extracellular proteinase from Enterococcus faecalis subsp. liquefaciens has been purified 780-fold by a method including gel filtration on Sephadex G-50 and affinity chromatography with gramicidin J as ligand. Approximately 15% of the original enzyme activity was recovered. A purification of 14,800-fold, with 11.4% yield, may be reached using chromatofocusing as final step in the purification procedure. The molar mass of the enzyme has been estimated to be approximately 30 kDa by Sephadex gel filtration and approximately 26 kDa by SDS-PAGE. The isoelectric point has been found to be 4.6. Maximum enzyme activity of the proteinase has been observed at pH 7.5 and 45 degrees C. The enzyme hydrolyzed bovine serum albumin, alpha-lactoalbumin, beta-lactoglobulin, casein and pork myofibrillar and sarcoplasmic proteins. The extracellular proteinase was very stable; the enzyme maintained its activity in cell-free extracts over a very wide range of temperatures (-25 to 37 degrees C) for at least 2 months. At 12 degrees C, it was stable in the pH range of 5.5 to 8.0.     

Identification of a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site

A myofibril-bound serine proteinase (MBSP) from the skeletal muscle of lizard fish (Saurida wanieso) was purified to homogeneity by a heating treatment followed by a series of column chromatographies on DEAE-Sephacel, Sephacryl S-200, Q-Sepharose, Hydroxyapatite and Benzamidine-Sepharose 6B, and characterized enzymatically. On SDS-poly-acrylamide gel electrophoresis (SDS-PAGE), the purified enzyme showed a band with molecular mass of approximately 29 kDa under reducing conditions, while 60 kDa under non-reducing conditions. The optimum temperature of the enzyme was 50 degrees C using t-butyloxycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a substrate. Substrate specificity analysis both using MCA-substrates and peptides showed that MBSP specifically cleaved at the carboxyl side of the arginine residue. Inhibitor susceptibility analysis revealed that MBSP was inhibited effectively by Pefabloc SC, soybean trypsin inhibitor (STI) and aprotinin, indicating the characteristic of a serine proteinase. When myofibril was incubated with the enzyme, it optically degraded myosin heavy chain at 55-60 degrees C, while alpha-actinin and actin were not at all hydrolyzed as detected by immunoblotting. The N-terminal amino acid sequence of MBSP was partially determined as IVGGAEXVPY- and was very homologous to other serine proteases.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

Purification and characterization of oxalate oxidase from wheat seedlings

Oxalate oxidase (OxO, EC 1.2.3.4.) was purified to homogeneity from wheat (Triticum aestivum) seedlings by sequential thermal treatment, ultrafiltration, Sephadex G-100 gel filtration and affinity chromatography with concanavalin A. The enzyme was purified 66.11-fold with a recovery of 21.97%. It showed a subunit molecular mass of 32.6 kDa on SDS-PAGE and a native molecular mass of 170 kDa on Sephadex G-150 filtration, suggesting that it is a pentamer. The wheat OxO had a maximum activity at pH 3.5. Its K _m for oxalate was 0.21 mM. Chemical modification revealed that cysteine, lysine and carboxylate residues were essential for OxO activity, whereas arginine, serine, threonine and tryptophane residues were not essential.