Cloning,Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus

An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68–80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.     

Primary structure of β-momorcharin,a ribosome-inactivating protein from the seeds of Momordica Charantia Linn (Cucurbitaceae)

The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S.aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 51 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.     

Brown Kidney Bean Bowman–Birk Trypsin Inhibitor is Heat and pH Stable and Exhibits Anti-proliferative Activity

A trypsin inhibitor with a molecular mass around 17 kDa was purified from the seeds of Phaseolus vulgaris cv. ‘brown kidney bean’. The purification protocol involved, in sequence, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Q-Sepharose and Mono Q, and gel filtration on Superdex 75. The molecular size and N-terminal amino acid sequence of the trypsin inhibitor resembled leguminous Bowman–Birk protease inhibitors (BBIs), signifying that brown kidney bean trypsin inhibitor is a BBI. Brown kidney bean trypsin inhibitor exhibited pronounced thermostability and pH stability. Its trypsin inhibitory activity was retained at all pH values (0–14) and up to 90 °C. The trypsin inhibitor also inhibited the proliferation of human breast cancer MCF7 cells with an IC₅₀ of 71.52 μM. On the other hand, it only slightly inhibited proliferation of hepatoma HepG2 and embryonic liver WRL68 cells at a concentration over 110 μM.     

PAS-cal: a Generic Recombinant Peptide Calibration Standard for Mass Spectrometry

We describe the design, preparation, and mass-spectrometric characterization of a new recombinant peptide calibration standard with uniform biophysical and ionization characteristics for mass spectrometry. “PAS-cal” is an artificial polypeptide concatamer of peptide cassettes with varying lengths, each composed of the three small, chemically stable amino acids Pro, Ala, and Ser, which are interspersed by Arg residues to allow site-specific cleavage with trypsin. PAS-cal is expressed at high yields in Escherichia coli as a Small Ubiquitin-like MOdifier (SUMO) fusion protein, which is easily purified and allows isolation of the PAS-cal moiety after SUMO protease cleavage. Upon subsequent in situ treatment with trypsin, the PAS-cal polypeptide yields a set of four defined homogeneous peptides in the range from 2 to 8 kDa with equal mass spacing. ESI-MS analysis revealed a conveniently interpretable raw spectrum, which after deconvolution resulted in a very simple pattern of four peaks with similar ionization signals. MALDI-MS analysis of a PAS-cal peptide mixture comprising both the intact polypeptide and its tryptic fragments revealed not only the four standard peptides but also the singly and doubly charged states of the intact concatamer as well as di- and trimeric adduct ion species between the peptides, thus augmenting the observable m/z range. The advantageous properties of PAS-cal are most likely a result of the strongly hydrophilic and conformationally disordered PEG-like properties of the PAS sequences. Therefore, PAS-cal offers an inexpensive and versatile recombinant peptide calibration standard for mass spectrometry in protein/peptide bioanalytics and proteomics research, the composition of which may be further adapted to fit individual needs.

A Novel Multifunctional α-Amylase from the Thermophilic Fungus Malbranchea cinnamomea: Biochemical Characterization and Three-Dimensional Structure

A novel α-amylase (McAmyA) from the thermophilic fungus, Malbranchea cinnamomea was purified, characterized and crystallized in the present study. McAmyA was purified to apparent homogeneity with a molecular mass of 60.3 kDa on SDS-PAGE. The enzyme exhibited maximal activity at pH 6.5 and was stable within pH 5.0–10.0. It was most active at 65 °C and was stable up to 50 °C. McAmyA was capable of hydrolyzing amylose, starch, amylopectin, pullulan, cyclodextrins and maltooligosaccharides. The full-length cDNA of an α-amylase gene (McAmyA) from the strain was cloned. McAmyA consisted of a 1,476-bp open reading frame encoding 492 amino acids. It displayed the highest amino acid sequence homology (less than 60 %) with the reported α-amylases. The crystal structure of McAmyA was solved at a resolution of 2.25 Å (PDB code 3VM7). The overall structure of McAmyA reveals three domains with ten α helices and 14 β strands, and the putative catalytic residues are positioned at domain A with somewhat different secondary structural circumstances compared with typical α-amylases.     

Purification and Characterization of Tyrosine Phenol Lyase from Citrobacter freundii

The purification and characterization of intracellular tyrosine phenol lyase from Citrobacter freundii has been carried out. The enzyme was purified 35-fold to homogeneity by ammonium sulphate precipitation and hydrophobic interaction chromatography. Its subunit molecular weight was found to be 52 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified tyrosine phenol lyase showed maximum activity in borate buffer (0.05 M at pH 8.5) at 45 °C after 20 min of incubation. The K _m and V _max values of purified enzyme were found to be 0.446 mm and 0.342 mM/min/mg. This enzyme exhibits t _1/2 of 10, 52 and 130 min at 55, 45 and 35 °C, respectively. The N-terminal amino acid sequence was determined as MET-ASN-TYR-PRO-ALA-GLU-PRO-PHE-ARG-ILE-TRP-TRP-VAL-GLY.     

Molecular Cloning and Heterologous Expression in Pichia pastoris of X-Prolyl-dipeptidyl Aminopeptidase from Basidiomycete Ustilago maydis

Dipeptidyl aminopeptidases are enzymes involved in the posttranslational control of bioactive peptides. Here we identified the gene dapUm in Ustilago maydis by homology with other fungal dipeptidyl aminopeptidases. Analysis of the dapUm-deduced amino acid sequence indicated that it encodes for membrane-type serine protease with a characteristic prolyl oligopeptidase catalytic motif triad: Ser, Asp, His. In order to overexpress the DapUm, the gene encoding for it was cloned and transformed into Pichia. Using this system, we observed a ～125-kDa recombinant protein with an optimal enzymatic activity at pH 6.0 and at 40 °C for the Ala-Pro-p-nitroanilide substrate and an experimental pH of 6.9. U. maydis DapUm was specifically inhibited by phenylmethylsulfonyl fluoride and Pefabloc, confirming the presence of a serine residue in the active site. To our knowledge, this study is the first report on the cloning and expression of a DPP IV dipeptidyl aminopeptidase from a basidiomycete organism. Moreover, the use of recombinant DapUm will allow us to further study and characterize this enzyme, in addition to testing chemical compounds for pharmaceutical purposes.     

Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

Identification and quantification of ricin in biomedical samples by magnetic immunocapture enrichment and liquid chromatography electrospray ionization tandem mass spectrometry

Ricin is a toxic protein derived from castor beans and composed of a cytotoxic A chain and a galactose-binding B chain linked by a disulfide bond, which can inhibit protein synthesis and cause cell death. Owing to its high toxicity, ease of preparation, and lack of medical countermeasures, ricin has been listed as both chemical and biological warfare agents. For homeland security or public safety, the unambiguous, sensitive, and rapid methods for identification and quantification of ricin in complicated matrices are of urgent need. Mass spectrometric analysis, which provides specific and sensitive characterization of protein, can be applied to confirm and quantify ricin. Here, we report a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method in which ricin was extracted and enriched from serum by immunocapture using anti-ricin monoclonal antibody 3D74 linked to magnetic beads, then digested by trypsin, and analyzed by LC-ESI-MS/MS. Among 19 distinct peptides observed in LC-quadrupole/time of flight-MS (LC-QTOF-MS), two specific and sensitive peptides, T_7A (⁴⁹VGLPINQR⁵⁶) and T_14B (¹⁸⁸DNCLTSDSNIR¹⁹⁸), were chosen, and a highly sensitive determination of ricin was established in LC-triple quadrupole-MS (LC-QqQ-MS) operating in multiple reaction monitoring mode. These specific peptides can definitely distinguish ricin from the homologous protein Ricinus communis agglutinin (RCA120), even though the amino acid sequence homology of the A-chain of ricin and RCA120 is up to ca. 93 % and that of B-chain is ca. 85 %. Furthermore, peptide T_7A was preferred in the quantification of ricin because its sensitivity was at least one order of magnitude higher than that of the peptide T_14B. Combined with immunocapture enrichment, this method provided a limit of detection of ca. 2.5 ng/mL and the limit of quantification was ca. 5 ng/mL of ricin in serum, respectively. Both precision and accuracy of this method were determined and the RSD was less than 15 %. This established method was then applied to measure ricin in serum samples collected from rats exposed to ricin at the dosage of 50 μg/kg in an intravenous injection manner. The results showed that ca. 10 ng/mL of the residual ricin in poisoned rats serum could be detected even at 12 h after exposure.     

ACE-Inhibitory and Antioxidant Activities of Peptide Fragments Obtained from Tomato Processing By-Products Fermented Using <Emphasis Type="Italic">Bacillus subtilis</Emphasis>: Effect of Amino Acid Composition and Peptides Molecular Mass Distribution

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC₅₀ values of hydrolysates for ACE-inhibitory and 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2 mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400 Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500–800 Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500–800 Da and 1200–1500 Da.     

Trypsin Inhibitor from Edible Mushroom Pleurotus floridanus Active against Proteases of Microbial Origin

Protease inhibitors can be versatile tools mainly in the fields of medicine, agriculture and food preservative applications. Fungi have been recognized as sources of protease inhibitors, although there are only few such reports on mushrooms. This work reports the purification and characterization of a trypsin inhibitor from the fruiting body of edible mushroom Pleurotus floridanus (PfTI) and its effect on the activity of microbial proteases. The protease inhibitor was purified up to 35-fold by DEAE-Sepharose ion exchange column, trypsin-Sepharose column and Sephadex G100 column. The isoelectric point of the inhibitor was 4.4, and its molecular mass was calculated as 37 kDa by SDS-PAGE and 38.3 kDa by MALDI-TOF. Inhibitory activity confirmation was by dot-blot analysis and zymographic activity staining. The specificity of the inhibitor toward trypsin was with Ki of 1.043?×?10^?10 M. The inhibitor was thermostable up to 90 °C with maximal stability at 30 °C, active over a pH range of 4–10 against proteases from Aspergillus oryzae, Bacillus licheniformis, Bacillus sp. and Bacillus amyloliquefaciens. Results indicate the possibility of utilization of protease inhibitor from P. floridanus against serine proteases.     

Purification and Characterization of Bowman‐Birk Protease Inhibitor from Rice Coleoptiles

A 15 kDa rice Bowman‐Birk inhibitor from fast elongating coleoptiles has been purified and identified using partial N‐terminal sequence, LC‐MS, and MALDI‐TOF MS as a 133 amino acid polypeptide (BBIrc 1). The kinetic study shows this protease inhibitor displays competitive inhibition toward trypsin with Ki of 4.0 × 10^?7 M and non‐competitive inhibition toward α‐chymotrypsin with Ki of 9.3 × 10^?6 M. The Western blotting results of the anti‐sera raised against this 15 kDa protein showed that this anti‐serum recognized two BBI proteins with molecular size around 15 kDa (BBIrc 1) and 25 kDa (BBIrc2) and the quantity of the expression of 15 kDa was nearly constant under both aerobic and hypoxia conditions; however, the 25 kDa expression was greatly up‐regulated when the fast elongating coleoptiles were transferred from hypoxia conditions to the aerobic conditions. The results indicate that the expression pattern of BBIs proteins correlated to the developmental stage in terms of morphological changes. The partial N‐terminal sequence of the first 9 amino acids of 25 kDa was AEAPPRPPK, which is the same as the amino acid sequence of 37th to 45th of RBBI3‐1 and LC‐MS study shows that several mass fragments fit to RBBI3‐1. The 25 kDa protein also shows specific binding to bovine trypsin. This expression pattern demonstrates for the first time that environmental factor, oxygen, can select and enhance specific BBI gene expression. The results of this study suggest BBI proteins might play multiple biological functions inside rice coleoptiles.     

Thermostable and Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars

Xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans was cloned in pET28a (+) vector and successfully expressed in Escherichia coli BL21 (DE3). The deduced amino acid sequence analysis revealed homology with that of glycosyl hydrolase (GH) 10 family with a high molecular mass (50 kDa). The purified recombinant xylanase is optimally active at pH 9.0 and 70 °C with T _1/2 of 10 min at 80 °C, and retains greater than 85 % activity after exposure to 70 °C for 180 min. The enzyme liberates xylose as well as xylooligosaccharides from birchwood xylan and agro-residues, and therefore, this is an endoxylanase. The xylan hydrolytic products (xylooligosaccharides, xylose, and xylobiose) find application as prebiotics and in the production of bioethanol. The xylanase being thermostable and alkalistable, it has released chromophores and phenolics from the residual lignin of pulps, suggesting its utility in mitigating chlorine requirement in pulp bleaching.     

Molecular Cloning, Expression, and Enzymatic Analysis of Cathepsin X from Starfish (Asterina pectinifera)

Cathepsin X, also known as cathepsin Z, is referred to as a “lysosomal proteolytic enzyme” and a member of the peptidase C1 family, which is involved in various biological processes such as immune response, cell adhesion, and proliferation. In the present study, the cDNA of starfish (Asterina pectinifera), which is known to cause serious damage to commercial shellfish mariculture, cathepsin X (ApCtX) was isolated through the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) methods for the application to find a way to reduce/control starfish densities. The full-length of ApCtX gene was determined to consist of the 2,240 bp nucleotide sequence, which encoded for a preproprotein of 296 amino acids with a molecular mass of about 32.7 kDa. The tissue type expression of ApCtX was determined in various tissues of A. pectinifera and was shown most abundantly in the liver. The cDNA encoding pro-mature enzyme of ApCtX was expressed in Escherichia coli BL21 (DE3) using the pGEX-4T-1 expression vector. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC. The optimal pH for the protease activity was 6.5. The enzymatic activity of proApCtX was reduced by antipain, NEM, EDTA, EGTA, and 1,10-phenanthroline, and the proApCtX enzyme was significantly inhibited by CuSO₄, HgCl₂, CoCl₂, and SDS whereas Triton X-100 and Brij 35 might have potentially acted as an activator. Here, we demonstrated for the first time that the structural features and enzymatic characteristics of Echinoderms cathepsin X are similar to those of the other mammalian and piscine cathepsin X except its pH optimum, and the results of tissue-specific expression might explain their importance in food digestion by hepatic cecain starfish.     

Cloning and Expression of a Novel Xylanase Gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris

A full-length complementary DNA (cDNA) of Auxyn11D, a gene that encodes a novel endo-??-1,4-d-xylanase of Aspergillus usamii E001 (abbreviated to AuXyn11D), was obtained using 3??- and 5??-rapid amplification of cDNA ends (RACE) methods. The cDNA sequence is 855?bp in length, containing 5??- and 3??-untranslated regions and a 696-bp open reading frame (ORF) that encodes a 32-aa signal peptide and a 199-aa mature peptide (namely AuXyn11D). Multiple homology alignment of amino acid sequences verified that AuXyn11D belongs to glycoside hydrolase family 11. Moreover, a mature peptide-encoding cDNA fragment of Auxyn11D was cloned and expressed in Pichia pastoris GS115. One P. pastoris transformant expressing the highest recombinant AuXyn11D (reAuXyn11D) activity of 15.0?U/mL, labeled as P. pastoris GSAuXyn4-16, was chosen by shake flask test. SDS?CPAGE assay demonstrated that the reAuXyn11D, a glycosylated protein with an apparent molecular mass of 32.0?kDa, was secreted into the medium. The purified reAuXyn11D displayed the highest activity at pH 4.5 and 55?°C. It was stable at a pH range of 3.5?C6.5 and at a temperature of 50?°C or below. Its activity was not significantly affected by most of metal ions tested and EDTA, but increased by Ca²⁺ and inhibited by Mn²⁺. The K _m and V _max of the reAuXyn11D towards birchwood xylan were 6.32?mg/mL and 391.6?U/mg, respectively.     

Purification and Biochemical Characterization of a Novel Alkaline (Phospho)lipase from a Newly Isolated Fusarium solani Strain

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH₂-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca²⁺ and bile salts. The specific activities of FSL were about 1,610 and 2,414 U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH 8.0 and 37 °C. The (phospho)lipase enzyme was stable in the pH range of 5–10 and at temperatures below 45 °C.     

Isolation,Purification and Characterisation of an Organic Solvent-Tolerant Ca2+-Dependent Protease from Bacillus megaterium AU02

A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl₂, and it retained 90 % activity at 50 °C for 3 h. The K _m and V _max values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH₂) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.     

Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90 % activity when incubated for 2 h at 60 °C and remain about 75 % and 44 % activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98 % with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.     

Mass spectrometric analysis of recombinant human α-2 interferon

Mass spectrometry (MS) was used for the characterization of recombinant human α-2 interferon (α-2 IFN) produced in E. coli. After purification by monoclonal antibody affinity chromatography, α-2 IFN showed two major peaks in reversed-phase liquid chromatography (RP- LC). Each component was digested with trypsin and Staphylococcus aureus protease V8, separately or in tandem, and the peptide mixture was analysed by MS without further purification. The first peak corresponded to the 165 amino acid sequence of human α-2 IFN and the main component of the second peak was the acetylated Cys₁ α-2 IFN. It was also possible to verify by MS the location of the SS bonds in α-2 IFN and the occurrence of incorrect SS bridges in the products of some renaturation processes. The best renaturation process for obtaining a product without adducts or scrambling of disulphide bonds could be found by using RP-LC and fast atom bombardment MS.     

The amino acid sequence of a double-headed trypsin inhibitor from the seeds of Momordica charantia Linn. Cucurbitaceae

A double-headed trypsin inhibitor (MCI-1) was isolated and purified from the seeds of Momordica charantia Linn. Cucurbitaceae, by using the trypsin-sepharose-4B affinity chromatography and CM-Sephadex-C50 ion exchange chromatography. It is composed of 77 amino acid residues: Asp₈ Thr₁ Ser₄ Glu₈ Pro₂ Gly₆ Ala₄ Cys₁₄ Val₂ Met₄ Ile₈ Leu₁ Phe₁ His₃ Lys₄ Arg₇. The amino acid sequence of MCI-1 was determined by sequencing the cyanogen bromide, tryptic and staphylococcus aureus V8 proteolytic peptides, then aligned by overlapped sequences. The result shows that MCI-1 contains 7 pairs of disulfide bonds, its sequence showed the high homology with those of “Bowman-Birk” inhibitors. About 50% trypsin inhibitory activity still remained after MCI-1 was cleavaged with cyanogen bromide.