In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors

Poly (A)⁺ RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I–IV. Polypeptides synthesized in vitro were labeled with (³⁵S)-cysteine and (³H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I–IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a 4.1k Da protein originally discovered in EAEC but known to be scattered in other diarrheagenic E. coli as well, possibly causing diarrhea in humans and animals. We report for the first time a method to express and purify EAST1 using the Glutathione S-transferase (GST) fusion system. The gst and astA genes were fused together on a pGEX-2T plasmid vector to produce a GST-EAST1 fusion protein. Using Glutathione Sepharose affinity chromatography and C(8) reverse phase high pressure liquid chromatography, EAST1 was purified to homogeneity with a yield of 0.29 mg/L of culture. The protein purified by this method was confirmed to be EAST1 by NH(2)-terminal sequencing and mass spectrometry. The molecular weight of EAST1 is 4104.0 Da, confirming a 38 amino acid peptide as predicted by the astA gene sequence. Mass spectrometry analysis of EAST1 and of two generated peptides established the presence and suggested the position of two disulfide bridges of EAST1 in the conformations C1-C2 and C3-C4. Polyclonal antibodies were raised against EAST1 in New Zealand white rabbits to a titer of 1:8000 using affinity-purified GST-EAST1 fusion protein and to a titer of 1:100 using HPLC-purified EAST1. The biological activity of various EAST1 preparations was tested using the suckling mouse assay with CD-1 and CFW mice strains, but failed to produce fluid accumulation in the intestine.     

Sequence of a new Bowman-Birk inhibitor fromTorresea acreana seeds and comparison withTorresea cearensis trypsin inhibitor (TcTI2)

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds ofTorresea acreana, one of the two known species ofTorresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears asM _r 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2,Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus

Growth and survival responses were determined for black field crickets Teleogryllus commodus (Walker) (Orthoptera: Gryllidae) on artificial diets containing a range of levels of dietary protein, and protease inhibitors (PI's) at 0.33% (weight volume, w:v). The effect on cricket gut enzyme activities of adding PI's to a high protein diet was measured. All PI's which had in vitro binding activity against either trypsin or elastase (the two major cricket gut endopeptidases) reduced growth, but those which bound to both enzymes had the greatest effect. None of the PI's acted as a source of nutritional protein. Cricket growth rate increased with the addition of casein up to 3% w:v, but not with a similar addition of wheatgerm. The impact of PI's on growth was greatest on a 1.5% casein diet. On a high protein (3% casein) diet, the gut activity of trypsin was increased by potato proteinase inhibitors 1 and 2 while the activity of elastase and leucine amino peptidase were increased by soybean trypsin inhibitor and potato proteinase inhibitor 2. Increasing dietary casein up to 3.3% improved cricket survival. The potential of PI's as plant resistance factors against crickets was confirmed.     

Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis

The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (k_cat/K_m) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0–9.0 and 50 °C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 °C and 60 °C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 °C to 75 °C and caused a prominent 31-fold increase in k_cat/K_m with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency.     

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.     

Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1α (stromal cell-derived factor-1α) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1α fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP–RANTES and MBP–SDF-1α was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1α.     

Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus

A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28 kDa, pH and temperature optima for this protease were 10 and 60 °C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu²⁺ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.     

Co-expression of a prophage system and a plasmid system in Bacillus subtilis

A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis. This dual expression system combines the phi105MU331 prophage system and a plasmid system within a single cell. Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive. Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models. Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system. Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system. Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities. Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production.     

原油污染土壤中生物表面活性剂菌株的筛选和产物鉴定

【目的】从原油污染的土壤中分离出产表面活性剂的枯草芽孢杆菌(Bacillus subtilis)SX-20,并对其产物进行提取及结构分析。【方法】采用氯化十六烷基吡啶和溴百里酚蓝混合溶液(cetylpyridinium chloride-bromothymol blue,CPC-BTB)显色反应结合血琼脂平板简单高效的筛选得到产脂肽的枯草芽孢杆菌。通过酸沉淀、甲醇萃取和旋转蒸发提取发酵所产生的粗产物,该产物对痤疮丙酸杆菌(Propionibacterium acnes)具有良好的抑制作用。运用傅里叶红光变换光谱(Fourier transform infrared spectroscopy,FTIR)、氨基酸分析和液相质谱联用(liquid chromatography-mass spectrometry,LC-MS)对粗产物的成分进行分析。【结果】筛选所得的菌株所产物质是含C15脂肪酸链和7个氨基酸形成的环状的脂肽表面活性剂。【结论】本研究为筛选脂肽类生物表面活性剂提供了一定的理论基础和技术路线,有利于后续获得高产的低成本的生物表面活性剂。     

Efficient secretory expression of an alkaline pectate lyase gene from Bacillus subtilis in E. coli and the purification and characterization of the protein

The gene encoding pectate lyase (PL) from Bacillus subtilis WSHB04-02 was amplified by PCR, fused with a periplasmic secretion signal peptide sequence, pelB, from pET22b(+), cloned and expressed in Escherichia coli cells using a temperature control vector, pHsh. The recombinant E. coil was grown in a 5 l fermentor. PL was secreted in broth at 22 U l⁻¹ after 20 h when temperature was increased from 30°C to 42°C. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. It was optimally active at pH 9.4 and 50°C over 30 min. Analysis of polygalacturonic acid (PGA) degradation products by electrospray ionization (ESI)-mass spectrometry (MS) indicated that PL produced a mixture of unsaturated oligo-galacturonides including unsaturated tri-galacturonic acid and unsaturated bi-galacturonic acid but not unsaturated mono-galacturonic acid.     

产胶原酶菌株的筛选鉴定、发酵优化及胶原酶纯化

【目的】从肉联厂附近土壤中筛选出新型的胶原酶菌种,并通过提高其产酶量后研究其胶原酶的纯化方法,进一步研究该胶原酶对胶原蛋白的降解效率。【方法】经形态特征、生理生化特征以及16S rRNA基因进化树分析鉴定该菌株,并对该菌株产酶发酵条件进行优化,最终利用强阴离子交换树脂对该菌株所产的胶原酶进行分离纯化。【结果】该菌株鉴定为蜡样芽孢杆菌(Bacillus cereus)。对该菌株的产酶发酵条件进行优化,结果表明最适碳源为2.0%葡萄糖、最适氮源为1.5%胰蛋白胨、最适无机盐为0.005%Ca~(2+);初始发酵液pH 7.5、发酵温度37℃,在最适条件下,该菌株发酵液的酶活为(65.81±2.06)U/m L,相比优化前(26.7±1.9)U/m L,酶活提高约1.5倍。对该菌株所产的胶原酶进行分离纯化,得到1个分子量约为100 k Da、纯度大于90%的胶原酶,其比酶活力约为(7615.0±78.7)U/mg。【结论】该研究所发现的胶原酶酶活较高,能够使胶原蛋白在短时间内被有效地降解为具有生物活性的胶原短肽,在食品、医疗、保健品、化妆品等领域具有较广泛的应用前景。     

Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein

Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to α-1,4-glucans.     

Analysis of the open and closed conformations of the GTP-binding protein YsxC from Bacillus subtilis

Genetic analysis has suggested that the product of the Bacillus subtilis ysxC gene is essential for survival of the microorganism and hence may represent a target for the development of a novel anti-infective agent. B.subtilis YsxC is a member of the translation factor related class of GTPases and its crystal structure has been determined in an apo form and in complex with GDP and GMPPNP/Mg2+. Analysis of these structures has allowed us to examine the conformational changes that occur during the process of nucleotide binding and GTP hydrolysis. These structural changes particularly affect parts of the switch I and switch II region of YsxC, which become ordered and disordered, respectively in the "closed" or "on" GTP-bound state and disordered and ordered, respectively, in the "open" or "off" GDP-bound conformation. Finally, the binding of the magnesium cation results in subtle shifts of residues in the G3 region, at the start of switch II, which serve to optimize the interaction with a key aspartic acid residue. The structural flexibility observed in YsxC is likely to contribute to the role of the protein, possibly allowing transduction of an essential intracellular signal, which may be mediated via interactions with a conserved patch of surface-exposed, basic residues that lies adjacent to the GTP-binding site.     

Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity

The aim of this study was to purify and characterise a novel protease inhibitor (PISC-2002) isolated from culture supernatants of Streptomyces chromofuscus. PISC-2002 was purified by anion-exchange chromatography, and RP-HPLC analysis. PISC-2002 had a molecular mass of 11.2 kDa and a high content of hydrophobic amino acids and proline. N-terminal sequence gave two sequences differing by one residue. The main sequence is ASLPAVSALVLTV and the shorter sequence is SLPAVSALVLTV. This shows its homology to Streptomyces subtilisin inhibitor family. Besides its large spectrum of powerful inhibitory activities against various serine proteases, PISC-2002 displayed significant antiviral effect against influenza virus A/Rostock/34 (H7N7).     

Heterologous expression of lipase in Escherichia coli is limited by folding and disulfide bond formation

Functional expression of lipase B from Pseudozyma antarctica (PalB) in the cytoplasm of Escherichia coli BL21(DE3) and its mutant derivative Origami B(DE3) was explored. Coexpression of DsbA was found to be effective in enhancing PalB expression. The improvement was particularly pronounced with Origami B(DE3) as a host, suggesting that both folding and disulfide bond formation may be major factors limiting PalB expression. Fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity, possibly by both enhanced solubility and correct disulfide bond formation. Our results suggest that PalB activity is closely associated with correct disulfide bond formation, and increased solubilization by PalB fusions does not necessarily result in activity enhancement.