Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli

Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.     

Cloning, functional expression, and characterization of cystatin in sesame seed

A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K(i) values of 7.89 x 10(-8) M and 2.77 x 10(-8) M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination.     

Minimizing the central hydrophobic domain in oleosin for the constitution of artificial oil bodies

Oleosin, a unique structural protein anchoring onto the surface of seed oil bodies by its central hydrophobic domain, stabilizes these lipid-storage organelles as discrete entities. Stable artificial oil bodies have been successfully constituted with native or recombinant oleosins. In this study, recombinant sesame oleosin with 12 residues stepwise truncated from its central hydrophobic domain of 72 residues was overexpressed in Escherichia coli, was purified to homogeneity, and was used for the constitution. Artificial oil bodies constituted by truncated oleosins with the central hydrophobic domain longer than 36 residues were as stable as native sesame oil bodies, and those constituted by truncated oleosins lacking more than half of the original central hydrophobic domain inclined to coalesce upon collision or aggregation.     

Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification

A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.     

Extracellular production of a functional soy cystatin by Bacillus subtilis

A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.     

Selective delivery of cargo entities to tumor cells by nanoscale artificial oil bodies

Artificial oil bodies (AOBs) are oil droplets that result from self-assembly of a mixture containing triacylglycerols, phospholipids, and membrane proteins of plant seeds. Owing to their small size, stability, hydrophobic core, biocompatibility, and biodegradability, AOBs were explored to examine their feasibility as a drug delivery carrier. This was approached by fusion sesame oleosin (Ole), the primary membrane protein of seed oil bodies, with a small domain consisting of the arginine-glycine-aspartic acid (RGD) motif. The resulting Ole-RGD fusion protein was overproduced in Escherichia coli and then isolated for reconstitution of AOBs. At the optimal condition, the size of stable AOBs was within the range of 100-400 nm. Furthermore, AOBs entrapped with a hydrophobic fluorescence dye were incubated with human tumor cells. As visualized by fluorescent microscopy and confocal microscopy, the RGD-tagged AOBs were able to selectively target cells expressing the αvβ3 integrin. Moreover, these AOBs were effectively internalized and the fluorescence dye that they carried was subsequently released inside the cells. The percentage of cells internalized by AOBs could reach 80% as analyzed by flow cytometry. Taken together, it illustrates a great promise of this proposed approach for targeted delivery of cargo entities to tumor cells.     

Constitution of stable artificial oil bodies with triacylglycerol, phospholipid, and caleosin

Seed oil bodies are lipid storage organelles of 0.5-2 microm in diameter and comprise a triacylglycerol matrix shielded by a monolayer of phospholipids and proteins. These proteins include abundant structural proteins, oleosins, and at least two minor proteins termed caleosin and steroleosin. This study examined if artificial oil bodies (AOBs) composed of triacylglycerol and phospholipid could be stabilized by oleosin, caleosin, or steroleosin. Our results showed that stabilization effects could be realized by oleosin or caleosin but not by steroleosin. The sizes of the AOBs constituted with oleosin (0.5-2 microm) or caleosin (50-200 nm) were similar to or 10 times smaller than those of the native oil bodies. Recombinant caleosin expressed in Escherichia coli also encapsulated AOBs with a size, topology, and stability comparable to those encapsulated with native caleosin. A proteinase K digestion indicated that caleosin anchored the AOBs via its central hydrophobic domain of approximately 4 kDa. Isoelectrofocusing revealed that the isoelectric point of the caleosin-stabilized AOBs was pH 4.0. Aggregation of AOBs was observed at a pH lower than 4.5; thus, their stability and integrity were presumably contributed by surface caleosin via electronegative repulsion and steric hindrance. The caleosin-stabilized AOBs were thermostable up to 70 degrees C and potentially useful for biotechnological applications.     

Purification and identification of a protease inhibitor from glassfish (Liparis tanakai) eggs

Two protease inhibitors of 67 and 18 kDa, respectively, were purified from glassfish, Liparis tanakai, eggs by affinity chromatography. The smaller protein was purified with a yield and purity of 0.25% and 49.69-fold, respectively, and was characterized for further study. The glassfish egg protease inhibitor exhibited stability between 50 and 65 degrees C in an alkaline environment (pH 8). It was shown to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 4.44 nM. Potent glassfish protease inhibitor with N-Val-Gly Ser-Met-Thr-Gly-Gly-Phe-Thr-Asp-C amino acid residues was synthesized and its inhibitory activity was compared. Moreover, the 18-kDa protein inhibited cathepsin, a cysteine protease, more effectively than did egg white protease inhibitor, whereas the reverse was true for papain. Glassfish egg protease inhibitor is classified as a member of the family I cystatins.     

Extraction and characterization of oil bodies from soy beans: a natural source of pre-emulsified soybean oil

Soybeans contain oil bodies that are coated by a layer of oleosin proteins. In nature, this protein coating protects the oil bodies from environmental stresses and may be utilized by food manufacturers for the same purpose. In this study, oil bodies were extracted from soybean using an aqueous extraction method that involved blending, dispersion (pH 8.6), filtration, and centrifugation steps. The influence of NaCl (0-250 mM), thermal processing (30-90 degrees C, 20 min) and pH (2-8) on the properties and stability of the oil bodies was analyzed using zeta-potential, particle size, and creaming stability measurements. The extracted oil bodies were relatively small ( d 32 approximately 250 nm), and their zeta-potential went from around +12 mV to -20 mV as the pH was increased from 2 to 8, with an isoelectric point around pH 4. The oil bodies were stable to aggregation and creaming at low (pH = 2) and high (pH >/= 6) pH values but were unstable at intermediate values (3 相似文献   

Plant seed cystatins and their target enzymes of endogenous and exogenous origin

Cystatins are protein inhibitors of cysteine proteinases of the papain family, and those of animal origin have long been studied from medical and physiological aspects. In the meantime, oryzacystatin cloned from rice seeds in 1987 was recognized as the first well-defined cystatin of plant origin. Cloning studies followed to disclose various plant cytstatins including those of corn and soybean origin, their similarities to and differences from animal cystatins being analyzed in detail. Plant seed cystatins are now understood as factors controlling germination by inhibition of endogenous cysteine proteinases. They can also recognize insect midgut proteinases as exogenous target enzymes to control. This paper discusses chemical and phytophysiological relationships between cystatins and their targets.     

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.     

Extraction of β‐Glucan from Oat Bran in Laboratory Scale

Effects of various enzymes and extraction conditions on yield and molecular weight of β‐glucans extracted from two batches of commercial oat bran produced in Sweden are reported. Hot‐water extraction with a thermostable α‐amylase resulted in an extraction yield of ≈76% of the β‐glucans, while the high peak molecular weight was maintained (1.6 × 10⁶). A subsequent protein hydrolysis significantly reduced the peak molecular weight of β‐glucans (by pancreatin to 908 × 10³ and by papain to 56 × 10³). These results suggest that the protein hydrolyzing enzymes may not be pure enough for purifying β‐glucans. The isolation scheme consisted of removal of lipids with ethanol extraction, enzymatic digestion of starch with α‐amylase, enzymatic digestion of protein using protease, centrifugation to remove insoluble material, removal of low molecular weight components using dialysis, precipitation of β‐glucans with ethanol, and air‐drying.     

Selective one-step extraction of Arabidopsis thaliana seed oleosins using organic solvents

Oleosins are hydrophobic proteins from oleaginous seeds, surrounding and stabilizing oil bodies. They are known to display interesting interfacial properties. Specific sera were raised against four different A. thaliana oleosins and used in dot-blot assays for oleosin quantification. These assays were used to set up extraction of oleosins from A. thaliana seeds. One mixture of chloroform/methanol gave optimal oleosin extraction. Extracted proteins represented 9% of seed proteins and were identified by immunoblot and proteomic analyses. Oleosins accounted for 79% of the extracted proteins. This simple one-step procedure allows selective extraction and concentration of oleosins from seeds without tedious oil body purification. Oleosin extract was indeed used to demonstrate the presence of the rare oleosin S5 in mature seeds. Moreover, this method will be useful to investigate the potential use of oleosins as emulsifier and to question their possible allergenicity.     

不同品种花生油脂体粒径电位和蛋白质组成的分析

为了探索不同品种花生油脂体的物理和化学性质差异,以5种（豫花23,豫花27,豫花9719,豫花9830和豫花9502）油脂含量不同的花生品种为原料,采用水剂法提取油脂体,并对提取后油脂体的粒径、ζ电位、氨基酸组成、蛋白质分子量分布进行分析比较。结果表明：提取后,5种花生油脂体粒径间存在一定差异,以豫花9719的粒径较大;花生油脂体均含有油脂体蛋白和贮藏蛋白,但不同品种间存在蛋白质种类的差异;5种花生油脂体在pH值为3.0时ζ电位为正值,在pH值为7.4和pH值为9.0时ζ电位为负值,盐浓度的增加会降低油脂体ζ电位的绝对值;5种花生油脂体的蛋白质均为极性带负电氨基酸质量分数均大于非极性带正电或不带电氨基酸,但氨基酸总量各不相同,以豫花27较低。该研究可为花生油脂体的品质特性研究和应用产品开发提供参考。     

Partial isolation and characterization of a cysteine proteinase inhibitor from Lima bean (Phaseolus lunatus)

Lima beans (Phaseolus lunatus) have been shown to contain cysteine proteinase inhibitor (CPI) activity, but the CPI has not been isolated or characterized. Accordingly, our objective was to isolate and partially characterize a CPI from lima bean. The isolation scheme included water extraction of lima bean flour followed by a chromatography series using DEAE Sepharose, Phenyl Sepharose, hydroxyapatite, and reversed-phase high performance liquid chromatography. This scheme resulted in the partial purification of a approximately 20 000-dalton protein with high inhibitory activity against papain. This isolated lima bean CPI had an N-terminal sequence homologous with other members of the cystatin class of CPIs. The protein was relatively heat labile; suggesting it could be inactivated with normal cooking, which is favorable for its use in transforming plants to create insect resistance.     

不同压榨工艺对芝麻油和芝麻饼品质的影响

为比较芝麻不同压榨工艺对芝麻油和芝麻饼品质的影响,分别以整籽带皮白芝麻和整籽脱皮白芝麻为原料,采用液压榨油机进行冷榨,对所得芝麻油和芝麻饼进行品质分析,并与热榨芝麻油、芝麻饼以及芝麻油国家标准进行对比.结果表明:冷榨芝麻油的色泽、酸价、过氧化值等质量指标都明显优于热榨芝麻油和芝麻香油国家标准;脱皮冷榨芝麻油较整籽冷榨芝...     

冷榨工艺对芝麻油和芝麻饼品质的影响

为比较芝麻不同压榨工艺对芝麻油和芝麻饼品质的影响,分别以带皮和脱皮白芝麻为原料,采用液压榨油机进行冷榨,对所得芝麻油和芝麻饼进行品质分析,并与热榨芝麻油、芝麻饼以及芝麻油国家标准进行对比。结果表明：冷榨芝麻油的色泽、酸价、过氧化值等质量指标都明显优于热榨芝麻油和芝麻香油国家标准;脱皮冷榨芝麻油较整籽冷榨芝麻油的酸价降低,但过氧化值有所升高;冷榨芝麻油较热榨芝麻油的VE含量、芝麻酚含量明显降低,芝麻素含量有所升高,氧化诱导时间明显缩短;脱皮冷榨芝麻油较整籽冷榨芝麻油的生育酚含量降低,芝麻素和芝麻酚含量有所升高,氧化诱导时间稍有延长。脱皮冷榨芝麻饼粗脂肪含量16.63%、粗蛋白含量54.09%、蛋白质溶解度NSI为23.50%。芝麻冷榨不仅可以得到色浅清亮的清香芝麻油,还可以得到蛋白质含量高且功能性好的食用芝麻蛋白。     

鸡毒霉形体黏附蛋白与鸡胚不同组织黏附特异性的研究

利用pGEX融合蛋白表达系统,将鸡毒霉形体黏附蛋白（pMGA）在大肠杆菌BL21中表达,经 GST·Bind Resin 纯化,用蛋白酶Thrombin切掉GST标签,获得纯度为96%的pMGA,Western-blot鉴定具有良好的免疫学活性。通过间接免疫荧光组织化学法（IFA）来检测pMGA与SPF鸡胚的气管、心脏、肝脏、肺脏、肾脏、腺胃、十二指肠、法氏囊、胸腺和脾脏等组织的特异性结合情况,结果表明,除脾脏外,其余组织与pMGA之间能产生特异性结合,说明这些组织中存在pMGA的受体蛋白。这为进一步研究pMGA的受体提供理论依据。     

Molecular cloning and characterization of oryzacystatin-III, a novel member of phytocystatin in rice (Oryza sativa L. japonica)

On the basis of cDNA sequences, we found that the calli of rice encodes an amino acid sequence that shares 56% and 89% identity, respectively, with oryzacystatin-I and oryzacystatin-II. This sequence differs from that of oryzacystatin-II in the N-terminal region (Gln(7)-Ala(19) in the oryzacystatin-III numbering), and this region contained a glycine residue (Gly(14)), which is evolutionarily conserved in the cystatin superfamily. We named this novel protein oryzacystatin-III. Nucleotide sequencing of the 5'-flanking region of the oryzacystatin-III gene showed that it is highly homologous to the oryzacystatin-II gene but distinct from the oryzacystatin-II locus. Oryzacystatin-III inhibited papain, ficin, and human cathepsin B. The inhibition constants for papain and ficin differ from those of oryzacystatin-I and -II, and cathepsin B activity is affected only by oryzacystatin-III, showing differences in the interaction of these inhibitors with enzymes. These data suggest that the above three inhibitors may play unique physiological roles in the regulations of rice cysteine proteinases.     

花生油体蛋白家族基因的克隆和表达分析

油体蛋白是一类覆盖在油体表面的碱性小分子蛋白.油体蛋白的存在对于维护油体的稳定性非常重要,油体蛋白也是种子作为生物反应器表达重组外源蛋白的重要载体.本研究通过构建花生(A rac his hypogaea L.)未成熟种子的全长cDNA文库,测序得到284条编码油体蛋白的EST.根据编码蛋白分子量的大小可将花生油体蛋白基因分为6个亚族:AhOLE O- 16.9、AhOLEO- 17.7、AhOLEO- 18.6、AhOLEO-22、AhOLEO- 18.4和AhOLEO- 14.3 (GenBank登录号分别为:EG372527、EG373122、EG373716、EG372719、EE125019和EE124234),其中AhOLEO- 16.9,AhOLEO- 17.7和AhOLEO- 14.3分别有2个成员,其它家族的各只有1个成员.本研究首次从花生中克隆了分子较大的AhOLEO-22.cDNA芯片和半定量PCR结果表明6个亚族的油体蛋白基因在花生的不同器官和在种子不同发育时期表达模式相似,在根、茎、叶、花中几乎检测不到油体蛋白基因的表达,在种子中表达量高,在果针入土15d内几乎检测不到油体蛋白基因的表达,随着种子发育成熟油体蛋白基因的表达量逐步提高.本研究为研究花生油体蛋白基因的家族构成和利用花生油体蛋白基因提高花生含油量、生产外源蛋白等提供了有用的信息.