A Facile Synthesis of 4,8-Dimethyldecanal,Aggregation Pheromone of Flour Beetles,and Its Analogues

A Rhodococcus erythropolis expression system for the bovine lactoferrin C-lobe was constructed. The DNA fragments encoding the BLF C-lobe were amplified and cloned into vector pTip LCH1.2. R. erythropolis carrying the pTip-C-lobe was cultured at 30 °C with shaking, and expression of the rBLF C-lobe was induced by adding 1 μg/ml (final concentration) thiostrepton. The rBLF C-lobe was isolated in native and denatured (8 M urea) form by Ni-NTA affinity chromatography. To obtain a bioactive rBLF C-lobe, the protein isolated in the denatured form was refolded by stepwise dialysis against refolding buffers. The antibacterial activity of the rBLF C-lobe was tested by the filter-disc plate assay method. The refolded rBLF C-lobe demonstrated antibacterial activity against selected strains of Escherichia coli.     

High-level Recombinant Expression and Refolding of Lysozyme from the Marine Shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

Shrimp lysozyme is as an antibacterial enzyme that participates in the innate defense against the invasion of bacterial pathogens. In this study, the lysozyme gene from hemocytes of the shrimp Marsupenaeus japonicus was isolated and characterized. The M. japonicus lysozyme (MjLys) encodes a polypeptide of 158 amino acids (aa) that includes an 18 aa signal peptide. The gene fragment encoding the mature MjLys protein was subcloned into the expression vector pET-32a(+) and transformed into E. coli BL21(DE3)pLysS, and the protein was strongly expressed in insoluble inclusion bodies. Following extraction using urea, the denatured recombinant protein was refolded by on-column Ni²⁺ affinity chromatography or dialysis with a gradient of decreasing urea concentration. Approximately 50% of the recombinant MjLys was successfully refolded into monomeric protein using urea gradient dialysis, while 30% was salvaged using on-column refolding. Purified MjLys exhibited significant antibacterial activity against Gram-positive bacteria Micrococcus lysodeikticus and Staphylococcus aureus. This efficient over-expression and refolding method can provide the large quantities of biologically active protein required for further biochemical and structural studies and potential biotechnological applications.     

Cycloamylose as an efficient artificial chaperone for protein refolding

High molecular weight cyclic alpha-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form.     

Expression,purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343–682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.     

Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli

The genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli. Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification. High-level expression of CPY and CPYPR-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His(6) was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His(6) was further purified to 90% purity by single-step immobilized metal ion affinity chromatography. The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His(6) at various concentrations. Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S. cerevisiae CPY.     

Isolation, amino acid analyses and refolding of subunits of pig heart succinyl-CoA synthetase.

For the first time, pig heart succinyl-CoA synthetase has been refolded from its isolated subunits after denaturation. Amino acid analyses of pig heart succinyl-CoA synthetase and its subunits were performed. Subunits were isolated by gel filtration in neutral 6 M-urea. The amino acid composition of the native enzyme bears a strong resemblance to that of the Escherichia coli enzyme. Application of the various methods for comparing amino acid compositions [Cornish-Bowden (1983) Methods Enzymol. 91, 60-75] shows that the degree of relatedness between the alpha-subunits of the pig heart and E. coli enzymes and between the beta-subunits of the two synthetases is intermediate between 'strong' and 'weak'. As for the E. coli synthetase, it is unlikely that the alpha-subunit arises from the larger beta-subunit by post-translational modification. The pig heart enzyme contains a single tryptophan residue, which is located in the beta-subunit. Excitation of the enzyme at 295 nm resulted in a typical tryptophan emission spectrum. Refolding of enzyme denatured in 6 M-guanidine hydrochloride or of alpha- and beta-subunits isolated in this solvent required the presence of either ethylene glycol or glycerol, optimally at 20-25% (v/v). GTP-Mg2+ did not stimulate reactivation of the enzyme, in contrast with the result obtained with ATP-Mg2+ in the reconstitution of the enzyme from E. coli. Yields of 60% and 40% were obtained in the refolding of denatured enzyme and isolated subunits respectively. The fluorescence spectrum of the refolded protein was essentially the same as that of native enzyme. Unrecovered activity could not be accounted for in the form of protein aggregates. The specific activity of refolded enzyme that had been separated from inactive protein on a Bio-Sil TSK 250 column was the same as that of native enzyme. Km values for GTP of 27 microM and 14 microM were determined for native and refolded enzyme respectively.     

尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征

将尖吻蝮蛇毒碱性磷脂酶A2 (A .aBPLA2 )基因克隆至温敏表达载体 pBLMVL2 ,在大肠杆菌RR1中成功诱导表达 .表达产物A .aBPLA2 约占细菌蛋白质总量的 2 0 % ,并以包涵体的形式存在 .纯化包涵体后 ,将产物变性、复性 ,然后用FPLCSuperoseTM12纯化 ,产物经过SDS 聚丙烯酰胺凝胶电泳检测只有单一条带 .对纯化后的表达A .aBPLA2 进行了酶活性、抑制血小板聚集活性和溶血活性的测定 .结果显示 ,表达A .aBPLA2的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A2 酶活性相近 ,具有类似变性后复性江浙蝮蛇碱性磷脂酶A2 的溶血活性 ,没有抑制血小板聚集活性 .最后对磷脂酶A2 的结构与这些活性的关系进行了讨论     

尖吻蝮蛇毒酸性磷脂酶A_2I的表达及其生化特征

将尖吻蝮蛇毒酸性磷脂酶 Ａ２ Ｉ（ Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ） 的基因克隆至表达载体ｐ Ｂ Ｌ Ｍ Ｖ Ｌ２ , 在大肠杆菌 Ｒ Ｒ１ 中成功表达。表达产物 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ约占细菌蛋白质总量的３０ ％ , 以包含体的形式存在。纯化包含体后, 将产物变性、复性, 然后用 Ｆ Ｐ Ｌ Ｃ Ｓｕｐｅｒｏｓｅ Ｔ Ｍ１２ 纯化, 产物经过 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 检测只有单一条带。对表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ进行了酶活性、抑制血小板聚集活性和溶血活性的测定。结果显示, 表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ的酶活性同变性后复性江浙蝮蛇酸性磷脂酶 Ａ２（ Ａ Ｐ Ｌ Ａ２） 的酶活性相近, 既具有抑制血小板聚集活性也具有溶血活性。最后对磷脂酶 Ａ２（ Ｐ Ｌ Ａ２） 的结构与这些活性的关系进行了讨论     

Thermostability of refolded ovalbumin and S-ovalbumin

Ovalbumin, a member of the serpin superfamily, is transformed into a thermostabilized form, S-ovalbumin, during storage of shell eggs or by an alkaline treatment of the isolated protein (DeltaT(m)=8 degrees C). As structural characteristics of S-ovalbumin, three serine residues (Ser164, Ser236 and Ser320) take the D-amino acid residue configuration, while the conformational change from non-thermostabilized native ovalbumin is very small. To assess the role of the structural characteristics on protein thermostabilization, ovalbumin and S-ovalbumin were denatured to eliminate the conformational modulation effects and then refolded. The denatured ovalbumin and S-ovalbumin were correctly refolded into the original non-denatured forms with the corresponding differential thermostability. There was essentially no difference in the disulfide structures of the native and refolded forms of ovalbumin and S-ovalbumin. These data are consistent with the view that the configuration inversion, which is the only chemical modification directly detected in S-ovalbumin so far, plays a central role in ovalbumin thermostabilization. The rate of refolding of S-ovalbumin was greater than that of ovalbumin, indicating the participation, at least in part, of an increased folding rate for thermodynamic stabilization.     

Evaluation of serodiagnosis of toxoplasmosis by using the recombinant nucleoside triphosphate hydrolase isoforms expressed in Escherichia coli

The nucleoside triphosphate hydrolase (NTPase) isoforms termed, NTPase-I and NTPase-II of Toxoplasma gondii, were expressed in Escherichia coli as inclusion bodies and purified under denaturing condition. Furthermore, NTPase-I was refolded as an active form and purified under non-denaturing condition. The purified NTPase isoforms, both denatured and refolded, were tested for their usefulness as antigens for the serodiagnosis of acute toxoplasmosis in immunocompetent humans. The test was conducted by using the recombinant NTPase isoforms and comparing the enzyme linked immunosorbent assay (ELISA) absorbances with the Sabin-Feldman dye test titer. Seventy-three sera from dye test-positive patients, and 30 sera from subjects with no T. gondii infection were examined. The total positive rates in dye test positive sera were: 82% (60/73) for denatured NTPase-I; 78% (57/73) for denatured NTPase-II; and 63% (46/73) for refolded NTPase-I. For all three antigen types of recombinant NTPase, the positive rates of sera of acute toxoplasmosis suspected patients were 93% (13/14). A moderate correlation between the ELISA absorbance using these antigens and the dye test titer was observed with the correlation coefficients, 0.583 (r2) for denatured NTPase-I, 0.472 (r2) for denatured NTPase-II, and 0.604 (r2) for refolded NTPase-I in the linear regression analysis. There was no significant difference observed in the antigenicity between refolded and denatured NTPase-I, nor between the isoforms.     

人血清Q型对氧磷酶在大肠杆菌中的表达

目的:应用大肠杆菌原核表达系统高效表达人血清Q型对氧磷酶(PON1)。方法:从pBlueScript-PON1重组质粒中通过PCR扩增得到了人PON1基因,将其亚克隆至原核表达载体pBV220中,构建了重组表达质粒pBV220-PON1,转化大肠杆菌,获得表达菌株。结果:rhPON1重组蛋白以不溶形式存在于包涵体中。凝胶扫描分析表明,重组蛋白表达量约占菌体总蛋白的18%。包涵体经分离、变性、复性等步骤处理后,产物未显示酶活性。结论:原核表达的rhPON1以包涵体形式存在,实现了人PON1在大肠杆菌中的高效表达。     

Respective importance of protein folding and glycosylation in the thermal stability of recombinant feruloyl esterase A

The thermal stability of four molecular forms (native, refolded, glycosylated, non-glycosylated) of feruloyl esterase A (FAEA) was studied. From the most to the least thermo-resistant, the four molecular species ranked as follows: (i) glycosylated form produced native, (ii) non-glycosylated form produced native, (iii) non-glycosylated form produced as inclusion bodies and refolded, and (iv) glycosylated form produced native chemically denatured and then refolded. On the basis of these results and of crystal structure data, we discuss the respective importance of protein folding and glycosylation in the thermal stability of recombinant FAEA.     

Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii.

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLF-related protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe.     

Catabolic pathway of gamma-caprolactone in the biocontrol agent Rhodococcus erythropolis

Gamma-caprolactone (GCL) is well-known as a food flavor and has been recently described as a biostimulant molecule promoting the growth of bacteria with biocontrol activity against soft-rot pathogens. Among these biocontrol agents, Rhodococcus erythropolis, characterized by a remarkable metabolic versatility, assimilates various γ-butyrolactone molecules with a branched-aliphatic chain, such as GCL. The assimilative pathway of GCL in R. erythropolis was investigated by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis. This analysis suggests the involvement of the lactonase QsdA in ring-opening, a feature confirmed by heterologous expression in Escherichia coli. According to proteome analysis, the open-chain form of GCL was degraded by β- and ω-oxidation coupled to the Krebs cycle and β-ketoadipate pathway. Ubiquity of qsdA gene among environmental R. erythropolis isolates was verified by PCR. In addition to a previous N-acyl homoserine lactone catabolic function, QsdA may therefore be involved in an intermediate degradative step of cyclic recalcitrant molecules or in synthesis of flavoring lactones.     

Reconstitution of Fragmentary Form of Thermostable Alanine Racemase

The fragmentary form of alanine racemase from Bacillus stearothermophilus is composed of two sets of two different polypeptides corresponding to the two domains of the subunit of wild-type enzyme. It was denatured with 6 M guanidium hydrochloride to be separated into pieces, and renatured by dilution with about 50% recovery of the activity. The two kinds of polypeptides (i.e., large and a small fragments) were isolated by gel filtration in the presence of 4 M guanidium hydrochloride. The CD spectra obtained by summation of the spectra of the refolded fragments were closely similar to that of the native fragmentary enzyme. The lysine residue to which PLP is bound in the wild-type enzyme occurs in the large peptide of the fragmentary enzyme containing the amino terminus of the wild-type enzyme. The visible spectrum of the large peptide refolded, however, indicated that PLP was not bound to it. The large peptide alone had no significant activity, but it was activated by incubation with the small peptide. Accordingly, co-existene of both peptide fragments is necessary for folding of a complex of the two kinds of peptide to form an active structure.     

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.     

人基质金属蛋白酶2催化区的克隆与表达

为从随机肽库中寻找具有基质金属蛋白酶 2 (MMP 2 )抑制活性的新型小肽抑制剂 ,应用PCR法从含有人MMP 2基因的质粒中扩增了人MMP 2的催化区 .序列分析结果表明无氨基酸突变 .然后构建人MMP 2催化区的表达载体pET MCD ,转化大肠杆菌BL2 1(DE3 ) ,经IPTG诱导表达人MMP 2催化区 .经包涵体分离、变性、金属螯合层析纯化和复性等过程 ,复性后的人MMP 2催化区具有较好的明胶水解活性 .     

On-column refolding of recombinant human interferon-gamma inclusion bodies by expanded bed adsorption chromatography

A refolding strategy was described for on-column refolding of recombinant human interferon-gamma (rhIFN-gamma) inclusion bodies by expanded bed adsorption (EBA) chromatography. After the denatured rhIFN-gamma protein bound onto the cation exchanger of STREAMLINE SP, the refolding process was performed in expanded bed by gradually decreasing the concentration of urea in the buffer and the refolded rhIFN-gamma protein was recovered by the elution in packed bed mode. It was demonstrated that the denatured rhIFN-gamma protein could be efficiently refolded by this method with high yield. Under appropriate experimental conditions, the protein yield and specific activity of rhIFN-gamma was up to 52.7% and 8.18 x 10(6) IU/mg, respectively.     

融合干扰素-BLA（IFN-BLA）的构建、表达、纯化及抗病毒活性测定

干扰素-BLA(IFN-BLA)由干扰素-beta-1b(IFN-beta-1b)和干扰素-alpha-2b（IFN-alpha-2b）通过连接肽-GGGS-融合而成。优化了其在大肠感菌BL21 CodonPlus (DE3)-RIL中的实验室表达条件,表达的目的蛋白占菌体总蛋白的35％以上并且主要以包涵体的形式存在。对包涵体的复性条件进行了摸索,建立了IFN-BLA的复性及纯化方法,纯化后的蛋白产量约为45 mg/L, 纯度在90％以上。抗病毒活性分析表明这一新的融合蛋白可能具有协同或加成活性。