单链抗体抑制乙肝病毒的研究

单链抗体已用于抗乙肝病毒(hepatitis B virus, HBV)的研究,目前已研制出作用于各种靶点,如HBV表面抗原pre-S1、核心蛋白(hepatitis B virus core antigen, HBc)、DNA聚合酶及X 蛋白的多种单链抗体。单链抗体对偶联的分子具有靶向定位作用,因此,对抗原的亲和性大小、对靶细胞内化(Internalization)的强弱及其自身结构的稳定性是影响单链抗体应用的主要因素。     

Protein refolding assisted by self-assembled nanogels as novel artificial molecular chaperone

Molecular chaperone-like activity for protein refolding was investigated using nanogels of self-assembly of cholesterol-bearing pullulan. Nanogels effectively prevented protein aggregation (i.e. carbonic anhydrase and citrate synthase) during protein refolding from GdmCl denaturation. Enzyme activity recovered in high yields upon dissociation of the gel structure in which the proteins were trapped, by the addition of cyclodextrins. The nanogels assisted protein refolding in a manner similar to the mechanism of molecular chaperones, namely by catching and releasing proteins. The nanogels acted as a host for the trapping of refolded intermediate proteins. Cyclodextrin is an effector molecule that controls the binding ability of these host nanogels to proteins. The present nanogel system was also effective at the renaturation of inclusion body of a recombinant protein of the serine protease family.     

The effects of arginine on refolding of aggregated proteins: not facilitate refolding,but suppress aggregation

Arginine is one of the universal reagents that are effective in assisting refolding of recombinant proteins from inclusion bodies. The mechanism of the effects of arginine on refolding has remained, however, to be elucidated. Here we show that arginine does not stabilize proteins against heat treatment, as demonstrated by little change in melting temperature. It does increase reversibility of thermal melting and reduce aggregation under thermal stress. The observations suggest that arginine may not facilitate refolding, but may suppress aggregation of the proteins during refolding.     

人基质金属蛋白酶-2C端片段PEX的高效表达及活性研究

将人的PEX基因克隆到表达载体pET-15b上,构建重组工程菌BL21(DE3)/pETPEX。采用5L发酵罐补料分批培养,当菌体密度增长到OD₆₀₀为35时,用IPTG诱导,PEX蛋白形成包涵体,最终OD₆₀₀达到90,PEX的表达水平达2.2g/L。包涵体蛋白经过纯化、复性后,获得生物活性。PEX能够有效抑制HUVECs的增殖,加药96h后,20nmol/L PEX对HUVECs生长有77.9%的抑制率;PEX能阻断bFGF诱导的鸡胚尿囊膜血管生成,抑制率为85%;小鼠实验表明,当给药剂量为5mg/kg时,PEX对S180肉瘤有31.4%的抑瘤率(P<0.05)。PEX是一个有效的抑制新血管生成和肿瘤生长的蛋白质因子。     

重组人成骨蛋白-1的离子交换及分子排阻色谱法纯化及其复性研究

经发酵大量表达重组人成骨蛋白-1(rhOP-1)。SDS-PAGE发现rhOP-1表达量占细菌总蛋白的35%。菌体经裂解、洗涤后,用8mol/L尿素溶解包涵体,离心后提取目的蛋白。经离子交换色谱法对变性状态下的目的蛋白进行纯化,绝大部分杂蛋白被去除,目的蛋白纯度达93%以上。为进一步提高目的蛋白浓度,采用分子排阻色谱法对目的蛋白进行再次纯化,纯度达98%以上。利用降低尿素梯度的方法对纯化的蛋白进行复性,二聚体的含量在50%以上。Westernblot证明了复性后的目的蛋白以单体和有活性的二聚体的形式存在。     

Expression,refolding, and characterization of recombinant thrombopoietin/stem cell factor fusion protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx) fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method. Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni²⁺ affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF.     

小檗碱对游离脂肪酸损伤的胰岛betaTC3 细胞的保护作用*

目的：观察小檗碱对棕榈酸损伤的胰岛betaTC3 细胞是否具有保护作用,并筛查出小檗碱起保护作用的有效浓度及合适的作 用时间。方法：以胰岛betaTC3 细胞为研究对象,用棕榈酸构建脂毒性模型,MTT 法筛选小檗碱起保护作用的有效浓度及时间;流式 细胞技术检测胰岛betaTC3 细胞凋亡情况。结果：①0.001-1 umol/L小檗碱作用茁TC3细胞24、48、72 h,对细胞增殖有不同程度促进 作用（与对照组比较P＜ 0.05）;随着作用时间的延长,低浓度小檗碱对betaTC3细胞的保护作用增加,而1 umol/L浓度保护作用下 降,10 umol/L及以上浓度出现细胞毒性作用（与对照组相比P＜0.01）,并呈浓度依赖性。②棕榈酸（0.2 -1.0 mmol/L)对betaTC3细胞 的损伤作用具有浓度及时间依赖性（与对照组比较P＜0.05）。③棕榈酸处理betaTC3细胞不同时间后,小檗碱治疗组较模型组的细 胞凋亡率显著降低（P<0.01)。结论：小檗碱对脂毒性损伤的胰岛betaTC3 细胞具有保护作用,且脂毒性作用时间越短,小檗碱的保护 作用越好,随着脂毒性作用时间的延长,小檗碱的保护作用明显减弱。因此,建议临床上在脂代谢紊乱早期给予小檗碱干预以减 轻甚至逆转游离脂肪酸导致的胰岛茁细胞凋亡。     

Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis

A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.     

The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 °C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 °C are more efficiently dissociated at 2.4 kbar than those produced at 47 °C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (−9 °C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 °C to 50 °C is proportional to the temperature, and a higher yield was obtained at 20 °C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 °C), of their dissociation at HHP (−9 °C) and of eGFP refolding (20 °C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.     

A screening system for the identification of refolding conditions for a model protein kinase, p38alpha

Protein kinases are key drug targets involved in the regulation of a wide variety of cellular processes. To aid the development of drugs targeting these kinases, it is necessary to express recombinant protein in large amounts. The expression of these kinases in Escherichia coli often leads to the accumulation of the expressed protein as insoluble inclusion bodies. The refolding of these inclusion bodies could provide a route to soluble protein, but there is little reported success in this area. We set out to develop a system for the screening of refolding conditions for a model protein kinase, p38α, and applied this system to denatured p38α derived from natively folded and inclusion body protein. Clear differences were observed in the refolding yields obtained, suggesting differences in the folded state of these preparations. Using the screening system, we have established conditions under which soluble, folded p38α can be produced from inclusion bodies. We have shown that the refolding yields obtained in this screen are suitable for the economic large-scale production of refolded p38α protein kinase.     

Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg

The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed.     

Addition of a polypeptide stretch at the N-terminus improves the expression, stability and solubility of recombinant protein tyrosine phosphatases from Drosophila melanogaster

The production of recombinant proteins in Escherichia coli involves substantial optimization in the size of the protein and over-expression strategies to avoid inclusion-body formation. Here we report our observations on this so-called construct dependence using the catalytic domains of five Drosophila melanogaster receptor protein tyrosine phosphatases as a model system. Five strains of E. coli as well as three variations in purification tags viz., poly-histidine peptide attachments at the N- and C-termini and a construct with Glutathione-S-transferase at the N-terminus were examined. In this study we observe that inclusion of a 45 residue stretch at the N-terminus was crucial for over-expression of the enzymes, influencing both the solubility and the stability of these recombinant proteins. While the addition of negatively charged residues in the N-terminal extension could partially rationalize the improvement in the solubility of these constructs, conventional parameters like the proportion of order promoting residues or aliphatic index did not correlate with the improved biochemical characteristics. These findings thus suggest the inclusion of additional parameters apart from rigid domain predictions to obtain domain constructs that are most likely to yield soluble protein upon expression in E. coli.     

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2(271-326))

Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2_271–326, a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidine-selected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2_271–326, was subsequently purified by reverse-phase HPLC and confirmed by SDS–PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2_271–326 in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% α-helical structure, with the remainder having β-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs.     

Stabilization of heat-induced changes in plant peroxidase preparations by ClpX, a bacterial heat shock protein

Peroxidases (PODs) are known to be quite stable at elevated temperatures. Moreover, partially denatured peroxidases are able to regain their catalytic activity during incubation at room temperature. In this paper, we describe the effects of some heat shock proteins on the self-reactivation of plant peroxidase preparations. Horseradish and artichoke peroxidases (HRP and ARP, respectively) were first heated (at 60 °C or 90 °C), then incubated at a slightly elevated temperature (30 °C). The heat-treatment resulted in a considerable loss of activity of both enzymes but the subsequent incubation allowed their reactivation. However, no reactivation could be detected when incubation was carried out in the presence of the molecular chaperone ClpX. Other chaperones that were tested (DnaK, DnaJ and GrpE) did not show the inhibitory effect. Electrophoretic analyses further indicated that the heat-treated horseradish peroxidase, but not the native enzyme, binds to ClpX eliminating the possibility of undesirable protein refolding that would result in aggregation.     

Increased expression of a molecular chaperone GroEL in response to unsaturated fatty acids by the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens

Butyrivibrio fibrisolvens is the most active bacterial species in the biohydrogenation of polyunsaturated fatty acids (PUFA) in the rumen. It needs to remove the unsaturated bonds in order to detoxify the PUFA to enable the growth of the bacterium. Here, we investigated the response of cell membrane-associated proteins in B. fibrisolvens to growth in the presence of PUFA. Numerous changes were observed in the cell membrane-associated proteome. One of the main modifications occurring when the 18:2 fatty acids, linoleic acid and conjugated linoleic acid, were added, was an increased expression of the molecular chaperone GroEL.     

Chaperone activity of tobacco HSP18, a small heat-shock protein, is inhibited by ATP

NtHSP18P (HSP18), a cytosolic class I small heat-shock protein from tobacco pollen grains, was expressed in Escherichia coli. The viability of these cells was improved by 50% at 50 degrees C, demonstrating its functionality in vivo. Purified recombinant protein formed 240 kDa HSP18 oligomers, irrespective of temperature. These oligomers interacted with the model substrate citrate synthase (CS) to form large complexes in a temperature-dependent manner. Furthermore, HSP18 prevented thermally induced aggregation of CS at 45 degrees C. The fluorescence probe bis-ANS revealed the exposure of HSP18 hydrophobic surfaces at this temperature. Reactivation of chemically denatured CS was also significantly enhanced by HSP18. Surprisingly, HSP18 function was inhibited (in contrast to the related chaperone alphabeta-crystallin and plant sHSPs studied so far) by the presence of ATP in a concentration-dependent manner. The conformational changes of HSP18 imposed by ATP binding were indicated by the difference in the quenching of intrinsic tryptophan fluorescence, and implied more compact structure with ATP. Fluorescence measurements with bis-ANS showed that the conformational shift of HSP18 is suppressed in the presence of ATP. Decreased chaperone activity of HSP18 in the presence of ATP is caused by the lower affinity of conformationally blocked HSP18 for the substrate, as demonstrated by a higher susceptibility of model substrate, malate dehydrogenase, to proteolytic cleavage. Our results suggest that the chaperone activity of some plant sHSPs could be regulated by the availability of ATP in the cytoplasm, which would provide a mechanism to monitor the cell environment, control biological activity of sHSPs, and coordinate it with other ATP-dependent chaperones such as HSP70.     

Expression, purification, and refolding of a novel immunotoxin containing humanized single-chain fragment variable antibody against CTLA4 and the N-terminal fragment of human perforin

Immunotoxins might be potential in treatment of cancer for their ability to kill selected cell populations. We constructed a novel immunotoxin hS83P34 by fusing N-terminal 34 amino acid fragment of human perforin to the C-terminus of humanized single-chain fragment variable antibody against CTLA4. The fusion protein was inductively expressed as inclusion bodies at a high level about 30% of total bacterial proteins. After washing with buffer containing 2 M urea, the purity of inclusion body was about 71%. The washed inclusion bodies were solubilized in 8 M urea and further purified to homogeneity (approximately 92% purity) by cation-exchange chromatography and Ni-agarose affinity chromatography under denaturing condition. The inclusion body refolding conditions were optimized following Pro-Matrix Protein Refolding Guide. After refolded in Tris buffer (pH 8.0) containing 1M urea, 0.8 M l-arginine, and 2 mM GSH:0.2 mM GSSG or 2 mM GSH:0.4 mM GSSG for 18h at 4 degrees C, over 90% proteins were recovered from inclusion bodies. In vitro dose-dependent cytotoxicity assay demonstrates that hS83P34 is only toxic to CTLA4-positive cells. IC(50) of hS83P34 for leukemic cells Raji and 6T-CEM are about 0.85 and 1.3 microM individually. Whereas, CTLA4-negative endothelial cell ECV-304 is resistant to hS83P34.     

Characterization of a sHsp of Schizosaccharomyces pombe, SpHsp15.8, and the implication of its functional mechanism by comparison with another sHsp, SpHsp16.0

There exist two small heat shock proteins (sHsps) in the fission yeast, Schizosaccharomyces pombe (S. pombe), whose expressions are highly induced by heat stress. We have previously expressed, purified, and characterized one of the sHsps, SpHsp16.0. In this study, we examined the other sHsp, SpHsp15.8. It suppressed the thermal aggregation of citrate synthase (CS) from porcine heart and dithiothreitol-induced aggregation of insulin from bovine pancreas with very high efficiency. Almost one SpHsp15.8 subunit was sufficient to protect one protein molecule from aggregation. Like SpHsp16.0, SpHsp15.8 dissociated into small oligomers and then interacted with denatured substrate proteins. SpHsp16.0 exhibited a clear enthalpy change for denaturation occurring over 60 degrees C in differential scanning calorimetry (DSC). However, we could not observe any significant enthalpy change in the DSC of SpHsp15.8. The difference is likely to be caused by the adhesive characteristics of SpHsp15.8. The oligomer dissociation of SpHsp15.8 and SpHsp16.0 and their interactions with denatured substrate proteins were studied by fluorescence polarization analysis (FPA). Both sHsps exhibited a temperature-dependent decrease of fluorescence polarization, which correlates with the dissociation of large oligomers to small oligomers. The dissociation of the SpHsp15.8 oligomer began at about 35 degrees C and proceeded gradually. On the contrary, the SpHsp16.0 oligomer was stable up to approximately 45 degrees C, but then dissociated into small oligomers abruptly at this temperature. Interestingly, SpHsp16.0 is likely to interact with denatured CS in the dissociated state, while SpHsp15.8 is likely to interact with CS in a large complex. These results suggest that S. pombe utilizes two sHsps that function in different manners, probably to cope with a wide range of temperatures and various denatured proteins.     

Inclusion body formation by interleukin-1β depends on the thermal sensitivity of a folding intermediate

We show that sequence and growth temperature effects on IB formation in the small, monomeric β-barrel protein interleukin-1β (IL-1β) can be quantitatively reproduced in an in vitro system in which IL-1β is refolded from denaturant at different temperatures. The results suggest that temperature and mutational effects on IB formation may be based on intrinsic properties of the protein sequence rather than interactions with chaperones or other cellular factors. We also report striking correlations of IB formation with mutation-dependent changes in residue hydrophobicity. The nature of these trends differs considerably with residue position, however, suggesting that they are mediated by particular local environments created by an ordered structure.     

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational,structure-guided Fv engineering

Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.