Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377 ± 162 U/ml for full-length rHu BChE and 574 ± 143 U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9 U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.     

Effect of polyethylene glycol on the thermal stability of green fluorescent protein

Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Some polymers, such as polyethylene glycol, are often used as modifiers of characteristics of biological macromolecules, to improve the biochemical activity and stability of proteins or drug bioavailability. The aim of this study was to evaluate the thermal stability of GFP in the presence of different PEG molar weights at several concentrations and exposed to constant temperatures, in a range of 70–95°C. Thermal stability was expressed in decimal reduction time. It was observed that the D‐values obtained were almost constant for temperatures of 85, 90, and 95°C, despite the PEG concentration or molar weight studied. Even though PEG can stabilize proteins, only at 75°C, PEG 600 and 4,000 g/mol stabilized GFP. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

重组L-门冬酰胺酶工程菌的表达和PEG的化学修饰

目的提高重组L-门冬酰胺酶(rL-ASP)工程菌的表达量,分离纯化rL-ASP并对之进行PEG化学修饰。方法将带有编码rL-ASP的基因的质粒(pKA)导入不同的宿主菌中,挑出高表达菌株,同时优化发酵培养基,分离纯化获得的高纯度rL-ASP再用PEG进行化学修饰,SDS-PAGE检测修饰效果。结果在pH7.0的条件下,宿主菌为JMl09的工程菌pKA/JMl09酶活力最高,三角瓶振摇培养的酶活力可达216×103IU/L;发酵罐发酵培养,酶活力达312×103IU/L。纯化后的rL-ASP比活力为220IU/mg,rL-ASP经过PEG化学修饰生成rL-ASP-PEG,分子量发生改变。结论改变目标蛋白表达的宿主菌和优化发酵工艺,提高了rL-ASP的表达量,纯化的rL-ASP经过PEG化学修饰后分子量增大。     

Chemical modification of recombinant human granulocyte colony-stimulating factor by polyethylene glycol increases its biological activity in vivo.

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Escherichia coli was chemically modified by polyethylene glycol (PEG) of molecular weights 4,500 or 10,000. The neutrophils observed at 32 hours after intravenous injection of the rHuG-CSF modified with PEG (4,500) or PEG (10,000) to mice were, respectively, 2.5 times and 5 times more than that observed after the injection of the unmodified rHuG-CSF. These results show that the covalent attachment of PEG to rHuG-CSF enhanced its pharmacological activity in vivo and that the modification with the larger PEG molecule is more effective to enhance the in vivo activity of rHuG-CSF.     

Effect of lysine modification on the stability and cellular binding of human amyloidogenic light chains

AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescence microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichroism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.     

An investigation into the protective effect of various salts on the acid inactivation of human serum butyrylcholinesterase (EC 3.1.1.8)

Human serum butyrylcholinesterase (EC 3.1.1.8) loses 100% of its activity toward butyrylthiocholine in 60 min atpH 3.0. This deactivation is retarded by 1.37 M ammonium sulfate to a loss of 40% after 60 min atpH 3.0. Reneutralization experiments suggest that the mechanism for this acid inactivation does not exclusively involve hydrolysis of peptide bonds or protonation of the enzyme's active site. Studies with different anions and cations demonstrate that the order of their effectiveness as protective agent against acid inactivation closely follows the Hofmeister series. No relationship was found between catalytic activation or inhibition by salt and protection from acid inactivation. Ultraviolet difference studies at 288 nm with enzyme brought topH 2.7 frompH 8.0 in the presence and absence of 1.37 M ammonium sulfate demonstrated no change in UV absorbance with ammonium sulfate present and approximately a 0.15 ODU rise in absorbance in the absence of ammonium sulfate. These results suggest that acidicpH conditions result in deactivating stereochemical changes in the active site of butyrylcholinesterase and that certain anions and cations, according to the Hofmeister series, are able to protect the enzyme from acid inactivation by stabilizing the active conformation of its active site.     

Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase.

Methoxypolyethylene glycols of 1900 daltons (PEG-1900) or 5000 daltons (PEG-5000) were covalently attached to bovine liver catalase using 2,4,6-trichloro-s-triazine as the coupling agent. Rabbits were immunized by the intravenous and intramuscular routes with catalase modified by covalent attachment of PEG-1900 to 43% of the amino groups (PEG-1900-catalase). The intravenous antiserum did not yield detectable antibodies against PEG-1900-catalase or native catalase, as determined by Ouchterlony and complement fixation methods, whereas the intramuscular antiserum contained antibodies to both PEG-1900-catalase and catalase. PEG-1900 did not react with either antiserum. Catalase was prepared in which PEG-5000 was attached to 40% of the amino groups (PEG-5000-catalase). This catalase preparation did not react with either antiserum. PEG-1900-catalase retained 93% of its enzymatic activity; PEG-5000-catalase retained 95%. PEG-5000-catalase resisted digestion by trypsin, chymotrypsin, and a protease from Streptomyces griseus. PEG-1900-catalase and PEG-5000-catalase exhibited enhanced circulating lives in the blood of acatalasemic mice during repetitive intravenous injections. No evidence was seen of an immune response to injections of the modified enzymes. Mice injected repetitively with PEG-5000-catalase remained immune competent for unmodieied catalase, and no evidence of tissue or organ damage was seen.     

Effect of ethylene glycol and polyethylene glycol on the acid-unfolded state of trypsinogen

Effect of increasing concentrations of two of the polyols, ethylene glycol (EG) and polyethylene glycol (PEG), was studied by near and far circular dichroism (CD), fluorescence emission spectroscopy, and binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show the transition of acid-unfolded trypsinogen from an unordered state to an intermediate state having ordered secondary structure. Interestingly, near-UV CD spectra show some amounts of stabilizing effect on the tertiary structure of the protein also. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on addition of EG and PEG. Maximum ANS binding occurs in presence of 80% EG and 90% PEG (v/v), suggesting the presence of an intermediate or molten globule-like state at high concentrations of the two polyols.     

Differential effect of sorbitol and polyethylene glycol on antioxidant enzymes in rice leaves

Polyethylene glycol (PEG) and sorbitol (ST) have each been used inosmotically induced water stress studies in plants, however, these osmotica maynot have equivalent effects in plants. The present study was designed to examinewhether antioxidant enzyme responses in rice leaves are different for PEG and STof osmotic potential –1.5 MPa. As judged by relative watercontent, PEG treatment resulted in a higher degree of water stress in riceleaves than ST treatment. PEG treatment markedly increased lipid peroxidation,judged by malondialdehyde content, in rice leaves. However, ST treatment had noeffect on lipid peroxidation. An increase in peroxidase (POX), ascorbateperoxidase (APX) and glutathione reductase (GR) activities was observed in riceleaves treated with ST. PEG treatment had no effect on POX and APX activitiesand decreased GR activity in rice leaves. The decrease in superoxide dismutaseactivity induced by PEG was more pronounced than by ST. Cycloheximide blockedthe enhanced activities of POX, APX and GR by ST, indicating de novo synthesisof the enzymes. Results suggest that ST but not PEG treatment can up-regulateantioxidant system in rice leaves.     

Activation in the family of Candida rugosa isolipases by polyethylene glycol

We have investigated activation of two isoenzymes (lip1 and lip3) from Candida rugosa in polyethylene glycol (PEG) media. Aqueous solutions of PEG 8000 and 20,000 activate lip3 but not lip1 from C. rugosa. Maximum activation (260%) of lip3 requires 6 h of pre-incubation with PEG 8000 (4%, w/v). PEG seems to shift the equilibrium between the open and the closed forms of lip3 towards the active conformation. Inhibition experiments demonstrate that ligands have easier access to the lip3 active site than to the lip1 active site, both in the presence and the absence of PEG.

The presence of PEG in the crystallization medium is responsible for reported differences in the crystal structures of lip1 and lip3. A comparative analysis of crystallographic models of lip1 and lip3 suggests a role for PEG in activation of lip3 and further stabilization of the activated/open form via dimerization in aqueous media.     

Effect of glycerol,polyethylene glycol and glutaraldehyde on stability of phenylalanine ammonia-lyase activity in yeast

Summary The polyhydric alcohols, glycerol and sorbitol, significantly increased the rate ofl-phenylalanine production from trans-cinnamic acid using whole cells ofRhodotorula rubra. Chloride ions and oxygen prevented the stimulatory effect of the polyhydric alcohols. Furthermore, the severe inhibition, of the biotransformation by high trans-cinnamic acid concentrations was alleviated in the presence of glycerol, and sorbitol. The rate of conversion could be manipulated still further, even with high trnas-cinnamic acid concentrations, by elevating the reaction pH to, 12 in the presence of polyhydric alcohol. When cells were also treated first with glutaradehyde (0.1% v/v) and then polyethylene glycol (15% v/v), although neither compound stimulated the actual rate of bioconversion, the reaction was markedly stabilised and gave a 73% yield after 28 days of continuous operation.     

Modifications of the fibroblast growth factor-2 gene led to a marked enhancement in secretion and stability of the recombinant fibroblast growth factor-2 protein

Progress in FGF-2 gene therapy has been hampered by the difficulty in achieving therapeutic levels of FGF-2 secretion. This study tested whether the addition of BMP2/4 hybrid secretion signal to the FGF-2 gene and mutation of cys-70 and cys-88 to serine and asparagine, respectively, would increase the stability and secretion of active FGF-2 protein in mammalian cells using MLV-based vectors. Single or double mutations of cys-70 and cys-88 to ser-70 and asp-88, respectively, markedly increased the amounts of FGF-2 protein in conditioned media and cell lysates, which may be due to glycosylation, particularly at the mutated asp-88 residue. Addition of BMP2/4 secretion signal increased FGF-2 secretion, but also suppressed FGF-2 biosynthesis. The combination of BMP2/4 secretion signal and double cys-70 and cys-88 mutations increased the total amount of secreted FGF-2 protein >60-fold. The modifications did not alter its ability to stimulate cell proliferation and Erk1/2 phosphorylation in marrow stromal cells or its ability to bind heparin in vitro, suggesting that the modified FGF-2 protein was functionally as effective as the unmodified FGF-2. An ex vivo application of rat skin fibroblasts (RSF) transduced with the modified FGF-2 vector in a subcutaneous implant model showed that rats with implants containing cells transduced with the modified FGF-2 vector increased serum FGF-2 level >15-fold, increased growth of the implant, and increased vascularization within the implant, compared to rats that received implants containing beta-galactosidase- or wild-type FGF-2-transduced control cells. This modified vector may be useful in FGF-2 gene therapy investigations.     

Action of polyethylene glycol on the fusion of human erythrocyte membranes

Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.     

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.     

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.     

Effect of circulatory occlusion on human muscle metabolism during exercise and recovery

To assess muscle metabolism and inorganic phosphate (P_i) peak splitting during exercise, 31-phosphorus nuclear magnetic resonance spectroscopy was performed during ramp incremental and submaximal step exercise with and without circulatory occlusion. Seven healthy men performed calf flexion in a superconducting magnet. There was no P_i splitting during ramp incremental exercise with the circulation present and phosphocreatine (PCr) decreased linearly by 0.07 (SEM 0.01) mmol · l⁻¹ · s⁻¹, while exercise with the circulation occluded caused the P_i peak to split into a high and a low pH peak. The rate of PCr decrease during exercise with the circulation occluded was 0.15 (SEM 0.03) mmol · l⁻¹ · s⁻¹ which with the efficiency of the adenosine 5′-triphosphate (ATP) hydrolysis reaction corresponded well to the mechanical energy. Both with and without occlusion of the circulation PCr decreased with some time lag which may reflect the consumption of residual oxygen. In submaximal step exercise PCr decreased exponentially at the onset of exercise with the circulation open whereas it decreased linearly by 0.15␣mmol · l⁻¹ · s⁻¹ when the circulation was occluded. After exercise, occlusion of the circulation was maintained for 1 min more and there was no PCr resynthesis. It is suggested that ATP synthesis was limited by the availability of oxygen. Accepted: 14 August 1996     

重组人尿激酶原半成品的稳定性观察

对干燥前尿激酶原半成品的稳定性进行了观察。将半成品分装为数管 (1 40 μl/管 ) ,分别放在 4℃ ,- 2 0℃和 - 70℃ ,用纤维蛋白平板溶解圈法和 SDS- PAGE扫描法观察其 3个月内的活性及纯度变化。结果表明在 4℃条件下 ,处于溶液中的尿激酶原稳定性最差 ,一周后活性由 5× 1 0 4IU/ml降至 2 .3 8× 1 0 4IU/ml,单链比例由 97.7%降至 88.3 %,二周后单链比例进一步下降至 43 .8%。贮存在 - 2 0℃和 - 70℃条件下活性稳定 ,但单链也要缓慢裂解 ,一周后单链比例由 97.7%分别下降到 93 .9%和 93 .5 %,3个月后分别为 83 .7%和 86 .2 %,两者无明显差别     

Effects of polyethylene glycol on stability of alpha-chymotrypsin in aqueous ethanol solvent

The effects of polyethylene glycol (PEG) of different molecular weights (400, 2000, 6000, 12,000, 20,000, and 35,000) on the conformational stability and catalytic activity of alpha-chymotrypsin in 60% ethanol were studied. The inactivation caused by the organic solvent was not influenced by PEG 400. However, the PEGs with higher molecular weights up to 35,000 increased the stability of the enzyme, but this alpha-chymotrypsin stabilizing effect was molecular weight-independent. With increase of the molecular weight of PEG, a more stable tertiary structure of the enzyme was observed.