Protein refolding is improved by adding nonionic polyethylene glycol monooleyl ethers with various polyethylene glycol lengths

Protein refolding from bacterial inclusion bodies is a crucial step for the production of recombinant proteins, but the refolding step often results in significantly lower yields due to aggregation. To prevent aggregation, chemical additives are often used. However, the ability of additives to effectively increase refolding yields are protein dependent, and therefore, it is important to understand the manner in which the substructures of additives confer suitable properties on protein refolding. We focused attention on nonionic detergents, the polyethylene glycol monooleyl ether (PGME) series, and systematically studied the influence of two to 90 polyethylene glycol (PEG) lengths of PGMEs on the refolding of pig muscle lactate dehydrogenase (LDH), hen egg white lysozyme, and yeast α‐glucosidase. PGMEs with longer PEG lengths such as PGME20, 50, and 90 suppressed aggregation, and increased refolding yields. Notably, PGME20 increased the LDH yield to 56.7% from 2.5% without additives. According to the refolding kinetic analysis of LDH, compared with PGME50 and 90, the refolding rate constant in PGME20 solutions remained relatively high at a broad range of concentrations because of its weaker steric hindrance of intramolecular interactions involved in folding, leading to a preference for refolding over aggregation. These findings should provide basic guidelines to identify appropriate PEG‐based nonionic detergents for protein refolding.     

Affinity extraction of lactate dehydrogenase by aqueous two-phase systems using free triazine dyes

Affinity partitioning of lactate dehydrogenase (LDH) was studied in polyethylene glycol (PEG) /salt and PEG / hydroxypropyl starch (PES) aqueous two-phase systems, using free triazine dyes as their affinity ligands. The free dyes showed one-sided partition to the top PEG-rich phase and thus enhanced the affinity partitioning effect in the systems. A two-step affinity extraction process has been discussed for large scale purification of LDH from rabbit muscle.Hu Lin is one of the cooperator of the experiment.     

Effects of methocel A15LV, polyethylene glycol, and polyvinyl alcohol on CD13 and CD33 receptor surface content and metabolism of HL60 cells cultured in stirred tank bioreactors

Flow cytometry was used to examine the effect of hydrodynamic forces in a stirred tank bioreactor on the CD13 and CD33 receptor surface content of HL60 (human promyelocytic leukemia) cells. A step increase in agitation rate from 80 to 400 rpm reduced the HL60 cell apparent growth rate and increased the CD13 receptor surface content per cell, on average, by 95%. In contrast, this step increase in agitation rate to 400 rpm decreased the CD33 receptor surface content per cell, on average, by 10%. The protective effects of 0.1% Methocel A15LV, polyethylene glycol (PEG), and polyvinyl alcohol (PVA) on CD13 and CD33 receptor surface content were examined under agitation at 300 rpm in parallel 2 L bioreactor runs. The average CD33 receptor surface content was unaffected by the presence of Methocel A15LV or PEG, while PVA had a slight protective effect. In contrast, in terms of CD13 receptor content, HL60 cells agitated at 300 rpm with Methocel A15LV, PEG, or PVA behaved like cells agitated at 80 rpm with no media additives (McDowell and Papoutsakis, 1998). That is, Methocel A15LV, PEG, and PVA prevented the transduction of mechanical forces which affect CD13 cell content. HL60 cells cultured with 0.1% A15LV, PEG or PVA under conditions of mild agitation (60 rpm) in spinner flasks exhibited glucose consumption and lactate production rates that were approximately 20% lower than values of cultures containing no additive. Under conditions of agitation at 300 rpm in the 2 L bioreactor, the presence of A15LV, PEG, and PVA reduced the HL60 glucose consumption and lactate production rates by approximately 50%. Thus, media additives can dramatically reduce lactate accumulation in agitated bioreactors due to cell growth, in addition to providing protection from cellular injury.     

Lipase-catalyzed synthesis of oleic acid esters of polyethylene glycol 400

Summary Quantitative esterification of polyethylene glycol (PEG) 400 using oleic acid and Lipozyme was achieved in hexane. The effects of temperature, nature of acyl donor, substrate ratio, enzyme quantity and reaction time upon PEG esterification were examined. Nearly selective production of either PEG monooleate or PEG dioleate was achieved. Lipozyme was still 80% active after five reaction cycles.     

Stabilizing effect of amphiphilic excipients on the freeze-thawing and freeze-drying of lactate dehydrogenase

The effects of amphiphilic excipients on the inactivation of lactate dehydrogenase (LDH) during freeze-thawing and freeze-drying were studied. Some amphiphilic excipients such as hydroxypropyl-beta-cyclodextrin (HP-beta-CD), CHAPS, polyethylene glycol (PEG) 3350, and sucrose fatty acid monoester prevented LDH inactivation during freeze-thawing and freeze-drying at a lower concentration than sugars and amino acids. Polyoxyethylene 9 lauryl ether and PEG 400 protected LDH during freeze-thawing but not during freeze-drying. The buffer concentration of the solution to be freeze-dried (10, 50, and 200 mM) affected the stabilizing effect of trehalose, but not that of HP-beta-CD. (c) 1994 John Wiley & Sons, Inc.     

Molecular mechanism of polyethylene glycol mediated stabilization of protein

The effect of different molar ratios of polyethylene glycol (PEG) on the conformational stability of protein, bovine serum albumin (BSA), was studied. The binding of PEG with BSA was observed by fluorescence spectroscopy by measuring the fluorescence intensity after displacement of PEG with chromophore ANS and had further been confirmed by measuring the intrinsic fluorescence of tryptophan residues of BSA. Co-lyophilization of BSA with PEG at optimum BSA:PEG molar ratio led to the formation of the stable protein particles. Circular dichroism (CD) spectroscopy study suggested that a conformational change had occurred in the protein after PEG interaction and demonstrated the highest stability of protein at the optimum BSA:PEG molar ratio of 1:0.75. Additional differential scanning calorimetry (DSC) study suggested strong binding of PEG to protein leading to thermal stability at optimum molar ratio. Molecular mechanism operating behind the polyethylene glycol (PEG) mediated stabilization of the protein suggested that strong physical adsorption of PEG on the hydrophobic core of the protein (BSA) along with surface adsorption led to the stability of protein.     

Large scale, liquid phase oligonucleotide synthesis by alkyl H-phosphonate approach

A new approach to the liquid phase synthesis of oligonucleotide is described, it is based on oxidative coupling using alkyl H-phosphonate synthon and polyethylene glycol (PEG5000) as a soluble support. Nucleoside alkyl H-phosphonate undergoes oxidative coupling in presence of NBS. The use of polyethylene glycol as a soluble polymeric support preserves some convenient features of the solid phase synthesis with new interesting advantages. This liquid phase method appears effective in terms of speed and coupling yield and can be evaluated for the production of large amount of oligonucleotide (100 microM).     

PEG引发紫花苜蓿和沙打旺种子的生理生态效应

牧草种子田间出苗率低是我国草业生产亟待解决的技术问题。采用较高渗透势和短时间的聚乙二醇引发技术(-0 .6MPa,2 4h) ,研究了引发对紫花苜蓿 (Med icago sativa )和沙打旺 (Astragalus adsurgens)不同质量种子萌发的促进作用及其生理生态效应。结果表明引发可显著 (p<0 .0 5)提高种子的早期发芽率和发芽指数 ,缩短达 3 0 %出苗的天数 ,但对最终发芽率无显著影响。引发效果因植物种、种批质量和萌发条件不同而异 ;其中 ,沙打旺大于紫花苜蓿 ,质量中等或中等偏下种批大于质量高或质量低的种批 ,在低温和干旱逆境条件下萌发大于适宜条件萌发。引发种子的丙二醛含量和水浸电导率极显著 (p<0 .0 1)低于对照 ;引发回干 2 4h种子的水浸电导率与引发种子的相当。引发种子在萌发吸水 0～ 3 0 h的吸水量极显著高于对照 ,在 2 4～ 72 h的可溶性糖和脯氨酸含量分别显著和极显著高于对照     

Fermentation of polyethylene glycol via acetaldehyde in Pelobacter venetianus

Summary Pelobacter venetianus, a strictly anaerobic bacterium recently isolated with polyethylene glycol (PEG) as substrate, ferments PEG's with molecular masses of 106–40000, as well as acetoin, ethanolamine, choline, and ethoxyethanol, to acetate and ethanol. Ethylene glycol (EG) and acetaldehyde were fermented in the same manner at limiting concentrations in continuous culture. Growth with glycolaldehyde led to acetate as sole fermentation product. Acetaldehyde appeared as byproduct of PEG fermentation, and accumulated to high concentrations during degradation of PEG 4000 and PEG 6000. Utilization of PEG's was constitutive, whereas acetoin degradation was inducible. Acetaldehyde was shown to be the primary product of EG degradation, and inhibited utilization of other substrates. Enzymes involved in the fermentation of PEG, EG, acetoin, and glycolaldehyde were demonstrated in cell-free extracts, except for the PEG degrading enzyme and EG dehydrase. These results demonstrate that acetaldehyde plays a central role in the metabolism of Pelobacter venetianus. A scheme of intermediary metabolism and PEG degradation is discussed.Abbreviations EG ethylene glycol - Di-EG diethylene glycol - PEG (20 000) polyethylene glycol (molecular weight 20 000)     

Factors affecting recombinant frequency in protoplast fusions of Streptomyces coelicolor.

The optimum concentration of polyethylene glycol 1000 (PEG) for the production of recombinants through protoplast fusion in Streptomyces coelicolor was about 50% (w/v). The addition of 14% (v/v) dimethyl sulphoxide to the fusion mixture enhanced recombination frequencies, but only at sub-optimal PEG concentrations. After treatment of protoplasts with 50% PEG for 1 min, the frequency of recombinants in a multi-factor 'cross' sometimes exceeded 20% of the total progeny. The frequency of recombinants in the progeny could be significantly enhanced by ultraviolet irradiation of the parental protoplast suspensions immediately before fusion.     

Enhancement of enzymatic production of cyclodextrins by adding polyethylene glycol or polypropylene glycol

Enzymatic production of cyclodextrins (CDs) from soluble starch was studied using either Bacillus macerans or Bacillus ohbensis cyclomaltodextrin glucanotransferase (CGTase). The production yield of CDs was found to be increased up to 1.5–2 times by the addition of low molecular weight polyethylene glycol (PEG 400) or polypropylene glycol (PPG 425) to the reaction medium. Such results were interpreted as being due to a conformational change of the substrate as well as reduction of hydrolytic activity of the enzyme in the presence of these additives.     

Effects of carbohydrate source, polyethylene glycol and gellan gum concentration on embryonal-suspensor mass (ESM) proliferation and maturation of maritime pine somatic embryos

Summary The influence of carbon sources and polyethylene glycol combined with 0.45 and 0.9% (w/v) of gellan gum on the maturation of maritime pine somatic embryos was tested. The effect of the carbon source and polyethylene glycol varied widely between lines. One out of the five lines tested showed a striking response to polyethylene glycol (PEG) treatment; the addition of this osmoticum limited the embryonal-suspensor mass (ESM) proliferation while it enhanced the maturation rate. Conversely, the ESM proliferation was stimulated by PEG in the other lines without subsequent improvement of the maturation rate. The use of a high concentration of gellan gum (0.9%) improved the maturation of the five ESM lines. It was concluded that the most efficient culture medium to recover cotyledonary embryos from all lines is one supplemented with sucrose at 6% (w/v) and gellan gum at 0.9% (w/v) without PEG. The determining factor in the maturation of maritime pine somatic embryos is the genotype and/or the quality of ESM. The possible relationship between maturation performances and ESM morphology, particularly the suspensor organization, is discussed.     

Refolding of lactate dehydrogenase by zeolite beta

We used zeolite beta as an adsorbing matrix to refold recombinant lactate dehydrogenase (LDH) protein collected as an insoluble aggregate from a bacterial expression system. The adsorption isotherm revealed that 1 g of zeolite adsorbed 200 mg of denatured LDH solubilized with a buffer containing 6 M of guanidine hydrochloride. The pH of the buffer had little effect on the adsorption, but this property was abolished by preincubation of the zeolite with polyethylene glycol (PEG) in a weight ratio of 1:10. These data suggest that the adsorption of LDH depends on the hydrophobicity of the zeolite surface, and that the adsorption of PEG to zeolite is sufficient to release LDH from its surface. LDH was thus released by refolding buffer containing PEG and arginine, and soluble LDH was obtained in its active enzymatic form. The addition of arginine dramatically increased the yield of LDH in a dose‐dependent manner. The overall refolding efficiency was optimized to 35%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Shifting product formation from xylitol to ethanol in pentose fermentations using Candida tropicalis by adding polyethylene glycol (PEG)

Summary When Candida tropicalis fermented xylose under oxygen limited conditions in the presence of increasing concentrations of polyethylene glycol (PEG), the ethanol production increased by a factor of two and the xylitol production was repressed by about 25%. Xylose assimilation and cell growth were not affected by the presence of PEG. The fermentation of glucose was not as strongly influenced by the presence of PEG as were xylose fermentations. The results are discussed in relation to the physico-chemical properties of a medium containing increasing concentrations of PEG. It is suggested that the presence of PEG might result in a fine-tuning of the teration in the medium, necessary for ethanol production from xylose with Candida tropicalis.     

Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10–14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.Abbreviations EG ethylene glycol - DiEG diethylene glycol - TriEG triethylene glycol - TeEG tetraethylene glycol - PEG polyethylene glycol (molecular mass indicated)     

Controlled concealment of exposed clearance and immunogenic domains by site-specific polyethylene glycol attachment to acetylcholinesterase hypolysine mutants

Cholinesterases are efficient scavengers of organophosphates and are currently being developed as drugs for treatment against poisoning by such compounds. Recombinant ChE bioscavengers have very short circular longevity, a limitation that can be overcome by complex post-translation manipulations or by chemical modification such as polyethylene glycol conjugation. Series of multiple Lys-Ala mutants of human acetylcholinesterase were prepared allowing the generation of homogenous and well defined polyethylene-glycol conjugated AChEs with either one, two, three, four, or five appended polyethylene glycol (PEG) moieties/molecule. The rank order of circulatory longevity of these molecules was dependent on the number of PEG appendages up to a certain threshold: 5 = 4 > 3 > 2 > 1 > 0. Hypolysine acetylcholinesterases (AChEs) carrying the same number of PEGs, and therefore with identical masses, allowed us to demonstrate that circulatory longevity correlates with the predicted extent of concealment of the AChE surface. Furthermore, circulatory profiles of high number and low number PEG-AChEs differing in their sialic acid contents demonstrate a direct relationship between PEG loading and the effective seclusion of AChE from the hepatic asialoglycoprotein receptor clearance system. Finally, an inverse relationship is found between the extent of PEG loading and the ability of the human acetylcholinesterase to elicit specific anti-HuAChE antibodies. In conclusion, these findings suggest that for the extension of circulatory longevity, protein surface domain concealment exerted by polyethylene glycol attachment is at least as important as its effect on size enlargement and highlights the role of PEG attachment in masking interactions between biomolecules and their cognate receptors.     

Bacterial oxidation of polyethylene glycol.

The metabolism of polyethylene glycol (PEG) was investigated with a synergistic, mixed culture of Flavobacterium and Pseudomonas species, which are individually unable to utilize PEGs. The PEG dehydrogenase linked with 2,6-dichlorophenolindophenol was found in the particulate fraction of sonic extracts and catalyzed the formation of a 2,4-dinitrophenylhydrazine-positive compound, possibly an an aldehyde. The enzyme has a wide substrate specificity towards PEGs: from diethylene glycol to PEG 20,000 Km values for tetraethylene glycol (TEG), PEG 400, and PEG 6,000 were 11, 1.7, and 15 mM, respectively. The metabolic products formed from TEG by intact cells were isolated and identified by combined gas chromatography-mass spectrometry as triethylene glycol and TEG-monocarboxylic acid plus small amounts of TEG-dicarboxylic acid, diethylene glycol, and ethylene glycol. From these enzymatic and analytical data, the following metabolic pathway was proposed for PEG: HO(CH2CH2O)nCH2CH2OH leads to HO(CH2CH2O)nCH2CHO leads to HO(CH2CH2O)nCH2COOH leads to HO(CH2CH2O)n-1CH2CH2OH.     

A simple procedure to overcome polyethelene glycol toxicity on whole plants

A procedure is described that can be used to minimize toxic effects of polyethylene glycol (PEG) to plants. The procedure is based on recycling nutrient solutions containing PEG-6000 through two plant cultures. Tomato plants grown in −0.3 megapascals PEG solutions used after two growth cycles exhibited minimal toxic effects. Long-term responses like dry matter production and chlorophyll content as well as short-term responses like CO₂ fixation rates and leaf conductance were severely inhibited by fresh PEG-6000 and only slightly reduced by recycled PEG-6000. Complete osmotic adjustment was obtained with tomatoes grown in recycled but not in fresh PEG solutions.     

Effects of Drought Stress Induced by Polyethylene Glycol on Pigment Content and Photosynthetic Gas Exchange of Pistacia Khinjuk and P. Mutica

Photosynthetica - The effects of drought stress induced by polyethylene glycol, PEG (molecular mass 6000) on some ecophysiological characteristics of two wild pistachio species, Mastic and Khinjuk...     

Effects of sodium chloride and polyethylene glycol on root-hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum

The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium leguminosarum strain 29d and root hairs of field bean (Vicia faba L. cv. Maris Bead) plants were investigated. Two levels each of NaCl (50 and 100 mol·m^–3) and PEG (100 and 200 mol·m^–3) were given at the time of root-hair formation. Scanning electron microscopy showed rhizobial attachment and colonization on root-hair tips. Adhesion of rhizobia in both lateral and polar orientation, sometimes associated with microfibrils, occurred mainly in crooks at the root-hair tips; most of the infections also occurred here. Bacterial colonization and root-hair curling were both reduced by stress treatments. Polyethylene glycol but not NaCl significantly reduced root-hair diameter. The proportion of root hairs containing infection threads was reduced by 30% under NaCl and by 52% under PEG. The structure of some of the root hairs, epidermal and hypodermal cells, as seen by light microscopy in ultrasections, was distorted as a result of NaCl and PEG treatments; cells showed plasmolysis and folded membranes. After three weeks of treatment, both NaCl and PEG inhibited nodule number by about 50% and nodule weight by more than 60%. It is concluded that the root-hair infection process in Vicia faba is impaired by NaCl and PEG treatments and this in turn results in fewer nodules being produced.Abbreviation PEG polyethylene glycol