Single step protocol to purify recombinant proteins with low endotoxin contents

Endotoxin is an unwanted by product of recombinant proteins purified from Escherichia coli. The inherent toxicity of endotoxins makes their removal an important step for the proteins' application in several biological assays and for safe parenteral administration. The method described in this paper is a one-step protocol which is effective at removing tightly bound endotoxin from over-expressed tagged proteins in E. coli. We combined affinity chromatography with a non-ionic detergent washing step, to remove most of the endotoxin contaminants from the end product. An endotoxin reduction of less than 4 to 0.2 EU mg(-1) was achieved with protein recovery close to a yield 100%. As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense.     

Endotoxin removal using a synthetic adsorbent of crystalline calcium silicate hydrate

A synthetic adsorbent of crystalline calcium silicate hydrate, the product LRA by Advanced Minerals Corp., has been studied for endotoxin removal from aqueous solutions. This adsorbent removes endotoxin effectively, and the removal is greatly enhanced by the presence of an electrolyte such as NaCl, Tris-HCl, or Na2HPO4. It has an endotoxin removal capacity as high as 6 million endotoxin units (EU) per gram. Its endotoxin removal kinetics is fast, and for instance, over 99.9% endotoxin in a 5000 EU/mL solution was removed by mixing for 2 min at an adsorbent usage of 10 g/L. Using the chromatographic column method to treat a 5000 EU/mL solution, an endotoxin log-reduction factor of 6.2 was achieved with a single pass. This adsorbent also demonstrated significantly better performance when compared to many commonly used endotoxin removal agents, such as ActiClean Etox Endotoxin Removal Resin, Affi-Prep Polymyxin Support, Detroxi-Gel Endotoxin Removing Gel, Q Sepharose Fast Flow Media, and Sigma Endotoxin Removal Solution. Furthermore, it demonstrated a high selective removal of endotoxin from a solution of lambda DNA. This adsorbent provides opportunities for developing disposable, scaleable, and cost-effective methods for endotoxin reduction in many biotechnological and pharmaceutical processes.     

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Optimization of the Endotoxin Removal Performance of Solid-Phase Conjugated S3E3 Antimicrobial Peptide Using Response Surface Methodology

S3E3 is a new variant of S3 antimicrobial peptide (AMP) derived from factor C of horseshoe hemolymph and features a high binding affinity for endotoxin. In this research, site-specific conjugated S3E3 AMP onto Sepharose 6% solid phase support (S3E3-S-Sepharose) was applied for endotoxin removal from protein solutions. The bovine serum albumin (BSA) was chosen as a protein model due to its acidic-sticky nature interfering with the endotoxin removal process. The batch process parameters including, endotoxin concentration, pH, and ionic strength of the sample were optimized by response surface methodology to reach maximum endotoxin binding capacity and protein recovery. The predicted optimal conditions for enhanced endotoxin removal performance were as follows: pH 4.5, 25 mM NaCl, and 68,500 EU/ml endotoxin leading to a maximum endotoxin binding capacity of 3.114?×?10⁺⁶ EU/ml of resin and a 95.89% protein recovery. S3E3-S-Sepharose could be applied as an efficient endotoxin removal affinity chromatography matrix at downstream processes of recombinant therapeutics due to its high capacity and protein recovery.

     

Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19–405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni–NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 μg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 μg/ml.     

Removal of Endotoxin during Purification of Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30°C was higher than 10⁴ EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev

Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins’ application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.     

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p<0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.     

Membrane cartridges for endotoxin removal from interferon preparations

The suitability of membrane cartridges for the removal of endotoxin from both distilled water and interferon preparations was examined. The endotoxin concentrations were reduced to 4.0 and 7.3 EU/ml, respectively, when about 4000 ml of distilled water with 20 and 28 EU/ml were passed through the deoxycholate and chitosan immobilized membrane cartridges. When 200 ml of interferon preparation with endotoxin concentration more than 80 EU/ml and pH 3.9 were applied to a deoxycholate immobilized membrane cartridge at a flow-rate of 9 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml. However, if an interferon preparation of 450 ml, with more than 80 EU/ml of endotoxin and pH 3.9 was applied to the chitosan immobilized membrane cartridge at a flow-rate of 18 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml.     

Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump

A parallel chromatographic procedure for the purification of milligram amounts of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quarternary ammonium group with respect to elution and regeneration properties and the 75 cm(2) Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimised. By introducing this step into the protocol, endotoxin levels could be reduced approximately 100-fold to 相似文献   

亲和介质及溶液条件对蛋白质溶液中内毒素去除的影响

生物制品中内毒素的去除是一项十分重要的工作。为了更好地去除各种生物制品中的内毒素,采用合成的多粘菌素B琼脂糖亲和介质,通过静态吸附的方法去除蛋白质溶液中的内毒素。重点考察了介质的间臂长度、配基密度以及各种溶液条件(pH值、盐种类和浓度、蛋白质种类和浓度、内毒素浓度、添加剂等)对内毒素去除率及蛋白质回收率的影响。分别采用动态浊度法和Lowry法检测内毒素含量和蛋白质浓度。结果表明该介质具有载量高、去除速度快、去除率高、可重复使用的特点。此外,配基密度、pH值、盐浓度和蛋白质特性(等电点和疏水性)对内毒素去除效果均有重要影响。在优化的条件下,血红蛋白、人血清白蛋白和溶菌酶的回收率分别达到87.2%、73.4%和97.3%,相应的内毒素去除率分别达到99.8%、97.9%和99.7%。阐明了各种因素对内毒素去除率和蛋白质回收率的影响规律,为生物制品中内毒素的高效去除提供了参考。     

Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study.

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.     

Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.     

Tritiation of endotoxin.

Tritiated endotoxin was synthesized by three different methods: (1) sodium boro[3H]hydride reduction of native endotoxin; (2) sodium boro[3H]hydride reduction of endotoxin that had been oxidized previously with sodium metaperiodate; and (3) exposure of dry endotoxin to 3H2 gas. Sodium borohydride reduces aldehyde groups and sodium metaperiodate oxidizes vicinal glycol groups to aldehydes. Chromatographic analysis of the three tritiated endotoxins, using agarose, revealed that the biological activity associated with each labeled product appeared at the void volume, and in each case the biological activity coincided with a peak in radioactivity. The labeled product of the first method had a specific radioactivity of 0.18 mCi/g and a biological activity equal to that of native endotoxin. The labeled products of the second and third methods had specific activities of 2.1 mCi/g and 60.0 mCi/g, respectively, while their biological activities were one hundred-fold less than native endotoxin, as determined by the Limulus amebocyte lysate assay. These three labeled endotoxins are potentially ueled endotoxin.     

Tightly bound calcium of adenosine triphosphatase in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcoplasmic reticulum vesicles were shown to possess a class of tightly bound calcium ions, inaccessible to the chelator, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid at 0 degrees C or 25 degrees C, amounting to 4.5 nmol/mg of protein (approximately 0.5 mol/mol (Ca2+,Mg2+)-ATPase). The calcium ionophores, A23187 and X537A, induced rapid exchange of tightly bound calcium in the presence of chelator. Chelator alone at 37 degrees C, caused irreversible loss of bound calcium, which correlated with uncoupling of transport from (Ca2+,Mg2+)-ATPase activity. Uncoupling was not accompanied by increased permeability to [14C]inulin. Slow exchange of tightly bound calcium with medium calcium was unaffected by turnover of the ATPase or by tryptic cleavage into 55,000- and 45,000-dalton fragments. Binding studies with labeled calcium suggested that tight binding involves a two-step process: Ca2+ + E in equilibrium K E . Ca2+ leads to E < Ca2+ where E and < Ca2+ represent the ATPase and tightly bound calcium, and K = 1.6 X 10(3) M-1. It is suggested that tightly bound calcium is located in a hydrophobic pocket in, or in close proximity to the ATPase, and, together with tightly bound adenine nucleotides (Aderem, A., McIntosh, D. B., and Berman, M. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3622-03632), is related to the ability of the ATPase to couple hydrolysis of ATP to vectorial transfer of calcium across the membrane.     

单克隆抗体纯化过程中内毒素去除方法分析

目的：分析单克隆抗体纯化过程中,去除内毒素的不同方法的应用效果,并探讨它们应用于中试规模的可行性。方法与结果：比较了多聚赖氨酸型的内毒素去除填料、20nm膜过滤、将单抗附着在蛋白A柱上后使用精氨酸和组氨酸溶液冲洗等3种方法的内毒素去除效果,发现3种方法都可以将内毒素水平大幅降低,可分别将内毒素去除70％、88％和97％。因为单抗分子等电点较高,所获得的最低内毒素含量为0．2～0-3EU／mg。结论：3种方法均具有一定的工艺放大潜力,进一步提高内毒素去除效果将需要综合使用不同机理的去除技术。     

Plasma endotoxin level of healthy donors

The plasma level of endotoxin was determined in 116 healthy blood donors. After a routine physical and laboratory investigations the endotoxin level was determined with Limulus amebocyte lysate assay (LAL-test) by the chromogenic kinetic method of Bio-Whittaker Co. (USA). Its sensitivity was 0.005-50 EU/ml. The plasma level of endotoxin in most of the healthy donors was less than 1 EU/ml (in the range of 0.01-1.0 EU/ml), but always measurable. The average +/- S.D. was 0.128 +/- 0.215 EU/ml. Because of the high standard deviation and high range of values, the data were distributed into two groups with the means of 0.05 +/- 0.022 EU/ml and 0.294 +/- 0.186 EU/ml. The difference between the groups was significant (p < 0.001). In conclusion, endotoxin can be measured in plasma of healthy individuals.     

Inhibition of uracil DNA glycosylase by an oxacarbenium ion mimic

We have investigated the inhibition of the DNA repair enzyme uracil DNA glycosylase (UDG) by an 11-mer oligonucleotide (AIA) containing a cationic 1-aza-deoxyribose (I) residue designed to be a stable mimic of the high-energy oxacarbenium ion reaction intermediate [Werner, R. M., and Stivers, J. T. (2000) Biochemistry 39, 14054-14064]. Inhibition kinetics and direct binding studies indicate that AIA binds weakly to the free enzyme (K(D) = 2 microM) but binds 4000-fold more tightly to the enzyme-uracil anion (EU) product complex (K(D) = 500 pM). The importance of the positive charge on the 1-nitrogen in binding is established by the observation that AIA binds >30 000-fold more tightly to the EU complex than the corresponding neutral tetrahydrofuran (F) abasic site product analogue (AFA). The unusual inhibition mechanism for AIA results in a time dependence that resembles slow-onset inhibition even though the apparent on-rate of the inhibitor for the EU(-) binary product complex is moderate (1 microM(-1) x s(-1)). Accordingly, the low K(D) of AIA for the EU complex is largely due its very slow off-rate (5 x 10(-4) x s(-1)). These results support previous kinetic isotope effect measurements that indicate UDG stabilizes a discrete oxacarbenium ion-uracil anion intermediate. This oxacarbenium ion mimic represents the tightest binding inhibitor of UDG yet identified.     

Probing the limits of electrostatic catalysis by uracil DNA glycosylase using transition state mimicry and mutagenesis

The DNA repair enzyme uracil DNA glycosylase (UDG) hydrolyzes the glycosidic bond of deoxyuridine in DNA by a remarkable mechanism involving formation of a positively charged oxacarbenium ion-uracil anion intermediate. We have proposed that the positively charged intermediate is stabilized by being sandwiched between the combined negative charges of the anionic uracil leaving group and a conserved aspartate residue that are located on opposite faces of the sugar ring. Here we establish that a duplex DNA oligonucleotide containing a cationic 1-aza-deoxyribose (I) oxacarbenium ion mimic is a potent inhibitor of UDG that binds tightly to the enzyme-uracil anion (EU(-)) product complex (K(D) of EU(-) = 110 pm). The tight binding of I to the EU(-) complex results from its extremely slow off rate (k(off) = 0.0008 s(-1)), which is 25,000-fold slower than substrate analogue DNA. Removal of Asp(64) and His(187), which are involved in stabilization of the cationic sugar and the anionic uracil leaving group, respectively, specifically weakens binding of I to the UDG-uracil complex by 154,000-fold, without significantly affecting substrate or product binding. These results suggest that electrostatic effects can effectively stabilize such an intermediate by at least -7 kcal/mol, without leading to anticatalytic stabilization of the substrate and products.     

Isolation and characterisation of a functional alpha beta heterodimer from the ATP synthase of Rhodospirillum rubrum.

An alpha beta heterodimer of the F1-ATPase of Rhodospirillum rubrum was isolated by extraction of chromatophores with LiCl. Each alpha beta heterodimer contains one tightly bound ADP, which is released upon removal of medium Mg2+. The dimer can be reversibly dissociated by removal of Mg(2+)-ions. The alpha beta heterodimer restores both ATP-synthetic and -hydrolytic activities to LiCl-treated chromatophores, saturation being achieved at approximately 2 mmol alpha beta.mol BChl-1. The heterodimer itself hydrolyses Mg-ATP with an activity distinct from RF1, being unaffected by azide or sulphite ions. The Vmax and Km (ATP) for this Mg(2+)-dependent activity were 110 +/- 10 nmol.min-1.mg protein-1 and 100 +/- 30 microM, respectively. The Km did not differ significantly from that of RF1.