Protein recovery from surfactant precipitation

The recovery of lysozyme from an aqueous solution containing precipitated lysozyme-AOT complexes formed by the direct addition of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) to a lysozyme solution was studied using both solvents, and a counterionic surfactant. Ethanol,methanol and solvent mixtures dissolved the surfactant precipitate and recovered lysozyme as a solid. Recovery efficiency and protein stability varied with the type of solvent used. An entirely different method of recovery was also evaluated using a counterionic surfactant: tri-octylmethylammonium chloride (TOMAC) which bound to AOT releasing lysozyme into solution.Complete recovery (100%) of lysozyme was achieved at a molar ratio of 2:1(TOMAC:AOT), and the original protein activity was maintained in the final aqueous phase.The recovered lysozyme retained its secondary structure as observed in circular dichroism(CD) spectra. Specific activity studies show that counterionic surfactant extraction does not alter the biological activity of the enzyme.     

Continuous capture of recombinant antibodies by ZnCl2 precipitation without polyethylene glycol

The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation‐based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl₂. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co‐precipitation agent. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid–liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl_2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.     

DNA purification by triple-helix affinity precipitation

Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)(7)), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.     

微生物成因的碳酸盐矿物研究进展

微生物诱导碳酸盐类矿物沉淀是地质微生物学的研究热点之一.微生物主要通过其代谢活动促进细胞周围微环境pH值及水体[CO32-]的升高,最终表现为碳酸盐类矿物饱和指数的增加.此外,微生物及其分泌的胞外聚合物可作为碳酸盐晶核的成核位点,为碳酸盐矿物晶体的生长进一步提供有利条件.微生物成因与纯化学成因的碳酸盐类矿物相比具有不同的矿物特征(如形貌、微量元素含量及碳同位素等).深入了解微生物诱导碳酸钙沉淀的行为对理解地质时期微生物活动及其在二氧化碳的地质封存中的潜在应用具有指导意义.本文综述了微生物诱导碳酸盐矿物沉淀的机理、代谢过程,总结了该领域的最新进展,探讨了生物成因以及化学成因碳酸盐矿物的区别,最后指出了该项研究在微生物岩以及CO2地质封存上的一些可能的拓展方向.     

In situ magnetic separation for extracellular protein production

A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production.     

Molecular confinement influences protein structure and enhances thermal protein stability

The sol-gel method of encapsulating proteins in a silica matrix was investigated as a potential experimental system for testing the effects of molecular confinement on the structure and stability of proteins. We demonstrate that silica entrapment (1) is fully compatible with structure analysis by circular dichroism, (2) allows conformational studies in contact with solvents that would otherwise promote aggregation in solution, and (3) generally enhances thermal protein stability. Lysozyme, alpha-lactalbumin, and metmyoglobin retained native-like solution structures following sol-gel encapsulation, but apomyoglobin was found to be largely unfolded within the silica matrix under control buffer conditions. The secondary structure of encapsulated apomyoglobin was unaltered by changes in pH and ionic strength of KCl. Intriguingly, the addition of other neutral salts resulted in an increase in the alpha-helical content of encapsulated apomyoglobin in accordance with the Hofmeister ion series. We hypothesize that protein conformation is influenced directly by the properties of confined water in the pores of the silica. Further work is needed to differentiate the steric effects of the silica matrix from the solvent effects of confined water on protein structure and to determine the extent to which this experimental system mimics the effects of crowding and confinement on the function of macromolecules in vivo.     

Some characteristics of protein precipitation by salts

The solubilities of lysozyme, alpha-chymotrypsin and bovine serum albumin (BSA) were studied in aqueous electrolyte solution as a function of ionic strength, pH, the chemical nature of salt, and initial protein concentration. Compositions were measured for both the supernatant phase and the precipitate phase at 25 degrees C. Salts studied were sodium chloride, sodium sulfate, and sodium phosphate. For lysozyme, protein concentrations in supernatant and precipitate phases are independent of the initial protein concentration; solubility can be represented by the Cohn salting-out equation. Lysozyme has a minimum solubility around pH 10, close to its isoelectric point (pH 10.5). The effectiveness of the three salts studied for precipitation were in the sequence sulfate > phosphate > chloride, consistent with the Hofmeister series. However, for alpha-chymotrypsin and BSA, initial protein concentration affects the apparent equillibrium solubility. For these proteins, experimental results show that the compositions of the precipitate phase are also affected by the initial protein concentration. We define a distribution coefficient kappa(e) to represent the equilibrium ratio of the protein concentration in the supernatant phase to that in the precipitate phase. When the salt concentration is constant, the results show that, for lysozyme, the protein concentrations in both phases are independent of the initial protein concentrations, and thus kappa(e) is a constant. For alpha-chymotrypsin and BSA, their concentrations in both phases are nearly proportional to the initial protein concentrations, and therefore, for each protein, at constant salt concentration, the distribution coefficient kappa(e) is independent of the initial protein concentration. However, for both lysozyme and alpha-chymotrypsin, the distribution coefficient falls with increasing salt concentration. These results indicate that care must be used in the definition of solubility. Solubility is appropriate when the precipitate phase is pure, but when it is not, the distribution coefficient better describes the phase behavior. (c) 1992 John Wiley & Sons, Inc.     

Rational design of a zinc phthalocyanine binding protein

Phthalocyanines have long been used as primary donor molecules in synthetic light-powered devices due to their superior properties when compared to natural light activated molecules such as chlorophylls. Their use in biological contexts, however, has been severely restricted due to their high degree of self-association, and its attendant photoquenching, in aqueous environments. To this end we report the rational redesign of a de novo four helix bundle di-heme binding protein into a heme and Zinc(II) phthalocyanine (ZnPc) dyad in which the ZnPc is electronically and photonically isolated. The redesign required transformation of the homodimeric protein into a single chain four helix bundle and the addition of a negatively charge sulfonate ion to the ZnPc macrocycle. To explore the role of topology on ZnPc binding two constructs were made and the resulting differences in affinity can be explained by steric interference of the newly added connecting loop. Singular binding of ZnPc was verified by absorption, fluorescence, and magnetic circular dichroism spectroscopy. The engineering guidelines determined here, which enable the simple insertion of a monomeric ZnPc binding site into an artificial helical bundle, are a robust starting point for the creation of functional photoactive nanodevices.     

Role of adipose differentiation-related protein in lung surfactant production: a reassessment

Based on data developed with the use of isolated lipid droplets from neonatal rat lung lipofibroblasts, we speculated previously that the droplet coat protein, adipose differentiation-related protein (ADFP), mediated the transfer of lipids into type 2 lung epithelial cells for the production of surfactant phospholipids. The present studies were designed to test the role of ADFP in this transfer with the use of ADFP-coated lipid droplets from CHO fibroblast cells and a cultured human lung epithelial cell line. We found no role for ADFP in the lipid transfer and conclude that a lipase associated with the lipid droplets hydrolyzes their core triacylglycerols, releasing fatty acids that are taken up by the epithelial cells.     

Efficient isolation and elution of cellular proteins using aptamer-mediated protein precipitation assay

Protein precipitation is one of the most widely used methods for antigen detection and purification in biological research. We developed a reproducible aptamer-mediated magnetic protein precipitation method that is able to efficiently capture, purify and isolate the target proteins. We discovered DNA aptamers having individually high affinity and specificity against human epidermal growth factor receptor (EGFR) and human insulin receptor (INSR). Using aptamers and magnetic beads, we showed it is highly efficient technique to enrich endogenous proteins complex and is applicable to identify physiologically relevant protein–protein interactions with minimized nonspecific binding of proteins. The results presented here indicate that aptamers would be applicable as a useful and cost-effective tool to identify the presence of the particular target protein with their specific protein partners.     

Purification of an acidic recombinant protein from transgenic tobacco

Tobacco has proven to be a promising alternative for the production of recombinant therapeutic proteins and offers numerous advantages over other plants as a host system. However, the recovery and purification steps needed to obtain a protein at high recovery and purity have not been well investigated. In this study, a process was developed to purify a model acidic protein, recombinant beta-glucuronidase (rGUS) from transgenic tobacco leaf tissue, in three main steps after extraction: polyelectrolyte precipitation, hydrophobic interaction chromatography (HIC), and hydroxyapatite chromatography (HAC). Using this three-step process, up to 40% of the initial rGUS activity could be recovered to near homogeneity as judged by SDS-PAGE. This work demonstrates that acidic recombinant proteins expressed in tobacco may be purified to high yield with high purity in a minimal amount of steps that are suitable for scale-up. Furthermore, the general steps used in this process may suggest that a wide variety of acidic recombinant proteins may be purified in a similar manner from transgenic tobacco or other leafy crops.     

Corynebacterium sarcosine oxidase: a unique enzyme having covalently-bound and noncovalently-bound flavins

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC, 1.5.3.1) was purified to homogeneity from Corynebacterium sp. U-96 by the use of ionic exchange chromatographies and gel filtrations. The enzyme contained two mol FAD per mol enzyme (one covalently-bound and one noncovalently-bound; mol. wt., 174,000). The “semiapoenzyme”, which contains the covalently-bound FAD alone, was prepared by the acid-ammonium sulfate treatment. The semiapoenzyme had practically no activity for sarcosine oxidation, but retained intact back-bone structure judging from the circular dichroic spectrum in the far ultraviolet region. On the contrary, the circular dichroic spectrum of the semiapoenzyme in the visible region (a large negative band around 443 nm) was quite distinct from that of the holoenzyme (positive bands at 387, 456 and 489 nm).     

Chromatographic capture of cells to achieve single stage clarification in recombinant protein purification

Recent advancements in cell culture engineering have allowed drug manufacturers to achieve higher productivity by driving higher product titers through cell line engineering and high-cell densities. However, these advancements have shifted the burden to clarification and downstream processing where the difficulties now revolve around removing higher levels of process- and product-related impurities. As a result, a lot of research efforts have turned to developing new approaches and technologies or process optimization to still deliver high quality biological products while controlling cost of goods. Here, we explored the impact of a novel single use technology employing chromatographic principle-based clarification for a process-intensified cell line technology. In this study, a 16% economic benefit ($/g) was observed using a single-use chromatographic clarification compared to traditional single-use clarification technology by improving the overall product cost through decreased operational complexity, higher loading capacity, increased product recovery, and higher impurity clearance. In the end, the described novel chromatographic approach significantly simplified and enhanced the cell culture fluid harvest unit operation by combining the reduction of insoluble and key soluble contaminants of the harvest fluid into a single stage.     

FtsZ polymer-bundling by the Escherichia coli ZapA orthologue, YgfE, involves a conformational change in bound GTP

Cell division is a fundamental process for both eukaryotic and prokaryotic cells. In bacteria, cell division is driven by a dynamic, ring-shaped, cytoskeletal element (the Z-ring) made up of polymers of the tubulin-like protein FtsZ. It is thought that lateral associations between FtsZ polymers are important for function of the Z-ring in vivo, and that these interactions are regulated by accessory cell division proteins such as ZipA, EzrA and ZapA. We demonstrate that the putative Escherichia coli ZapA orthologue, YgfE, exists in a dimer/tetramer equilibrium in solution, binds to FtsZ polymers, strongly promotes FtsZ polymer bundling and is a potent inhibitor of the FtsZ GTPase activity. We use linear dichroism, a technique that allows structure analysis of molecules within linear polymers, to reveal a specific conformational change in GTP bound to FtsZ polymers, upon bundling by YgfE. We show that the consequences of FtsZ polymer bundling by YgfE and divalent cations are very similar in terms of GTPase activity, bundle morphology and GTP orientation and therefore propose that this conformational change in bound GTP reveals a general mechanism of FtsZ bundling.     

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC–MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm × 50 mm, 3 μm) using a mobile phase of ACN/H₂O (80/20, v/v) containing 10 mM NH₄Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408 → 235 for sitagliptin and m/z 412 → 239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 μL of plasma is processed. The linear calibration range is 1–1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n = 6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.     

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.     

Conformation of the abortifacient protein pinellin: A circular dichroic study

The conformation of pinellin was studied by circular dichroism, which showed a minimum at 223 nm and a double maximum at 198–200 nm. The protein was rich in -sheet (about 40%) with little -helix, based on current CD analyses. It was stable betweenpH 4 and 10 beyond which it unfolded reversibly, but in alkaline solution, prolongly stored at, say,pH 12, it became irreversibly denatured. Thermal denaturation indicated a transition between 55° and 68°C; the solution at 80°C was partially renatured upon air-cooling back to room temperature. Addition of sodium dodecyl sulfate caused a sharp increase in -helix, which leveled off at 0.25 mM surfactant.     

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.     

Folding studies on a knotted protein

YibK is a 160 residue homodimeric protein belonging to the SPOUT class of methyltransferases. Proteins in this group all display a unique topological feature; the backbone polypeptide chain folds to form a deep trefoil knot. Such knotted structures were completely unpredicted, it being thought impossible for a protein to fold efficiently in this way. However, they are becoming more common and there are now a growing number of examples in the Protein Data Bank. These intriguing knotted structures represent a new and significant challenge in the field of protein folding. Here, we present an initial characterisation of the folding of YibK, one of the smallest knotted proteins to be identified. This is the first detailed folding study on a knotted protein to be reported. We have established conditions under which the protein can be denatured reversibly in vitro using urea, thereby showing that molecular chaperones are not required for the efficient folding of this protein. A series of equilibrium unfolding experiments were performed over a 400-fold range of protein concentration. Both secondary and tertiary structural probes show a single, protein concentration-dependent unfolding transition, and data are most consistent with a three-state equilibrium denaturation model involving a monomeric intermediate. Thermodynamic parameters obtained from the fit of the data to this model indicate that the intermediate is a stable species with appreciable secondary and tertiary structure; whether the topological knot remains in the intermediate state is still to be shown. Together, these results demonstrate that, despite its complex knotted structure, YibK is able to fold efficiently and behaves remarkably similarly to other dimeric proteins under equilibrium conditions.