Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.     

Detachment of multi species biofilm in circulating fluidized bed bioreactor

In this study, the detachment rates of various microbial species from the aerobic and anoxic biofilms in a circulating fluidized bed bioreactor (CFBB) with two entirely separate aerobic and anoxic beds were investigated. Overall detachment rate coefficients for biomass, determined on the basis of volatile suspended solids (VSS), glucose and protein as well as for specific microbial groups, i.e., for nitrifiers, denitrifiers, and phosphorous accumulating organisms (PAOs), were established. Biomass detachment rates were found to increase with biomass attachment on carrier media in both beds. The detachment rate coefficients based on VSS were significantly affected by shear stress, whereas for protein, glucose and specific microbial groups, no significant effect of shear stress was observed. High detachment rates were observed for the more porous biofilm structure. The presence of nitrifiers in the anoxic biofilm and denitrifiers in the aerobic biofilm was established by the specific activity measurements. Detachment rates of PAOs in aerobic and anoxic biofilms were evaluated.     

Biotransformation of R-2-hydroxy-4-phenylbutyric acid by D-lactate dehydrogenase and Candida boidinii cells containing formate dehydrogenase coimmobilized in a fibrous bed bioreactor

R-2-hydroxy-4-phenylbutyric acid (R-HPBA) is an important intermediate in the manufacture of angiotensin converting enzyme inhibitors. In this work, a recombinant D-lactate dehydrogenase (LDH) was used to transform 2-oxo-4-phenylbutyric acid (OPBA) to R-HPBA, with concomitant oxidation of beta-nicotinamide adenine dinucleotide (NADH) to NAD(+). The cofactor NADH was regenerated by formate dehydrogenase (FDH) present in whole cells of Candida boidinii, which were pre-treated with toluene to make them permeable. The whole cells used in the process were more stable and easier to prepare as compared with the isolated FDH from the cells. Kinetic study showed that the reaction rate was dependent on the concentration of cofactor, NAD(+), and that both R-HPBA and OPBA inhibited the reaction. A novel method for co-immobilization of whole cells and LDH enzyme on cotton cloth was developed using polyethyleneimine (PEI), which induced the formation of PEI-enzyme-cell aggregates and their adsorption onto cotton cloth, leading to multilayer co-immobilization of cells and enzyme with high loading (0.5 g cell and 8 mg LDH per gram of cotton cloth) and activity yield ( > 95%). A fibrous bed bioreactor with co-immobilized cells and enzyme on the cotton cloth was then evaluated for R-HPBA production in fed-batch and repeated batch modes, which gave relatively stable reactor productivity of 9 g/L . h and product yield of 0.95 mol/mol OPBA when the concentrations of OPBA and R-HPBA were less than 10 g/L.     

Characterization of a continuous supermacroporous monolithic matrix for chromatographic separation of large bioparticles

A continuous supermacroporous monolithic chromatographic matrix has been characterized using a capillary model, experimental breakthrough curves, and pressure drop experiments. The model describes the convective flow and its dispersive mixing effects, mass transfer resistance, pore size distribution, and the adsorption behavior of the monolithic matrix. It is possible to determine an effective pore size distribution by fitting the capillary model to experimental breakthrough curves and pressure drop experiments. The model is able to describe the flow rate dependence of the experimental breakthrough curves. Mass transport resistance was due to: (i) dispersive mixing effects in the convective flow in the pores; and (ii) slow diffusion in the stagnant film covering the surface within each pore, under adsorption conditions. The monolithic matrix can be described by a very narrow pore size distribution, illustrating one of the advantages of the gel. A broader pore size distribution results in increased band broadening. This can be studied easily using the model developed in this investigation.     

Isomaltose formation by free and immobilized dextransucrase

The acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL-B512F with glucose as acceptor is of technical interest for isomaltooligosaccharide (IMOs) synthesis. Different experimental conditions were investigated for free and immobilized enzyme. The data for oligosaccharide formation up to a degree of polymerization 4 were correlated with a model developed earlier, and optimal reaction conditions for immobilized dextransucrase design and application were identified for later continuous application. Furthermore, stability was investigated for free and immobilized enzyme including stabilization by sugars.     

Stability and reproducibility of low-temperature anaerobic biological wastewater treatment

The reproducibility of low-temperature anaerobic biological wastewater treatment trials was evaluated. Two identical anaerobic expanded granular sludge bed bioreactors were used to treat synthetic volatile fatty acid-based industrial wastewater under ambient conditions (18-20 degrees C) and to investigate the effect of various environmental perturbations on reactor performance and microbial community dynamics, which were assessed by chemical oxygen demand removal or effluent volatile fatty acid determination and terminal restriction fragment length polymorphism analysis, respectively. Methanogenic activity was monitored using specific methanogenic activity assays. Reactor performance and microbial community dynamics were each well replicated between Reactor 1 and Reactor 2. Archaeal dynamics, in particular, were associated with reactor operating parameters. Terminal restriction fragment length polymorphism data suggested dynamic acetoclastic and hydrogenophilic methanogenic populations and were in agreement with temporal specific methanogenic activity data. Putative psychrophilic populations were observed in anaerobic bioreactor sludge for the first time.     

Screen-less expanded bed column: new approach for the recovery and purification of a malaria transmission blocking vaccine candidate from Pichia pastoris

An experimental malaria transmission blocking vaccine antigen, Pfs25H, expressed and secreted from Pichia pastoris was recovered and purified using a screenless expanded bed column equipped with a rotating fluid distribution system. This column was able to accommodate feed stock, containing 30% biomass, at a flow rate of 300–400 cm/h without affecting column stability. This capability is three times higher than the capability of the expanded bed column currently in use, which is equipped with a perforated plate fluid distribution system; this design could accommodate biomass concentrations of only up to 10%. The screen-less design did not affect the binding capacity, purification level or process yield and, therefore, shorten the process. Purified Pfs25H of 6.4 g were recovered from 37 l of Pichia pastoris culture in one step.     

Impact of long-term partial aeration on the removal of 2,4,6-trichlorophenol in an initially methanogenic fluidized bed bioreactor

A fluidized bed bioreactor (FBBR) was operated for more than 1000 days under two regimes, Methanogenic (M) and Methanogenic-Aerobic (M-A), to remove 2,4,6-trichlorophenol (TCP) and phenol (Phe) from a synthetic wastewater, containing different amounts of TCP and Phe, using different aeration flow-rates (0, 2.13, and 1.06 NL O(2)/L.day). M conditions (80:20 mg/L of TCP:Phe, 0 NL O(2)/L.day) showed similar TCP and Phe removal (>95%). Nevertheless accumulation of 4-chlorophenol (4CP) up to 16 mg/L and Phe up to 4 mg/L was observed, while in M-A conditions (80:20 mg/L of TCP:Phe, 2.13 NL O(2)/L.day) TCP and Phe removal achieved 99.9(+)% and after 70 days no accumulation of intermediates were detected. The increase of TCP and Phe in the influent under M-A conditions from 80:20 to 120:30 mg/L of TCP:Phe did not negatively affect the removal of TCP, intermediates and Phe; in fact, they were similar to those in previous M-A conditions. The decrease in the oxygen flow rate from 2.13 to 1.06 NL O(2)/L.day had no negative effect on pollutant removals, which were as high as in previous two M-A conditions. The specific methanogenic activity of bioparticles of the fluidized bed decreased with long-term partial aeration, starting from 1.097 mmol CH(4)/h.g(TKN) in the M regime (day 60) to <0.02 mmolCH(4)/h.g(TKN) at day 1050, suggesting aerobic regime in the bioreactor rather than an M-A regime. In conclusion, complete removal of TCP and less chlorinated intermediates could be achieved in an initially methanogenic FBBR under conditions of partial aeration, although long-term operation seemed to negatively affect the methanogenic activity of biomass. It is also likely that after extended aeration the microbial community was finally enriched with strains with the ability to attack 2,4,6-TCP under aerobic conditions. This report represents the first evidence of a long exposure to oxygen of an anaerobic microbial consortium that efficiently remove TCP.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

Biosorption and desorption of lanthanum(III) and neodymium(III) in fixed-bed columns with Sargassum sp.: perspectives for separation of rare earth metals

Rare earth (RE) metals are essentials for the manufacturing of high-technology products. The separation of RE is complex and expensive; biosorption is an alternative to conventional processes. This work focuses on the biosorption of monocomponent and bicomponent solutions of lanthanum(III) and neodymium(III) in fixed-bed columns using Sargassum sp. biomass. The desorption of metals with HCl 0.10 mol L(-1) from loaded biomass is also carried out with the objective of increasing the efficiency of metal separation. Simple models have been successfully used to model breakthrough curves (i.e., Thomas, Bohart-Adams, and Yoon-Nelson equations) for the biosorption of monocomponent solutions. From biosorption and desorption experiments in both monocomponent and bicomponent solutions, a slight selectivity of the biomass for Nd(III) over La(III) is observed. The experiments did not find an effective separation of the RE studied, but their results indicate a possible partition between the metals, which is the fundamental condition for separation perspectives.