Improvement of bilirubin adsorption capacity of cellulose acetate/polyethyleneimine membrane using sodium deoxycholate

Bilirubin (BR) adsorbents have low removal efficiency because of the tight binding of BR with human albumin (HA) in a complicated blood system. Sodium deoxycholate (SDC) was selected as an adsorption promoter to improve the BR adsorption capacity of a cellulose acetate (CA)/polyethyleneimine (PEI) membrane. Static adsorption experiments show that the maximum BR adsorption capacity of the membrane with SDC in BR–HA mock solution is 100–200% at the molar ratio of SDC to HA ranging from 8 to 12, higher than that without SDC. It is also found that SDC is more efficiently adsorbed by the membrane than BR and HA. Absorption, circular dichroism, and zeta potential studies demonstrate that SDC can be bound with the BR–HA complex to form a ternary BR–HA-SDC_m complex. On the basis, the facilitated adsorption mechanism of BR with SDC was proposed that SDC aggregates or micelles form a quasi-multilayer adsorption on the membrane, increase approachable binding sites, and prolong the distance between the BR–HA complex and the membrane. Thus, SDC as a spacer reduces the influence of the steric hindrance of HA, resulting in an enhanced BR adsorption capacity. Dynamic adsorption results further evidence the facilitated adsorption mechanism.     

Recovery and purification of plant-made recombinant proteins

Plants are becoming commercially acceptable for recombinant protein production for human therapeutics, vaccine antigens, industrial enzymes, and nutraceuticals. Recently, significant advances in expression, protein glycosylation, and gene-to-product development time have been achieved. Safety and regulatory concerns for open-field production systems have also been addressed by using contained systems to grow transgenic plants. However, using contained systems eliminates several advantages of open-field production, such as inexpensive upstream production and scale-up costs. Upstream technological achievements have not been matched by downstream processing advancements. In the past 10 years, the most research progress was achieved in the areas of extraction and pretreatment. Extraction conditions have been optimized for numerous proteins on a case-by-case basis leading to the development of platform-dependent approaches. Pretreatment advances were made after realizing that plant extracts and homogenates have unique compositions that require distinct conditioning prior to purification. However, scientists have relied on purification methods developed for other protein production hosts with modest investments in developing novel plant purification tools. Recently, non-chromatographic purification methods, such as aqueous two-phase partitioning and membrane filtration, have been evaluated as low-cost purification alternatives to packed-bed adsorption. This paper reviews seed, leafy, and bioreactor-based platforms, highlights strategies for the primary recovery and purification of recombinant proteins, and compares process economics between systems. Lastly, the future direction and research needs for developing economically competitive recombinant proteins with commercial potential are discussed.     

Separation of recombinant human vasoactive intestinal peptide–humanin fusion protein via three different approaches

Both vasoactive intestinal peptide (VIP) and humanin (HN) can provide neuroprotection against β-amyloid toxicity and are believed to be beneficial in the treatment of Alzheimer's disease. For the sake of tandem co-expression of recombinant human VIP and HN(rhVIP-HN) fusion protein, new VIP-HN gene is constructed and cloned into the plasmid pET28a(+) and is expressed in Escherichia coli BL21(DE3). Since the fusion protein was present as inclusion bodies, three different preparation approaches are employed to obtain the hexa-histidine tagged rhVIP-HN (His₆-rhVIP-HN) fusion protein by Ni²⁺ chelating resin chromatography. The comparison of the results obtained from the three approaches reveals that renaturation during the separation process (Approach III) is the most efficient for large-scale preparation of His₆-rhVIP-HN. Ultimately, 46 mg of the target protein was obtained from one gram of the inclusion body with purity up to 90%. Since there is not separated step in renaturation procedure, Approach III could be more time-saving, buffer-saving and easy to operate than the other two approaches. These results can be useful for preparation of rhVIP-HN in large scale.     

Morphological characterization of <Emphasis Type="Italic">Cupriavidus necator</Emphasis> DSM 545 flocs through image analysis

Flocculating agents are used as auxiliary to recover bacterial cells in downstream processes for polyhydroxyalkanoate production. However little is known about the Curpiavidus necator flocs. In this work a new procedure for floc characterization through digital image analysis is presented and validated using the batch settling test. Average diameter, particle size distribution and morphological characteristics of the microbial aggregates were obtained from the flocculation/sedimentation process of the Cupriavidus necator DSM 545 cells by the use of tannin as flocculating agent. The experimental results demonstrated that the proposed method is adequate to determine the average floc diameter with values around 150 μm in accordance with the value obtained from the batch settling test. Nevertheless a morphological characterization of Cupriavidus necator DSM 545 bioaggregates in terms of size distribution and regularity could only be performed by an image analysis procedure. The procedure allowed us to describe the regularity of bacterial flocs through the quantification of morphological parameters of Euclidean [convexity (Conv) and form factor (FF)] and fractal geometry [surface fractal dimension (D _BS)], which are important factors to be considered in the settling efficiency of aggregates.     

Microfluidic bio-particle manipulation for biotechnology

Microfluidics and lab-on-a-chip technology offers unique advantages for the next generation devices for diagnostic therapeutic applications. For chemical, biological and biomedical analysis in microfluidic systems, there are some fundamental operations such as separation, focusing, filtering, concentration, trapping, detection, sorting, counting, washing, lysis of bio-particles, and PCR-like reactions. The combination of these operations led to the complete analysis systems for specific applications. Manipulation of the bio-particles is the key ingredient for these applications. Therefore, microfluidic bio-particle manipulation has attracted a significant attention from the academic community. Considering the size of the bio-particles and the throughput of the practical applications, manipulation of the bio-particles is a challenging problem. Different techniques are available for the manipulation of bio-particles in microfluidic systems. In this review, some of the techniques for the manipulation of bio-particles; namely hydrodynamic based, electrokinetic-based, acoustic-based, magnetic-based and optical-based methods have been discussed. The comparison of different techniques and the recent applications regarding the microfluidic bio-particle manipulation for different biotechnology applications are presented. Finally, challenges and the future research directions for microfluidic bio-particle manipulation are addressed.     

Purification effect of artificial chaperone in the refolding of recombinant ribonuclease A from inclusion bodies

Artificial chaperone (AC) containing cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD) has been used to refold recombinant ribonuclease A (RNase A) from inclusion bodies (IBs). At low urea concentration (0.8 M), the AC could enhance the refolding yield of RNase A by effectively suppressing its intermolecular interaction-induced aggregation. As a result, 0.9 mg/mL RNase A could be 77% refolded, which was a 57% increase as compared to that without the AC. At high protein concentration range (0.9–2.3 mg/mL in total protein concentrations) and 1.6 M urea, CTAB selectively precipitated contaminant proteins distinctly, so a purification effect was achieved. For example, 1.5 mg/mL RNase A could be 62% refolded and recovered at a purity of 87%, which was a 34% increase in purity as compared to that in IBs (65%). The precipitation selectivity was considered due to the differences in the hydrophobicity of the proteins. The work indicates that by using the AC, RNase A could be efficiently refolded at low urea concentration and purified at high urea concentration from IBs at high protein concentrations.     

Peptide disulfide RKCGCFF facilitates oxidative protein refolding by mimicking protein disulfide isomerase

This communication reports a new design of peptide disulfide, RKCGCFF, for facilitating oxidative protein refolding. The new design mimics the properties of protein disulfide isomerase (PDI) by introducing hydrophobic and positively charged patches into the two terminals of disulfide CGC. RKCGCFF was found more effective than the traditional oxidant oxidized glutathione (GSSG) as well as its counterpart, RKCGC, in facilitating the oxidative refolding of lysozyme. More importantly, RKCGCFF could improve lysozyme refolding yield at a high concentration (0.7 mg/mL). The research proved that incorporation of hydrophobic and charged patches into the CGC disulfide made the oxidant more similar to PDI in structure and properties.     

Enrichment in axial direction of aqueous foam in continuous foam separation

Estimation of overhead production enrichment in continuous foam separation was conducted with a surfactant: sodium n-dodecylbenzenesulfonate (SDBS) and soluble proteins: ovalbumin (OA) and hemoglobin (HB). Axial profiles of the volumetric flow rate and the concentration of the collapsed foam liquid within the column were measured, and the enrichment ratio and the liquid holdup in axial direction were determined experimentally. The proposed model was fitted to the experimental results obtained with various experimental conditions (superficial gas velocity, feed concentration and pH) and was in reasonable agreement with the experimental data by using the least square regression. The present model makes it possible to estimate the foamate concentration at a desired foam height.     

Rational design of a host cell protein heat precipitation step simplifies the subsequent purification of recombinant proteins from tobacco

The benefits of plants for the production of biopharmaceutical proteins include the ease of scale-up and the low pathogen burden, but the purification of target proteins is often affected by low expression levels and the abundance of host cell proteins in leaf extracts. These factors can significantly diminish the competitiveness of plant-based expression systems. We therefore compared three different heat precipitation steps that remove most host cell proteins while leaving a heat-stable candidate malaria vaccine protein in solution. A design-of-experiments approach was used to test each method and analyze the resulting data, allowing us to model and optimize the corresponding process. The heat treatment of leaves was superior to both methods for the heat treatment of extracts in terms of process-scale implementation, energy consumption and speed. We discuss additional benefits and drawbacks of these methods compared to current strategies for the purification of biopharmaceutical proteins produced in plants.