Charles E. Glatz

Fusion tails for the recovery and purification of recombinant proteins

Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.

Considerations for the Recovery of Recombinant Proteins from Plants

The past 5 years have seen the commercialization of two recombinant protein products from transgenic plants, and many recombinant therapeutic proteins produced in plants are currently undergoing development. The emergence of plants as an alternative production host has brought new challenges and opportunities to downstream processing efforts. Plant hosts contain a unique set of matrix contaminants (proteins, oils, phenolic compounds, etc.) that must be removed during purification of the target protein. Furthermore, plant solids, which require early removal after extraction, are generally in higher concentration, wider in size range, and denser than traditional bacterial and mammalian cell culture debris. At the same time, there remains the desire to incorporate highly selective and integrative separation technologies (those capable of performing multiple tasks) during the purification process from plant material. The general plant processing and purification scheme consists of isolation of the plant tissue containing the recombinant protein, fractionation of the tissue along with particle size reduction, extraction of the target protein into an aqueous medium, clarification of the crude extract, and finally purification of the product. Each of these areas will be discussed here, focusing on what has been learned and where potential concerns remain. We also present details of how the choice of plant host, along with location within the plant for targeting the recombinant protein, can play an important role in the ultimate ease of recovery and the emergence of regulations governing plant hosts. Major emphasis is placed on three crops, canola, corn, and soy, with brief discussions of tobacco and rice.

Propionic acid production by extractive fermentation. I. Solvent considerations

Solvent selection for extractive fermentation for propionic acid was conducted with three systems: Alamine 304-1 (trilaurylamine) in 2-octanol, 1-dodecanol, and Witcohol 85 NF (oleyl alcohol). Among them, the solvent containing 2-octanol exhibited the highest partition coefficient in acid extraction, but it was also toxic to propionibacteria. The most solvent-resistant strain among five strains of the microorganism was selected. Solvent toxicity was eliminated via two strategies: entrapment of dissolved toxic solvent in the culture growth medium with vegetable oils such as corn, olive, or soybean oils; or replacement of the toxic 2-octanol with nontoxic Witcohol 85 NF. The complete recovery of acids from the Alamine 304-1/Witcohol 85 NF was also realized with vacuum distillation.

Membrane-based extractive fermentation to produce propionic and acetic acids: Toxicity and mass transfer considerations

A series of microporous membranes and polymer films was examined for mass transfer performance in the membrane-based extraction of propionic and acetic acids. A range of organic solvents and acid-complexing carriers was screened for toxic effects on a strain of Propionibacterium acidipropionici. Based on these results, more extensive studies were made of the extraction kinetics of tri-n-octylphosphine oxide in the diluents decane and kerosene. After the supported liquid membrane configuration showed problems with stability, the TOPO/kerosene in Celgard X-20 microporous membrane system was chosen for use in an extractive fermentation with the membrane between the fermentation broth and bulk extractant in a hollow fiber module.

Mechanisms of Aqueous Extraction of Soybean Oil

Aqueous extraction processing (AEP) of soy is a promising green alternative to hexane extraction processing. To improve AEP oil yields, experiments were conducted to probe the mechanisms of oil release. Microscopy of extruded soy before and after extraction with and without protease indicated that unextracted oil is sequestered in an insoluble matrix of denatured protein and is released by proteolytic digestion of this matrix. In flour from flake, unextracted oil is contained as intact oil bodies in undisrupted cells, or as coalesced oil droplets too large to pass out of the disrupted cellular matrix. Our results suggest that emulsification is an important extraction mechanism that reduces the size of these droplets and increases yield. Protease and SDS were both successful in increasing extraction yields. We propose that this is because they disrupt a viscoelastic protein film at the droplet interface, facilitating droplet disruption. An extraction model based on oil droplet coalescence and the formation of a viscoelastic film was able to fit kinetic extraction data well.

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production byPropionibacterium acidipropionici

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor withPropionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 g/l propionic acid and 12.8 g/l acetic acid were obtained in about 146 h in laboratory medium with 79 g/l glucose added over five feeding periods. The highest concentration of propionic acid, 45 g/l, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 g/l propionic acid and 11 g/l acetic acid were produced in 108 h from 59.4 g/l total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30–37 g/l propionic acid and 13 g/l acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.

Extraction of protein from distiller’s grain

We have investigated the feasibility of extracting the oil and protein from distillers grain (DG) to obtain a higher-valued protein-rich product and a carbohydrate-rich residue better suited for conversion to fermentable sugars. Protein extractions based on aqueous ethanol, alkaline-ethanol, and aqueous enzyme treatments were compared. Three of the methods extracted a significant amount of the protein from dried, defatted DG (DDDG). The enzymatic extraction decreased the crude protein content in the solid phase for both milled and unmilled DDDG from 41% (dry weight) to approximately 10% (dry weight) protein in the residual solid; this corresponded to extraction of 90% of the protein in the original DDDG. The alkaline-ethanol extraction was similarly effective for milled but not unmilled DG. Simple extraction with alcohol was not as effective. Amino acid analysis of each protein extract was consistent with it consisting mainly of zeins. For the protease-assisted extractions, 95% the proteins were in the form of peptides smaller than 10kDa.

Effects of pH and ionic strength on microfiltration of C. glutamicum

Abstract The effects of pH and ionic strength on microfiltration of Corynebacterium glutamicum (C. glutamicum) were evaluated. The filtration performance was characterized by the specific cake resistance (α) calculated from measurement of dead-end filtration flux using Ruths equation. When the pH of the cell slurry was adjusted from 6.3 to 2.0, α decreased by a factor of 5 which meant that the total flux was improved five times; the effect of ionic strength was much less. Measurements of zeta potential and hydrophobicity showed the cells to be more hydrophobic and have lower surface charge at pH 2 than at neutral pH. Microscopic observation of cross-sections of the cell cake layer showed cells aggregated at pH 2, but not at pH 5. The aggregation behavior is consistent with the reduction of surface repulsion at pH 2 and the larger voids between aggregates account for the larger flux.

Aggregate breakage in protein precipitation

Abstract Particle breakage establishes limits on both size and shape in a variety of aggregation processes. The mechanisms by which breakage of isoelectrically precipitated protein aggregates occurs and models to describe the size and shear dependence of breakage rates are the subjects of this work. The analyses are based on experiments performed in a turbine-agitated vessel in which various concentrations of protein aggregates prepared under standard precipitation conditions were subjected to a range of agitation (and hence, shear) levels. The data collected were the aggregate size distributions as functions of time of exposure. Maximum aggregate size decreased with agitation level but not in the fashion predicted by past models based on maximum stable size. In contrast, a statistical model, assuming similarity of the breakage function, did give useful insights. Such analysis showed that a power law description of breakage frequency required two such functions to cover the entire distribution with a higher exponent for the larger sizes. Further, it showed that the daughter size distribution shifts from a more thorough to a more erosive mode as the aggregate volume increases. However, the relatively small reduction in size observed in these experiments did not allow conclusive proof of the similarity assumption. Mechanistic models for the kinetics of the size reduction proved the most useful. They show the expected increase in rate as the shear rate increases. The total extent of breakage depends on the solids concentration in the suspension. This is best described as a second order process involving collision of a breakage-susceptible aggregate with another aggregate. Expressions are given both for the rate of decrease in mean size and for the rate of removal by breakage of numbers of particles from the large end of the size distribution.

Suitability of immobilized metal affinity chromatography for protein purification from canola.

This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his(6)-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co(2+) with iminodiacetate (IDA) as the chelating ligand, beta-glucuronidase-his(6) (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu(2+) < Ni(2+) < Zn(2+) < Co(2+) in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his(6) in forming a tridentate with Me(2+)-IDA than in forming a bidentate with Me(2+)-NTA. The more flexible binding allows for multisite interactions over the protein.