Robert R. Beitle

Preparation of magnetic immobilized metal affinity separation media and its use in the isolation of proteins

A new method of pseudobiospecific protein isolation is developed and tested, which employs both metal affinity and magnetism as the basis for isolation. The chelating group iminodiacetic acid (IDA) has been coupled to the surface of magnetic agarose, and when charged with metal ions (Cu2+ or Zn2+) is capable of binding model proteins which display metal affinity, and of separating protein mixtures. Magnetic properties of the medium facilitated the batch recovery of the adsorbent, as losses are minimized by concentrating and retaining the separation medium with the aid of a magnet. Model proteins were used to characterize protein adsorption, capacity, and stability of IDA magnetic agarose. Recovery from a cell lysate was demonstrated by protein isolation from extracts of E. coli containing a target protein. Overall, this study effectively illustrates the engineering of separation media which combine several desired properties for the development of a new branch of metal affinity-based bioseparation.

Characterization of metal affinity of green fluorescent protein and its purification through salt promoted, immobilized metal affinity chromatography.

Immobilized metal affinity chromatography (IMAC) was investigated as a method of recovery for green fluorescent protein (GFPuv). It was found that in the absence of genetic modification to enhance metal affinity, GFPuv displayed strong metal affinity to Cu(II) and Ni(II), and weak or negligible affinity to Zn(II) and Co(II). Changes in the mobile phase NaCl concentration during Ni(II)-IMAC strongly affected purity and yield of GFPuv, with fine resolution under higher NaCl concentrations. Finally, IMAC via Cu(II) and Zn(II) with intervening diafiltration was used to recover GFPuv with high yield and purity.

Protein purification via aqueous two-phase extraction (ATPE) and immobilized metal affinity chromatography. Effectiveness of salt addition to enhance selectivity and yield of GFPuv.

This study illustrates the compatibility and complementary nature of aqueous two‐phase extraction (ATPE) and immobilized metal affinity chromatography (IMAC) in a general recovery scheme. The purification of green fluorescent protein (GFPuv) from extracts of Eschericia coli was investigated using a combination of these two techniques. High molarity of sodium chloride was found effective in increasing selectivity, with the promotion of hydrophobic interaction the probable mechanism that drove the target protein to a particular phase in ATPE, as well as that which enhanced GFPuv adsorption in IMAC. Moreover, the similar salt condition allows the direct application of the GFPuv‐containing phase to the IMAC column without additional adjustment step. A simple screen of conditions was therefore performed to generate a favorable two‐step purification scheme for GFP leading to an overall high purity.

Use of proteomics for design of a tailored host cell for highly efficient protein purification

After some initial optimization, a downstream process comprised of one or several chromatography steps removes the majority of the host proteins and achieves a reasonable degree of purification. The separation of remaining contaminant proteins from the target protein could become very difficult and costly due to their similar physicochemical properties. In this paper we describe a highly efficient strategy, based on proteomic analysis and elution chromatography, by which a protein of interest may be isolated from copurifying contaminants. Mutant strains of Escherichia coli were prepared that are deficient in three prevalent host proteins found in a strategic fraction of an elution profile of nickel immobilized affinity chromatography. Recombinant green fluorescent protein (GFPuv) served as a model protein and its elution was directed to this optimized fraction with an N-terminus hexahistidine tag (his(6)), thereby easing its recovery. We demonstrate that proteomic data can facilitate the rational engineering of host cell expressing the target protein and the design of an efficient process for its purification.

Application of Wafer-Enhanced Electrodeionization in a Continuous Fermentation Process to Produce Butyric Acid with Clostridium tyrobutyricum

Butryic acid is an organic acid which can be produced from Clostridium tyrobutyricum. This organic acid has many applications in food and perfumes as well serving as the first step in a two-step butanol production process. However, the fermentation is product inhibited as well as producing by-products of acetic acid and lactic acid. Electrodialysis and electrodeionization were explored as ways to separate butyric acid from a fermentation broth, in situ. For butyric acid system, the current resistance for ED was almost half that of EDI indicating that ED might be a better option for a pure component separation. However, in a simulated broth containing butyric, lactic, and acetic acid, the opposite trend was found to be true. These results were used to design a fermentation experiment where ED and EDI were used to separate butyric acid from an actual fermentation. It is shown that the percentage of butyric acid (compared to other organic acids) is 84%, 85%, and 92% for no separation, ED, and EDI, respectively. Further, the productivity rate is enhanced by almost 220% when comparing EDI to no separation indicating that it may make sense to use EDI for the separation of butyric acid from a Clostridium tyrobutryicum fermentation.

Synergistic Effect of Abrasive and Sonication for Release of Carbohydrate and Protein from Algae

While algae have demonstrated significant potential as a raw material due to the high intracellular concentration of carbohydrates and proteins, a primary limitation as a biofuel feedstock is due to the fact that an economically feasible method of extraction has yet to be offered. Algae samples, acquired from a local waste treatment facility, were combined with abrasive materials and subjected to ultrasonication for specific time intervals. It was found that the synergistic effect of sonication in the presence of abrasive material, such as silicon beads or sand, could extract adequate protein and carbohydrate to be utilized in fermentation processes.

Identification and characterization of native proteins of Escherichia coli BL-21 that display affinity towards Immobilized Metal Affinity Chromatography and Hydrophobic Interaction Chromatography Matrices.

The purpose of this study was to identify and characterize Escherichia coli proteins which display affinity towards both Immobilized Metal Affinity Chromatography (IMAC) and Hydrophobic Interaction Chromatography (HIC). Co(II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Results provided insight on this rather small pool of E. coli proteins. Nine out of the ten have isoelectric values less than six, and half are considered nonessential. These data indicate that the combination of IMAC and HIC could be developed as a potent method for the purification of recombinant proteins by judicious choice of the salt concentration used to promote HIC, the development of E. coli strain(s) deficient in certain genomic proteins, and the design of an IMAC-HIC affinity tail for recombinant protein isolation based on the very proteins deleted from the genome.

Real-time monitoring of recombinant bacterial proteins by mass spectrometry

Genetically altered bacteria manipulated to express green fluorescent protein (GFP) were used in an investigation of real‐time monitoring for recombinant protein expression in cell by matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight (TOF) mass spectrometry (MS). A significant advantage to whole cell MALDI MS is its ability to analyze bacterial cultures without pretreatment other than concentration. This paper describes the simultaneous analysis of overexpressed GFP recombinant Escherichia coliJM101 by MALDI‐TOF MS and standard fluorescence measurements. Cells were harvested from liquid culture media during a 12 h GFP induced expression cycle to demonstrate the feasibility of near real‐time monitoring of induced protein expression. The results show that although MALDI MS is not as sensitive as fluorescence measurements, expression levels of the targeted protein can easily be determined. Data available only through MALDI MS measurements reveal the presence of both native GFP and GFP‐(histidine)6 proteins. Additionally, biochemical processes not yet fully understood are observed in the presence and absence of ribosomal protein constituents. Thus, the work presented here demonstrates the ability of MALDI MS to monitor and characterize in real time the expression of targeted and unexpected genetically recombinant proteins in active cell cultures.

Analysis of a Microbial Community Oxidizing Inorganic Sulfide and Mercaptans

Successful treatment of refinery spent‐sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio‐Sep). The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources. The starting cultures for the enrichment consisted of several different Thiobacillispp. (T. thioparus, T. denitrificans, T. thiooxidans, and T. neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester. Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms. Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR‐amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis). Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands),Gram‐positive cocci (Micrococcineae), α proteobacteria (3 bands), and an unidentified β proteobacterium. Culturable microbes included several genera of fungi as well as various Gram‐positive and Gram‐negative heterotrophic bacteria not seen in techniques using direct DNA extraction.

Biotreatment of Refinery Spent-Sulfidic Caustic Using an Enrichment Culture Immobilized in a Novel Support Matrix

Sodium hydroxide solutions are used in petroleum refining to remove hydrogen sulfide (H2S) and mercaptans from various hydrocarbon streams. The resulting sulfide-laden waste stream is called spent-sulfidic caustic. An aerobic enrichment culture was previously developed using a gas mixture of H2S and methylmercaptan (MeSH) as the soleenergy source. This culture has now been immobilized in a novel support matrix, DuP ont BIO-SEPTM beads, and is used to biotreat a refinery spent-sulfidic caustic containing both inorganic sulfide and mercaptans in a continuous flow, fluidized-bed column bioreactor. Complete oxidation of both inorganic and organic sulfur to sulfate was observed with no breakthrough of H2S and <2 ppmv of MeSH produced in the bioreactor outlet gas. Excessive buildup of sulfate (>12 g/L) in the bioreactor medium resulted in an upset condition evidenced by excessive MeSH breakthrough. Therefore, bioreactor performance was limited by the steady-state sulfate concentration. Further improvement in volumetric productivity of a bioreactor system based on this enrichment culture will be dependent on maintenance of sulfate concentrations below inhibitory levels.