RESUMEN
One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling beta-sheet forming peptide P(11)-2 in fusion to thioredoxin. Homogenate was heat treated (55 degrees C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P(11)-2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250 mM NaCl to homogenate to prevent P(11)-2 from partitioning to the precipitate. This process structure gave recombinant P(11)-2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials.
Asunto(s)
Biotecnología/métodos , Fraccionamiento Químico , Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Péptido Hidrolasas/metabolismo , Péptidos/genética , Péptidos/metabolismo , Multimerización de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tiorredoxinas/genéticaRESUMEN
Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states.
Asunto(s)
Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Tensoactivos/aislamiento & purificación , Tensoactivos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Fraccionamiento Químico , Cromatografía de Afinidad , Medios de Cultivo/química , Endopeptidasas/metabolismo , Escherichia coli/genética , Proteínas de Unión a MaltosaRESUMEN
Peptides have recently attracted interest as building blocks for the assembly of novel functional materials including switchable surfactants, nanocoatings, hydrogels and aqueous vesicles. We expressed a beta-sheet forming peptide that has been widely studied in self-assembly processing, P(11)-2, as a monomer, dimer, tetramer and nonamer fused to an insoluble expression partner, ketosteroid isomerase, using minimal media. Expression was followed by whole cell extraction and isolation of the fusion protein to greater than 90% purity via a single immobilised metal affinity chromatography (IMAC) step. Peptides were chemically cleaved from each other and from the fusion partner, followed by acetone precipitation of the contaminating protein fragments. Pure peptide was recovered by reversed-phase HPLC. The expression level of the fusion protein decreased as the peptide concatamer number increased, as did the efficiency of the chemical cleavage, making the single-peptide process the most efficient overall. Applying this laboratory process to the single-peptide fusion protein nevertheless resulted in a pure peptide yield of greater than 30% of the expressed peptide.
Asunto(s)
Cromatografía de Afinidad/métodos , Escherichia coli/fisiología , Nanoestructuras/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección/métodos , Péptidos/genéticaRESUMEN
The MixAlco process is a patented technology that converts any biodegradable material (e.g., sorted municipal solid waste, sewage sludge, industrial biosludge, manure, agricultural residues, energy crops) into mixed alcohol fuels containing predominantly 2-propanol, but also higher alcohols up to 7-tridecanol. The feedstock is treated with lime to increase its digestibility. Then, it is fed to a fermentor in which a mixed culture of acid-forming microorganisms produces carboxylic acids. Calcium carbonate is added to the fermentor to neutralize the acids to their corresponding carboxylate salt. The dilute (approximately 3%) carboxylate salts are concentrated to 19% using an amine solvent that selectively extracts water. Drying is completed using multi-effect evaporators. Finally, the dry salts are thermally converted to ketones which subsequently are hydrogenated to alcohols. All the steps in the MixAlco process have been proven at the laboratory scale. A techno-economic model of the process indicates that with the tipping fees available in New York (126 dollars/dry tonne), mixed alcohol fuels may be sold for 0.04 dollars/L (0.16 dollars/gal) with a 60% return on investment (ROI). With the average tipping fee in the United States rates (63 dollars/dry tonne), mixed alcohol fuels may be sold for 0.18 dollars/L (0.69 dollars/gal) with a 15% ROI. In the case of sugarcane bagasse, which may be obtained for about 26 dollars/dry ton, mixed alcohol fuels may be sold for 0.29 dollars/L (1.09 dollars/gal) with a 15% ROI.