High-Throughput and High-Yield Purification of Recombinant Proteins Expressed in Escherichia coli.
Poster Feb 03, 2006
Chiann-Tso Lin, Priscilla A. Moore, Deanna L. Auberry, Kristin D. Victry, Frank R. Collart and Vladimir Kery
AbstractMany biotechnologically important microbial processes, drug and biomarker discovery require understanding of protein function in cellular networks. Deciphering protein function creates high demand for production of large number of purified proteins of yield sufficient for protein crystallography, mapping of protein – protein interactions or developing affinity reagents (antibodies, aptamers) towards proteins of interest.
Therefore, robust high throughput protein expression and purification processes need to be developed to meet this demand. We have developed a high throughput expression of recombinant proteins containing hexahistidine affinity tag in Escherichia coli followed by one step affinity purification of proteins on Ni2+ agarose beads.
Genes of interest were cloned using automated high throughput cloning. Proteins were expressed using producing strains of E. coli in the scale of 800 ml/clone using Bactolift™ (Lofstrand) or 25 ml/clone in 50 ml Falcon tubes. Proteins were then purified by IMAC on Ni2+ agarose from crude lysates either using filtration based low throughput – higher yield automated process on BioRobot 3000 (Qiagen) or using magnetic separation based high throughput - lower yield automated process on Biomek FX™ robot (Beckman Coulter).
Thus process parameters of protein purification of sets of 12 or 96 proteins could be compared, respectively. Although both processes provided proteins with comparable purity the Biomek-based process was technically much easier, much higher -throughput and less error-prone. Using those processes for automated high throughput production of milligram quantities of purified recombinant proteins is discussed.
Psychiatric Risk Gene Cacna1c and Early Life Stress: Potential Gene-Environment interactions?Poster
Early life stress (ELS) is highly associated with development of psychopathology
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Novel Role of the Innate Immune DNA Sensor IFI16 (Interferon Gamma Inducible Protein 16) as a Major Epigenetic Modulator During KSHV Infection and Lytic ReactivationPoster
Studies have shown that IFI16 acts as an antiviral restriction factor against a number of DNA viruses, by inhibiting viral replication or transcription through epigenetic modifications. However, till date, no specific epigenetic function of IFI16 has been identified. Here, we have discovered that IFI16 recruits two histone methyltransferases on the KSHV episome leading to altered Histone H3K9 methylation, thus regulating its lifecycle.READ MORE
A new method for generating arrayed RNAi screening tools for any organismPoster
RNA interference (RNAi) using small interfering RNAs (siRNAs) is an important technology for down-regulation of gene expression and a powerful tool to study cellular processes and pathways. Previously, large collections of siRNAs were available only for traditional experimental model systems, such as human and mouse, and predominantly provided as chemically synthesized libraries.READ MORE