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.
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Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
P450 Induction in Cryopreserved Hepatocytes from PXR and CAR Nuclear Receptor Knock-out RatsPoster
The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s), phase II metabolizing enzymes and transporter genes in response to xenobiotics, including prescription drugs.READ MORE
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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