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.
Performance of the D5000 and High Sensitivity D5000 ScreenTape Assays for the 4200 TapeStation SystemPoster
Here, we focus on quantification, sizing, and sensitivity of both D5000 ScreenTape assays.READ MORE
CiPA Phase 2 Study: validation of an automated microelectrode array (MEA) assay of hiPSC-derived cardiomyocyte electrophysiology for cardiac safety evaluationPoster
These results support the use of hSC-CM and MEA technology for preclinical assessment of proarrhythmic risk within the proposed CiPA paradigm, and, more generally, demonstrate that automation of the CM-MEA assay can achieve high reliability and throughput for cardiac risk assessment in vitro.READ MORE
Comments | 0 ADD COMMENT
Diet Therapy Could Be Effective at Starving Cancer
News Apr 24, 2017