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.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE