Published on January 24, 2008
Slide1: Expression of Authentic Human Proteins in Human Cells HumanZyme Inc. 2201 W. Campbell Park Drive, Chicago, IL 60612 Abstract The increasing demand for milligram quantities of human recombinant proteins for drug discovery and diagnostic kit development justifies the need for fast, economic and scalable expression system. We have developed and optimized a highly efficient transient transfection method as well as a stable transfection process for producing recombinant human proteins in an engineered human cell. The cell lines grow in chemically defined medium and in suspension yielding expression levels of mg/L for cytosolic and secreted proteins. In addition, scale-up to 30 liter reactors is possible for producing large quantities of recombinant proteins. Our processes have been used repeatly to produce a large number of protein kinases, phosphatases, coagulation factors and other proteins. Side-by-side comparisons demonstrate that the specific activities of these proteins are significantly higher than those produced in other, non-human expression systems. Production of Difficult-to-Express Kinases in Human Cells Introduction Functional activities of human proteins highly depend on proper folding, phosphorylation, disulfide bridge formation, and proteolytic processing or adequate glycosylation. Ideally, recombinant human proteins are produced in human cells that are likely more authentic in terms of both physical properties and biochemical functions. HumanZyme Technologies HumanZyme has developed a high yield and cost-effective system to increase protein production and reduce cost, including: Engineered human cell line adopted to suspension and serum-free medium Expression vectors driven by strong promoter Large scale transient expression with small quantity of DNA Rapid creation of stable human cell line and adoption to serum-free medium Short time to scale up production 1mg to 100 mg Efficient purification Recombinant Proteins Are Transiently Expressed Large size 370 kDa Difficult to express in non-human cells Successful expression in Human Cells Can be scaled up to mg quantity for drug screening Serum-free suspension culture: adaptation within one week Cost effective transfection process: μg DNA vs mg DNA; High expression level: mg/L Fast timeline: deliverables within four weeks Recombinant Proteins Are Stably Expressed Stable cell line selection: 4 weeks Serum-free suspension culture High expression level: mg/L Easily purified from crude extract Highly active with authentic post-translational modification Fast timeline: deliverables within eight weeks Authentic Human Kinases HumanZyme has successfully produced a large number of highly active human protein kinases and phosphatases. The purity of the recombinant protein is at least 80%. As shown in the tables, side-by-side comparisons of JNK1 and PTEN demonstrate that the specific activities of our proteins are the highest among the commercial products. Services and Products We provide highly active and purified recombinant human proteins ranging from standard lab packs (μg) to 100 mg. Customized production of bulk volume highly active secreted and cytosolic proteins for drug screening, pre-clinical testing, crystallization and diagnostic antibody development Authentic human proteins as retail products for value-added resellers.
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