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Tutorials : Apr 15, 2008 ( )
Producing Proteins for New Drug Development
Authentic Reagents Can Be Cost-Effectively Derived from Human Cells
Recombinant proteins (r-proteins) have increasingly become important in fundamental research as well as in preclinical and clinical work. Because many r-proteins are of human origin, cultivated human cells are the optimal choice to ensure authentic biophysical properties and functional activities, which depend on proper folding, phosphorylation, disulfide bridge formation, proteolytic processing, and glycosylation (Table 1).
Current expression systems, however, are inefficient, and it is often a daunting task to produce a sufficient amount of quality proteins (1–100 mg) at an acceptable cost. To address this deficiency, HumanZyme has developed HumaXpress™ to produce active recombinant human proteins in a human HEK-derivative cell line in serum-free and chemically defined media.
Using this technology, the company’s scientists have produced a large number of active and authentic human cytokine and kinase reagents designed to meet the needs of researchers. Comparative studies indicate that these recombinant human proteins from human cells are superior to those produced in nonhuman cell systems.
Most human cytokines are glycoproteins less than 30 kD in size. Due to their central role in the immune system, cytokines are involved in a variety of immunological, inflammatory, and infectious diseases and are widely used in research, diagnostics, and therapeutics. Currently, these proteins are predominantly produced in nonhuman cell-expression systems (e.g., E. coli, SF9, or CHO) and therefore lack authenticity due to the absence of physiologically relevant glycosylation.
HumanZyme has produced VEGF165HuXp from human cells adapted to chemically defined media. As shown in Figure 1A, the molecular mass of the E. coli-expressed protein in monomer is 18 kD. This compares with the VEGF165HuXp that migrates as a band of 28 kD due to glycosylation.
Cytokines produced in E. coli are not glycosylated and may expose cryptic or normally hidden epitopes. Hence, antibodies may have different affinities for native human proteins compared to the E. coli-produced proteins.
Indeed, Western blot analysis shows that mAbs raised against a full-length protein from insect cells recognize the VEGF165 protein from E. coli as well as other highly reactive species that may correspond to micro-aggregates (Figure 1C). In contrast, only one band is seen with the human-cell version.
Due to their critical role in intracellular communication, dysregulation of protein kinases has been implicated in as many as 400 human diseases including cancer, diabetes, heart disease, neurological disorders, and rheumatoid arthritis. Hence, protein kinases are important for drug design and screening.
The Km.ATP for p38aHuXp is 109 ± 12 µM while the Km was 212 ± 26 µM for the Vendor A preparation. The Km of 120 µM was found with Vendor B enzyme. The IC50 values were determined for 14 known kinase inhibitors (Table 2). While the IC50 values for SB-202190 (the known p38a-selective inhibitor) for both p38a preparations were similar (0.02 µM and 0.03 µM respectively), there is clearly a difference in the sensitivity to the inhibitors between p38aHuXp and Vendor A preparations (Figure 3 and Table 2).
The Vendor A preparation was only sensitive to AMP-PNP (a nonhydrolysable ATP analog). Yet, the protein was sevenfold less sensitive than p38aHuXp, which is consistent with its higher Km. p38aHuXp, on the other hand, had measurable IC50 values against staurosporine, K252a, Ro 31-8220, KT5720, and SB-202190. The inhibition profile of Vendor A kinase is comparable to that of Vendor B (Table 2).
Taken together, the current study demonstrates that the properties of the human cytokines and protein kinases produced in human cells are distinct from those produced in nonhuman-cell systems. Ever since Genentech scientists produced the first recombinant human protein in E. coli in 1977, r-protein expression in heterologous hosts has played a critical role in the launch of the entire modern biotechnology industry.
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