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.
Additionally, a number of important cytokines such as those belonging to the TGF-b superfamily are difficult to produce in sufficient quantity due to inadequate proteolytic processing, protein folding, or other post-translational modifications that occur in the nonhuman cell-expression systems.
HumanZyme’s work with VEGF165 and IL-4 demonstrates that the recombinant cytokines from human cells are differentiated from the nonhuman-cell version and can be used as preferred reagents for research and antibody development.
VEGF165 plays a prominent role in normal and pathological angiogenesis. It has been demonstrated that inhibition of VEGF activity by treatment with a mAb specific for VEGF can suppress tumor growth in vivo. Currently, commercially available VEGF165 protein reagents are produced from nonhuman cells including E. coli and insect cells.
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.
The bioactivity of VEGF165 was determined by its ability to induce proliferation of human umbilical vein endothelial cells. This indicated that VEGF165HuXp is sixfold more active than the E. coli-expressed protein (Figure 2A).
IL-4 plays a critical role in the development of allergic inflammation and asthma. Commercially available IL-4 protein reagents are produced from E. coli with a molecular mass of 14 kD (Figure 1B). This compares with the IL-4HuXp from human cells, which migrates as a major band of 19 kD due to glycosylation. The biological activity of IL-4 was determined by the dose-dependent stimulation of the proliferation of human TF-1 cells. As shown in Figure 2B, IL-4HuXp has fourfold higher potency than the E. coli expressed cytokine.
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.
The mAbs raised against a full-length protein from E. coli recognize the IL-4 protein from E. coli under both reducing and nonreducing conditions (Figure 1D). In contrast, only the protein under nonreducing conditions is detected 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.
Currently, kinases are predominantly produced in nonhuman cells (e.g., E. coli or insect cells), many of which require protein truncation and/or in vitro activation steps due to the limitations of the expression system. HumanZyme has expressed more than 100 recombinant human protein kinases that are full length and in vivo activated including difficult to produce ATM and mTOR kinases.
Using p38a as an example, HumanZyme demonstrated that the properties and inhibition profiles of the human protein kinases produced in human cells are differentiated from versions of the same kinase that were produced in nonhuman-cell systems. This will allow researchers to avoid pursuing false-negative leads and missing promising targets.
p38aHuXp was produced and activated in human cells in the presence of arsenite. Sample kinases from Vendor A and B were expressed and purified from E. coli, in vitro activated by MKK6, and repurified. SDS PAGE analysis shows that p38a produced in the human cell-expression system is pure with a dominant band of 60 kD and minor band of endogenous human GST of 23 kD. This was confirmed by MS analysis, and no other contaminant proteins were found.
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.
With the widespread availability of authentic and cost-effective human protein reagents from human cells, we are now at the point of a paradigm shift where authentic proteins will be widely used as the preferred research and diagnostic reagents as well as for drug screening and antibody development, thereby greatly improving the speed and quality of both basic research and pharmaceutical development.
Soon Seog Jeong, Ph.D., is director of research, Hui Feng, Ph.D., and Gabriel Cubberley are research scientists, and Maria Borovilos is research associate at HumanZyme. Web: www.humanzyme.com. E-mail: email@example.com.