Human Protein Diversity

Coussens, L., Parker, P.J., Rhee, L., Yang-Feng, T.L., Chen, E., Waterfield, M.D., Francke, U., and Ullrich, A., 1986, Multiple, distinct forms ofbovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233 859-866. 

In the phosphorylation assay, we used 33P-ATP to radiolabel those elements of the array that were substrates for the relevant kinase the arrays were then read using a phosphorimager (6). We have also shown that by manipulating the experimental conditions in array-based protein-protein interaction assays, we can readily distinguish true from false positives. For example, we studied the interaction of calmodulin with a diverse array of human proteins in the presence and absence of calcium (2+) ions. Because calmodulin binding should be calcium dependent, we were able to deduce true from false positives based on the array data (Fig. 11) (unpublished data). 

Fig. 11. Binding of calmodulin to an array of a diverse set of 144 human proteins enables novel, calcium-dependent interactions to be identified. (A) Binding of Cy3-labeled anti-His tag antibody to the array allows the relative amount of protein in each spot to be determined. (B) Binding of Cy3-labeled calmodulin allows potential interacting partners to be identified. (C) Histogram showing the relative amount of calmodulin bound per unit protein in the presence of calcium ions. (D) Histogram showing the relative amount of calmodulin bound per unit protein in the presence of calcium ions and a high concentration of a divalent metal ion chelator, ethylenediamine tetra-acetic acid.

Similar to human CAR, alternative splicing of CAR is evolutionarily conserved and has been reported in other species such as mouse [51], rat [68, 69], and chimp [60], indicating their role in providing protein diversity to CAR. 

The first successful therapeutic protein made in a transgenic system was human tissue plasminogen activator regulated by a milk-directed promoter for accumulation in mouse milk [5]. Human growth hormone, which was one of the first proteins produced using recombinant microbial systems in the early 1980s, became the first human protein expressed in plants (tobacco cells) in 1986 [6]. Since then, over 200 biotherapeutics of diverse origin. 

Proteins have many functions in the body. They serve as transporters of hydrophobic compounds in the blood, as cell adhesion molecules that attach cells to each other and to the extracellular matrix, as hormones that carry signals from one group of cells to another, as ion channels through lipid membranes, and as enzymes that increase the rate of biochemical reactions. The unique characteristics of a protein are dictated by its linear sequence of amino acids, termed its primary structure. The primary structure of a protein determines how it can fold and how it interacts with other molecules in the cell to perform its function. The primary structures of all of the diverse human proteins are synthesized from 20 amino acids arranged in a linear sequence determined by the genetic code. 

Fig. 8. Array-based qualitative analysis of immobilisation efficiencies. Flere an array of 48 diverse BCCP-tagged human proteins was printed in triplicate, with 4 replica arrays per slide. Western blot-style analysis, probing with an anti-c-myc antibody, reveals that all proteins are immobilised as expected apparent gaps on the array are due to the printing pattern used here.

Autoantibodies react with the conserved sequence and conformational elements of their cognate antigens these features have made them useful regents in the cloning of cDNAs of expressed proteins from cDNA libraries from a variety of species. However, because of their reactivity with the human protein, they have been used primarily to clone the cDNAs and characterize the primary stmc-tures of numerous human cellular proteins. The diversity of the targets that have been exploited by this approach is illustrated in Tables I and II. 

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