| Autor: |
John M. Richardson, Nancy B. Rankl, John E. Hale, Gerald W. Becker, Lisa M. Churgay |
| Rok vydání: |
1997 |
| Předmět: |
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| Popis: |
Publisher Summary Baculovirus infected insect cells represent an effective system for the overexpression of heterologous proteins. The level of soluble K-ras produced by these cells is quite high as seen in the chapter. Kirsten-ras is the most frequently activated oncogene in human tumors. Activated K-ras is bound to GTP and possesses intrinsic GTPase activity that leads to its inactivation. Determination of the crystal structure of K-ras is likely to aid in the rational design of anticancer therapeutics. Truncated K-ras is expressed in baculovirus infected insect cells that has been suggested as an appropriate system for its production. K-ras is expressed in abundance from these cells and is purified to apparent homogeneity evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Analytical Diethylaminoethyl (DEAE) chromatography and electrospray-ionization mass spectrometry (ESI-MS) of the purified protein indicated substantial heterogeneity. The different proteins have been characterized by tryptic mapping utilizing LC-MS. This analysis indicated the presence of at least four different N-terminal variants of K-ras and additional heterogeneity because of dissociation of bound nucleotide, indicating that unwanted cellular processing of proteins may occur in baculovirus infected cells. The study described in this chapter includes discussion on the utility of analytical DEAE coupled with ESI-MS in the determination of bound nucleotide. This technique may be useful in evaluation of K-ras protein that has been loaded with GTP analogues to activate it. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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