Functional expression of mammalian adenosine cyclic monophosphate-dependent protein kinase in Saccharomyces cerevisiae
| Autor: | M J, Zoller, K E, Johnson, W M, Yonemoto, L, Levin |
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| Rok vydání: | 1991 |
| Předmět: |
Transcription
Genetic Macromolecular Substances Blotting Western Genetic Vectors Molecular Sequence Data Alcohol Dehydrogenase DNA Saccharomyces cerevisiae Recombinant Proteins Kinetics Mice Escherichia coli Animals Electrophoresis Polyacrylamide Gel Amino Acid Sequence Cloning Molecular Promoter Regions Genetic Protein Kinases Plasmids |
| Zdroj: | Methods in enzymology. 200 |
| ISSN: | 0076-6879 |
| Popis: | The heterologous expression of protein kinases in E. coli has proved difficult and unpredictable. Although the v-abl protein kinase is successfully expressed in E. coli, our experiments on expression of yeast C subunits in E. coli produced large amounts of predominantly insoluble and inactive protein. Attempts to refold the protein proved unsuccessful. In contrast, a major fraction of mouse C alpha expressed in E. coli is soluble and the enzyme in the soluble fraction is active; however, certain mutant forms have proved to be unstable, difficult to purify, or insoluble. In addition, the E. coli system cannot be used to study the biological role of posttranslational modifications specific to eukaryotic systems. Several protein kinases have been expressed in soluble form in insect cells using baculovirus, suggesting that this system is generally more reliable than E. coli. However, the presence and nature of posttranslational modifications in insect cells may be different from that found in the natural source and may affect the biochemical function. In addition, baculovirus expression is not particularly useful for studying biological questions. Mouse C alpha and C beta have been overexpressed in NIH3T3 cells. This approach is useful in characterizing the biochemical properties of C alpha versus C beta, but it may not be an ideal system for studying mutant proteins since wild-type C subunits are still expressed from the chromosomal copies in this genetic background. This small level of wild type may make it difficult to analyze weakly functional mutants, which have activities less than 10% that of wild type. Several cell lines with altered subunits of cAMP-dependent protein kinase have been identified but a strain completely devoid of C subunit has not been adequately characterized for protein structure/function studies. Disruption of the genes encoding cAMP-dependent protein kinase in mammalian cells has not yet been accomplished. This chapter describes a method to express a C subunit of mammalian cAMP-dependent kinase in yeast. We have demonstrated that the mouse C alpha subunit can substitute for its yeast counterpart. Since at least one functional C subunit is required for viability, these results suggest that the yeast substrates important for viability are recognized by the mammalian C subunit. Although the sequence conservation between yeast and mouse C subunit is only about 50%, these results demonstrate that heterologous proteins with relatively low sequence conservation with their yeast counterparts can be functional in yeast.(ABSTRACT TRUNCATED AT 400 WORDS) |
| Databáze: | OpenAIRE |
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