The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult ...The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult task for structure-function studies. Based on codon optimization for protein expression in Escherichia coli, we have been able to produce, in soluble form, the C-terminal domains of Inscuteable and Staufen as chimeras with N-terminal maltose binding protein tag that contains a rigid linker between them for feasible crystallization. In addition, using an optimized synthetic gene, corresponding to the amino acid region 250 - 623 of Inscuteable fused to glutathione-S-transferase, low-scale expression experiments showed production of soluble protein. Finally, eukaryotic expression of Inscuteable in the methylothropic yeast Pichia pastoris failed to produce the Drosophila protein at detectable amounts, reinforcing the fact that E. coli still was the microorganism of choice for high-yield protein expression.展开更多
文摘The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult task for structure-function studies. Based on codon optimization for protein expression in Escherichia coli, we have been able to produce, in soluble form, the C-terminal domains of Inscuteable and Staufen as chimeras with N-terminal maltose binding protein tag that contains a rigid linker between them for feasible crystallization. In addition, using an optimized synthetic gene, corresponding to the amino acid region 250 - 623 of Inscuteable fused to glutathione-S-transferase, low-scale expression experiments showed production of soluble protein. Finally, eukaryotic expression of Inscuteable in the methylothropic yeast Pichia pastoris failed to produce the Drosophila protein at detectable amounts, reinforcing the fact that E. coli still was the microorganism of choice for high-yield protein expression.
