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
To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in E. coli, fusion protein of SPV Vp2 was expressed in E. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacter...To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in E. coli, fusion protein of SPV Vp2 was expressed in E. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacterial protein extraction reagent was used to extract the protein. Detergents with different characteristics were used to solubilize the fusion protein, and metal chelating resin （ProBond） with a continuous elusion polyacrylamide gel electrophoresis procedure was employed to purify the fusion protein. SDS-PAGE gel stained with coomassie blue and Western-blotting probed with anti-thio and anti-SPV Vp2 antibodies were used to identify the specificity of the expressed and purified fusion proteins. It was found that the SPV Vp2 protein expressed in E. coli was highly insoluble, and could not be solublized by the commonly used detergent. However, 6 M urea could solubilize the fusion proteins and was then employed for the further purification procedure, but metal chelating resin could not be used for this procedure, because of the loss of the tertiary structure of HP-thiaoredoxin and the metal-binding domain. The technique with continuous elusion polyacrylamide gel electrophoresis yielded a homogenous protein with a single band on the gel stained with coomassie blue and retained reactivity with anti-thio or anti-SPV Vp2 antibodies. It is evident that this technique with successful purification of SPV Vp2 protein has practical significance for the further investigation on the simian parvovirus infection.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.
文摘To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in E. coli, fusion protein of SPV Vp2 was expressed in E. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacterial protein extraction reagent was used to extract the protein. Detergents with different characteristics were used to solubilize the fusion protein, and metal chelating resin （ProBond） with a continuous elusion polyacrylamide gel electrophoresis procedure was employed to purify the fusion protein. SDS-PAGE gel stained with coomassie blue and Western-blotting probed with anti-thio and anti-SPV Vp2 antibodies were used to identify the specificity of the expressed and purified fusion proteins. It was found that the SPV Vp2 protein expressed in E. coli was highly insoluble, and could not be solublized by the commonly used detergent. However, 6 M urea could solubilize the fusion proteins and was then employed for the further purification procedure, but metal chelating resin could not be used for this procedure, because of the loss of the tertiary structure of HP-thiaoredoxin and the metal-binding domain. The technique with continuous elusion polyacrylamide gel electrophoresis yielded a homogenous protein with a single band on the gel stained with coomassie blue and retained reactivity with anti-thio or anti-SPV Vp2 antibodies. It is evident that this technique with successful purification of SPV Vp2 protein has practical significance for the further investigation on the simian parvovirus infection.
