Cloning, expression and purification of the general stress protein Yhbo from Escherichia coli
Jad Abdallah, Renee Kern, Abderrahim Malki, Viola Eckey, Gilbert Richarme
Abstract
We cloned, expressed and purified the Escherichia coli yhbO gene product, which is homolog to the Bacillus subtilis general stress protein 18 (the yfkM gene product), the Pyrococcus furiosus intracellular protease PfpI, and the human Parkinson disease protein DJ-1. The gene coding for YhbO was generated by amplifying the yhbO gene from E. coli by polymerase chain reaction. It was inserted in the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21(DE3) E. coli strain transformed with the YhbO-expression vector pET-21a-yhbO, accumulates large amounts of a soluble protein of 20 kDa in SDS-PAGE that matches the expected YhbO molecular weight. YhbO was purified to homogeneity by HPLC DEAE ion exchange chromatography and hydroxylapatite chromatography and its identity was confirmed by N-terminal sequencing and mass spectrometry analysis. The native protein exists in monomeric, trimeric and hexameric forms.
Create a lesson
Related papers
Learning Interpretable Tumor Microenvironment Representations by Fitting Pan-Cancer Cell State-Niche Correlation
Xiao Xiao, Jiashu He, Shiyang Zhang et al.
Optimizing RNA yield using deep neural networks coupled to massively parallel screening
Dinghai Zheng, Justin Hong, Jun Wang et al.
A Conditional Structure-Aware Generative Transformer for Multi-Objective Design of m1Ψ-Modified RNA 5' UTRs
Narges Zarnaghinaghsh, Ahmadreza Mofayezi, Byung-Jun Yoon
mLS-GKM: Efficient Multi-class Regulatory Sequence Classification with Gapped k-mer SVMs
Kieran Howard, Nathan Harmston
DNA Methylation Profiling in Melanoma: From Lesion Classification to Therapeutic Stratification
Jana T. Winterstein, Lukas Heinlein, Günter Raddatz et al.
Hepatitis C Virus Genotyping with a Transformer Neural Network
Ariella Aro, Taimá Furuyama, Marcelo R. S. Briones et al.