Tail proteins of phage T5: Investigation of the effect of the His6-tag position, from expression to crystallisation

• Automation allowed cloning 8 phage genes with N and Cter His6Tag and no-tag sequences.
• Automated expression tests allowed identifying constructs producing soluble protein.
• Position of the His6-tag can influence protein expression or protein solubility.
• Position of the His6-tag can influence protein oligomerisation or crystal quality.

Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality.

Figure optionsDownload as PowerPoint slide