Difference between revisions of "Part:BBa K3117026"

Revision as of 17:02, 21 October 2019

scFv against GPA33 with SpyCatcher codon optimized for CHOs

The part BBa_K3117026 is a fusion protein of an anti-GPA33 single-chain variable fragment (scFv) and SpyCatcher (BBa_K3117016.

Usage and Biology

The sequence contains a C-terminal His-tag (BBa_K3117005) for easy purification and detection. Secretion of the protein is ensured by an Igk leader (BBa_K3117006), which directs the produced protein into the secretory pathway. When the protein passes the membrane, the leader segment is cleaved off. By connecting the variable regions of the heavy and the light chain of an anti-GPA33 antibody with a short, flexible GGGGS linker (BBa_K3117028), the scFv retains its antigen-binding ability and is much smaller than a conventional antibody.

The SpyCatcher attached to the scFv belongs to the SpyTag/SpyCatcher system, of which the sequence is derived of the FbaB protein in the bacteria Streptococcus pyogenes. Once it comes into contact with its corresponding other part, the SpyTag (BBa_K3117015), they bind covalently (Hatlem, Trunk, Linke, & Leo, 2019). This allows our part to be used in a modular manner in combination with other molecules carrying the SpyTag.

Since the scFv targets GPA33, which is expressed on more than 95% of colon cancers (Rageul et al., 2009), our part is expected to be optimal for specifically directing its fusion partner (e.g. an effector protein) to colon cancer cells.

Therefore, it was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection.

Sequence and Features

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Characterization and measuring of the composite part