The holy grail of bioseparation for biotechnology is the desire to recover a maximum amount of desired valuable protein, i.e. protein, at high purity with minimum losses during production and downstream processing. One approach is to reduce the number of recovery steps while increasing product purity and decreasing yield losses. Fusion proteins with appended binding domains (e.g. maltose binding domain) and external cleavage control (e.g. inteins) offer an opportunity to do this. However, for this approach to be successful, the desired protein should not have unnatural residues at a terminus and cleavage control should occur with an external trigger and only when desired. To date, such external control is unavailable. Inteins are proteins which autocatalytically perform peptide cleavage and ligation reactions, allowing traceless excision from within the context of a host protein. Though inteins have been effectively used as a self-cleaving affinity tag in single-step purification platforms in bacteria, it has been difficult to adapt inteins to mammalian and other complex cell systems due to a lack of control over premature cleavage and the need for a residual cysteine at the N-terminus of the desired protein in these systems. We have engineered an intein with a pH switch, that undergoes aberrant chemistry and forms a thiazoline ring at the N-terminal splicing junction. This ring prevents cleavage chemistry and traps precursor when present, but can be hydrolyzed at low pH. The result is an intein switch with robust pH and temperature sensitivity that produces a high yield of precursor during cellular production conditions, but undergoes efficient autocatalytic affinity-tag cleavage under selected post-purification conditions. A pH cycling strategy to induce N-terminal cleavage is proposed, which may be of interest for bioprocessing and other biotechnological applications requiring a splicing activity switch. Also, by locating the desired protein at the N-terminus in place of the natural N-extein, no residual cysteine is required for the specific Mtu RecA intein. These two new developments - an intein switch and N-terminal cleavage - now allow for single step protein purification platforms.

Engineering an intein with cleavage control for single step purification platforms

255th National Meeting (2018)

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