Patrick Stancombe

Isolation of the gene and large-scale expression and purification of recombinant Erythrina cristagalli lectin

Using polymerase chain reaction, the coding sequence for Erythrina cristagalli lectin (ECL) has been cloned and expressed in Escherichia coli. The amplified DNA sequence of ECL is highly homologous to that previously reported for Erythrina corallodendron lectin (ECorL), confirming the absence of introns in the ECL gene. The polypeptide sequences of ECL and ECorL have been compared and five amino acids have been identified that differentiate the two proteins. Recombinant E. cristagalli lectin (recECL) was expressed in E. coli from a genomic clone encoding the mature E. cristagalli lectin gene. Constitutive expression localised recombinant protein in inclusion bodies, which were solubilised, and recECL, subsequently refolded and purified by lactose affinity chromatography. Significant advantages were observed for purification from inclusion bodies rather than from a clone optimised to express soluble protein. A large-scale purification scheme has been developed that can prepare functional recECL from inclusion bodies with a yield of 870 mg/l culture. By the range of characterisation methods employed in this study, it has been demonstrated that recECL is functionally equivalent to native ECL obtained from the E. cristagalli plant. In addition, characterisation of the binding of radiolabelled recECL to cultured dorsal root ganglia demonstrated that recECL binds to a single pool of receptors.

Engineering botulinum neurotoxin domains for activation by toxin light chain

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50‐kDa endopeptidase light chain and translocation domain (N‐terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single‐chain proteins and then cleaved post‐translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self‐activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross‐activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled‐coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions.

Structures of Engineered Clostridium Botulinum Neurotoxin Derivatives

Targeted secretion inhibitors (TSIs) are a new class of engineered biopharmaceutical molecules derived from the botulinum neurotoxins (BoNTs). They consist of the metalloprotease light chain (LC) and translocation domain (Hn) of BoNT; they thus lack the native toxicity towards motor neurons but are able to target soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) proteins. These functional fragment (LHn) derivatives are expressed as single-chain proteins and require post-translational activation into di-chain molecules for function. A range of BoNT derivatives have been produced to demonstrate the successful use of engineered SNARE substrate peptides at the LC-Hn interface that gives these molecules self-activating capabilities. Alternatively, recognition sites for specific exoproteases can be engineered to allow controlled activation. Here, the crystal structures of three LHn derivatives are reported between 2.7 and 3.0 Å resolution. Two of these molecules are derivatives of serotype A that contain a SNARE peptide. Additionally, a third structure corresponds to LHn serotype B that includes peptide linkers at the exoprotease activation site. In all three cases the added engineered segments could not be modelled owing to disorder. However, these structures highlight the strong interactions holding the LHn fold together despite the inclusion of significant polypeptide sequences at the LC-Hn interface.