By: Caroline Pearson
Membrane proteins are notoriously difficult to produce recombinantly due to their hydrophobicity but a research team from Karolinska Institutet in Sweden have taken inspiration from nature to help overcome this difficulty. Spider silk is composed of large aggregation prone proteins called spidroins. Despite their aggregation prone nature these proteins can be produced at high concentrations without aggregation, before being passed through a narrow duct and converted into spider silk. The amphiphilic nature of spidroins with hydrophilic N- and C-terminal domains flanking hydrophobic centres allows them to arrange into micellar structures with hydrophilic terminal domains sequestering the hydrophobic and aggregation prone regions inside the micelle.
Previous research found that the hydrophilic N-terminal domain (NT) was highly conserved and mediates solubility of spidroins proteins above pH 6.5. However, as the proteins move towards the spinning duct the surrounding pH is decreased and NT form into antiparallel bundles through dipolar interactions which interconnect the proteins, allowing them to be spun into silk. In order to use spiroidins NT domain as a pH insensitive solubility tag, the research team produced a charge-reversed NT with Asp40 and Lys65 swapped in their positions to prevent antiparallel bundle formation. The novel NT mutant (named NT*) was shown to persist as a monomer over a wide pH range through size exclusion chromatography, and NMR spectroscopy revealed that the structure of the NT* was comparable to that of monomeric wild type NT. Promisingly this NT* also showed improved solubility, stability and refolding capacity.
The team then investigated whether NT* could enhance heterologous production of pharmaceutically relevant proteins that had previously been difficult to produce due to their high aggregation tendency. One such protein, SP-C, is an important surfactant protein that allows lungs to re-inflate after expiration of breath. It is also used to prevent respiratory distress in premature infants with insufficient amounts of lung surfactants. It is “perhaps the most hydrophobic peptide isolated from mammals”. Currently, surfactant preparations have to be extracted from animal lungs as recombinant production has proven difficult due to its hydrophobic nature.
N-terminal fusion with wild type NT and NT* enhanced recombinant production of an SP-C analogue compared to other fusion peptides used for comparison. Specifically, NT* resulted in production of mainly soluble fusion protein whereas wild type NT fusion produced mainly insoluble SP-C analogue. These results, along with successful production of a range of other pharmaceutically relevant peptides, suggest NT* as a novel solubility enhancing fusion tag with the potential to allow production of a wide variety of peptides that have previously been refractory to recombinant production. With ~60% of currently available pharmaceutical drugs targeting membrane associated proteins this is a positive step towards improved membrane protein research in drug development.
Source: Nina Kronqvist, Médoune Sarr, Anton Lindqvist, Kerstin Nordling, Martins Otikovs, Luca Venturi, Barbara Pioselli, Pasi Purhonen, Michael Landreh, Henrik Biverstål, Zigmantas Toleikis, Lisa Sjöberg, Carol V. Robinson, Nicola Pelizzi, Hans Jörnvall, Hans Hebert, Kristaps Jaudzems, Tore Curstedt, Anna Rising, Jan Johansson. Efficient protein production inspired by how spiders make silk. Nature Communications, 2017; 8: 15504