Abstract
Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
Highlights
Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins
Considering that wild-type N-terminal domain (NT) (NTwt) confers solubility primarily in its monomer conformation above pH 6.5, we designed a charge-reversed double mutant with the intention to increase the useful pH range by preventing dimerization, while still maintaining the ability to arrange into micelle-like particles in fusion with a TM peptide (Fig. 1b), in agreement with how the nonpolar parts of spidroins are protected within micellar structures (Fig. 1a)
We showed that NT* allows for soluble expression of the TM peptides rSP-C33Leu and rKL4 as well as of the surfactant protein fragment rfhSP-D and the cholecystokinin peptide recombinant production of human CCK-58 (rCCK-58), all which previously have been reluctant to recombinant production in E. coli
Summary
Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. Studies propose that the unusually high solubility is attributed to the amphiphilic nature of spider (and silkworm) silk proteins, allowing them to arrange into micellar structures with the hydrophilic terminal domains sequestering the more hydrophobic repeat regions from the aqueous surrounding, preventing premature b-sheet formation[26,27] (Fig. 1a). SP-C33Leu and KL4 recapitulate the function of native surfactant peptides, including transmembraneous insertion[52,53], but are less prone to aggregate[49] and are feasible to produce for development of synthetic surfactant preparations. KL4 surfactant is approved by the FDA for prophylactic treatment of premature infants[54,55], and CHF5633 based on chemically synthesized SP-C33Leu is in clinical trials for neonatal RDS56
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