Engineered Hydrophobin as a Crystallization Inhibitor for Flufenamic Acid.

Hydrophobins are multifunctional, highly surface-active proteins produced in filamentous fungi. Due to their surface-active properties, resistance to degradation, and potential immunological inertness, hydrophobins have been used in many applications such as protein purification, increasing implant biocompatibility, increasing water solubility of insoluble drugs, and foam stabilizers for food products. To further explore surface-active and self-assembly properties of hydrophobins, we evaluated an engineered, recombinant hydrophobin (class II type 1, HFB1) as a potential crystallization inhibitor for maintaining drug supersaturation for an amorphous drug delivery system. A supersaturation-precipitation method was employed utilizing an ultraviolet (UV) fiber optic system for tracking precipitation kinetics of a model drug, flufenamic acid (FA), that was selected due to its low aqueous solubility in its crystalline form. The effectiveness of HFB1 as a crystallization inhibitor was compared with commonly used pharmaceutical grade polymeric crystallization inhibitors. The following polymers were selected to compare with HFB1: methocel (A4C grade), methocel (K15M grade), Kollidon vinylpyrrolidone-vinyl acetate (VA64), and hydroxypropyl methylcellulose acetate succinate (HPMCAS) (MF grade). The supersaturation-precipitation experiments concluded that HFB1 outperformed all polymers tested in this study and can potentially be used as a crystallization inhibitor at significantly lower concentrations in amorphous drug delivery systems. Dynamic light scattering (DLS) and circular dichroism (CD) results suggest a crystallization inhibition mechanism in which HFB1 functions differently depending on whether flufenamic acid is molecularly dispersed but supersaturated relative to its crystalline solubility or it has exceeded its amorphous solubility limit and exists as a phase-separated drug-rich colloid.

Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris.

Hydrophobins are small highly surface-active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression host Pichia pastoris. One-, 2-, and 3-copy-HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction. Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression of PDI1 or ERO1 caused insignificant changes in HFBI secretion in 1- and 2-copy strains, but a statistically significant HFBI secretion increase in 3-copy strain. KAR2 overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3-copy-HFBI secreting 22±1.6 fold more than the 1-copy-HFBI/no chaperone strain. The highest increase was seen in 3-copy-HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1-copy-HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small-scale format used in this study.

Hydrophobins: multifunctional biosurfactants for interface engineering.

Hydrophobins are highly surface-active proteins that have versatile potential as agents for interface engineering. Due to the large and growing number of unique hydrophobin sequences identified, there is growing potential to engineer variants for particular applications using protein engineering and other approaches. Recent applications and advancements in hydrophobin technologies and production strategies are reviewed. The application space of hydrophobins is large and growing, including hydrophobic drug solubilization and delivery, protein purification tags, tools for protein and cell immobilization, antimicrobial coatings, biosensors, biomineralization templates and emulsifying agents. While there is significant promise for their use in a wide range of applications, developing new production strategies is a key need to improve on low recombinant yields to enable their use in broader applications; further optimization of expression systems and yields remains a challenge in order to use designed hydrophobin in commercial applications.

A Structural and Functional Role for Disulfide Bonds in a Class II Hydrophobin.

Hydrophobins are multifunctional, highly surface active proteins produced in filamentous fungi and can be identified by eight conserved cysteine residues, which form four disulfide bridges. These proteins can be subdivided into two classes based on their hydropathy profiles, solubility, and structures formed upon interfacial assembly. Here, we probe the structural and functional roles of disulfide bonds for a class II hydrophobin in different interfacial contexts by reducing its disulfides with 1,4-dithiothreitol and blocking the free thiols with iodoacetamide and then examining the protein secondary structure, emulsification capability, hydrophobic surface wetting, and solution self-assembly. Changes in circular dichroism spectra upon reduction and blocking of disulfides are consistent with an increase in the level of random coil secondary structure. Emulsification of octane in water using reduced and unreduced forms of class II hydrophobin showed a substantial loss of emulsification ability without disulfides and stable emulsion formation for hydrophobin with disulfides. Additionally, water contact angle measurements performed on polytetrafluoroethylene treated with solutions of reduced and unreduced hydrophobin showed efficient wetting of the hydrophobic surface for unreduced samples only. Lastly, Förster resonance energy transfer (FRET) was used to assess the role of disulfides in self-assembly in solution, and near complete loss of the FRET signal is consistent with a model in which solution self-assembly does not occur after reduction and blocking of the disulfides. From this, we conclude that, in contrast to class I hydrophobins, the disulfides of this class II hydrophobin are required for protein structural stability, surface activity at both liquid-liquid and solid-liquid interfaces, and solution self-assembly.