title: Non-chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules.
abstract: Inspired by nature, synthetic mineralizing proteins have been developed to synthesize various structures of silica-based nanomaterials under environmentally friendly conditions. However, the development of bioprocesses able to assist in the translation of these new materials has lagged the development of the materials themselves. The development of cost-effective and scalable bioprocesses which minimize reliance on chromatography to recover biomolecules from microbial cell factories remains a significant challenge. This paper reports a simplified purification process for a recently reported recombinant catalytic modular (D4S2) protein (M(DPSMKQLADS-LHQLARQ-VSRLEHA)4EPSRKKRKKRKKRKKGGGY; M 13.3 kDa; pI 10.9), which combines a variant of the established designer biosurfactant protein DAMP4 with a new biomimetic sequence (RKKRKKRKKRKKGGGY), providing for a bi-modular functionality (emulsification and biosilicification). The four-helix bundle structure of the protein has been demonstrated to remain stable and soluble under high temperature and high salt conditions, which confers simplified bioprocessing character. However, the high positive charge on the biosilification sequence necessitates removal of DNA contaminants from crude cell-extract at an early stage in the process by adding poly(ethyleneimine) (PEI). In this process, cellular protein contaminants were selectively precipitated by adding Na2SO4to the protein mixture up to a high concentration (1 M) and mixed at high temperature (90°C, 5 min) where D4S2 remained stable and soluble due to its four-helix bundle structure. Further increase of the Na2SO4concentration to 1.8 M precipitated, thus separated, D4S2 from residual PEI. The overall yield of the protein D4S2 was 28.8 mg per 800 mL cells (final cultivation OD600∼2) which gives an approximate 79% D4S2-protein yield. In comparison with the previously reported chromatographic purification of D4S2 protein (Wibowo et al., 2015), the final yield of D4S2 protein is increased fourfold in this study. The bio-produced protein D4S2 was proved to retain it emulsification and biosilicification functionalities enabling the formation of oil-core silica-shell nanocapsules at near-neutral pH and room temperature without the use of any toxic organic solvents, confirming no adverse effects due to bioprocess simplification. This work demonstrates that, through proper bioprocess engineering including the removal of critical contaminants such as DNA, a more efficient, simple, and scalable purification process can be used for the high-yield bio-production of a recombinant templating protein useful in the synthesis of bio-inspired nanomaterials. This simplified process is expected to be easily adapted to recover other mineralizing helix bundle-based functional proteins from microbial cell factories. Biotechnol. Bioeng. 2017;114: 335-343. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Wibowo
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
authors.forename: Guang-Ze
authors.initials: GZ
authors.lastname: Yang
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
authors.forename: Anton P J
authors.initials: AP
authors.lastname: Middelberg
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
authors.forename: Chun-Xia
authors.initials: CX
authors.lastname: Zhao
mesh_terms: D057005:Bioengineering / Q000379:methods*; D005591:Chemical Fractionation; D004926:Escherichia coli / Q000235:genetics; D053769:Nanocapsules / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry / Q000378:metabolism
chemical_list: D053769:Nanocapsules; D011994:Recombinant Proteins; D012822:Silicon Dioxide
keywords: mineralizing proteins; nanocapsules; non-chromatography; recombinant; selective precipitation; silica