Cry Protein Crystal-Immobilized Metallothioneins for Bioremediation of Heavy Metals from Water

Abstract

Cry proteins have been the subject of intense research due to their ability to form crystals naturally in Bacillus thuringiensis (Bt). In this research we developed a new strategy that allows for the removal of cadmium and chromium from wastewater by using one Cry protein, Cry3Aa, as a framework to immobilize tandem repeats of the cyanobacterial metallothionein SmtA from Synechococcus elongatus (strain PCC 7942). SmtA is a low molecular weight cysteine-rich protein known to bind heavy metals. A series of Cry3Aa-SmtA constructs were produced by the fusion of one, three, or six tandem repeats of SmtA to Cry3Aa. Overexpression of these constructs in Bt resulted in the production of pure Cry3Aa-SmtA fusion crystals that exhibited similar size, crystallinity, and morphology to that of native Cry3Aa protein crystals. All three Cry3Aa-SmtA constructs exhibited efficient binding to cadmium and chromium, with the binding capacity correlated with increasing SmtA copy number. These results suggest the potential use of Cry3Aa-SmtA crystals as a novel biodegradable and cost-effective approach to the removal of toxic heavy metals from the environment.

Genetically Encoded Multienzyme Particles for the Biosynthesis of Putrescine from l-Arginine

“…32,51 Indeed, over the course of developing the Cry3Aa immobilization technology, we have observed the general trend that only proteins of less than 50 kDa could be successfully expressed as highly active Cry3Aa fusion crystals in vivo. 35,36,39,51 We thus sought another strategy to promote its immobilization. Since Cry1Ab is much larger than Cry3Aa (Figure S3B−D), we wondered whether it might be better suited for the immobilization of large proteins such as ODC with respect to preserving the enzyme activity.…”

Emerging Solutions for in Vivo Biocatalyst Immobilization: Tailor-Made Catalysts for Industrial Biocatalysis

“…The previously identified double mutant PML VG was entrapped within the crystals via coproduction, and washing the crystals with buffers at pH values between 4 and 9, or high concentrations of sodium chloride, did not result in substantial release of the enzyme. Furthermore, the strategy has found use in bioremediation, where a metallothionein from Synechococcus elongatus was fused to Cry3Aa . Here, the smtA gene encoding the metallothionein was cloned in up to six tandem repeats for the fusion to Cry3Aa, which generated crystals with similar sizes and morphologies and bound chromium and cadmium with efficiencies positively correlating with the smtA copy number.…”

“…Furthermore, the strategy has found use in bioremediation, where a metallothionein from Synechococcus elongatus was fused to Cry3Aa. 85 Here, the smtA gene encoding the metallothionein was cloned in up to six tandem repeats for the fusion to Cry3Aa, which generated crystals with similar sizes and morphologies and bound chromium and cadmium with efficiencies positively correlating with the smtA copy number. Notably, when nine copies of SmtA were fused to Cry3Aa, the yield was low, and the resulting crystals were less stable, pointing toward the limitation of the approach for incorporating large POIs.…”

Engineering of an in-cell protein crystal for fastening a metastable conformation of a target miniprotein

“…26,[28][29][30][31][32][33][34][35][36][37][38] The high porosity and stability of in-cell protein crystals permit the accumulation of foreign synthetic molecules and proteins. 24,[39][40][41][42][43] These advantageous properties will promote the collection of diffraction data for the target molecules in the crystals. While the structures of metal complexes immobilized in the pores have been determined, 44,45 the structures of exogenous proteins and peptides have not yet been reported because procedures for fixation of the proteins and peptides have not been established.…”