In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

Abstract

Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the in vivo self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable ␣-amylase from Bacillus licheniformis, N-acetyl-D-neuraminic acid aldolase (NanA) from Escherichia coli, and organophosphohydrolase (OpdA) from Agrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The ␣-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert N-acetylmannosamine and pyruvate to Nacetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.

GFP Scaffold-Based Engineering for the Production of Unbranched Very Long Chain Fatty Acids in Escherichia coli With Oleic Acid and Cerulenin Supplementation

“…Previous studies have developed a mutated version of GFP, termed split-GFP or self-assembly GFP, where the gene encoding the first 10 beta sheets (GFP1-10) and the gene encoding the last beta sheet (GFP11) are expressed separately but are able to self-assemble and fluoresce (Cabantous et al, 2005;Cabantous and Waldo, 2006). Selfassembly GFP has been previously reported to enhance protein solubility, reduce formation of bacterial inclusion bodies and for the immobilization of various enzymes in recombinant E. coli BL21 (DE3) (Cabantous and Waldo, 2006;Venning-Slater et al, 2014). It has also been applied for topology analyses of membrane-bound enzymes in N. benthamiana, without affecting their biological functionality (Xie et al, 2017).…”

“…We focus on the expression of four enzymes involved in the plantbased VLCFA biosynthesis of A. thaliana using a synthetic polycistronic expression cassette in combination with a selfassembly GFP system. The application of the self-assembly GFP system is a highly innovative method to enable simultaneous solubilization and guided interaction of the plant-derived fatty acid biosynthesis enzyme systems (Cabantous et al, 2005;Cabantous and Waldo, 2006;Venning-Slater et al, 2014;Xie et al, 2017). Furthermore, oleic acid and cerulenin were required to initiate VLCFA biosynthesis.…”

Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

“…These values compared favourably with the previously reported activities of immobilised NanA between 2.5–36 U/mg protein. Moreover, an alternative GFP fusion protein particle approach for immobilisation of NanA resulted in specific NanA activity of 76 mU/mg [51].…”

“…The direct comparison of specific enzymes immobilised to PHA beads and immobilised to GFP fusion protein particles is particularly relevant because of the similarity in fusion protein construction. In all three cases, NanA, α-amylase, and OpdA the performance under the same reaction conditions is less for the GFP fusion protein particles when compared to the equivalent PHA bead [51]. This is likely to be due to differences in the way the different particles form and the resultant orientation and surface exposure of the respective enzyme.…”

An Optical Biosensor from Green Fluorescent Escherichia coli for the Evaluation of Single and Combined Heavy Metal Toxicities

“…Segami et al , [ 21 ] reported the dynamics of vacuoles and H + -pyrophosphatase visualized by monomeric GFP in Arabidopsis. Several applications of GFP including enzyme immobilization in food processing and biotechnological industries [ 22 ] and screening of multiple membrane proteins in E. coli [ 23 ]. In short, GFP-modified cells were utilized as a biomarker because they can be employed without the need of exogenous substrate.…”