Pull-Down Into Active Inclusion Bodies and Their Application in the Detection of (Poly)-Phosphates and Metal-Ions.

Inclusion bodies are typically ignored as they are considered unwanted protein waste generated by prokaryotic host cells during recombinant protein production or harmful protein inclusions in human cell biology. However, these protein particles may have applications for in vivo immobilization in industrial biocatalysis or as cell-tolerable protein materials for the pharmaceuticals industry and clinical development. Thus, there is a need to in vivo "pull-down" (insolubilize) soluble enzymes and proteins into inclusion bodies. Accordingly, in this study, sequences from the short-chain polyphosphatase ygiF were used to design pull-down tags capable of detecting (poly)-phosphates and metal ions. These tags were compared with the entire CHAD domain from Escherichia coli ygiF and SACS2 CHAD from Saccharolobus solfataricus. The results demonstrated that highly soluble green fluorescent protein variants could be pulled down into the inclusion bodies and could have modified sensitivity to metals and di-/tri-inorganic phosphates.

Insoluble Protein Applications: The Use of Bacterial Inclusion Bodies as Biocatalysts.

Biocatalysis and biotransformations have a broad application in industrial synthetic chemistry. In addition to the whole cell catalysis, purified recombinant enzymes are successfully used for biocatalysis of specific chemical reactions. In this contribution, we report characterization, immobilization, and application of several model target enzymes (D-amino acid oxidase, sialic acid aldolase, maltodextrin phosphorylase, polyphosphate kinase, UDP-glucose pyrophosphorylase) physiologically aggregated within inclusion bodies retaining their biological activity as immobilized biocatalysts.

Magnetization of active inclusion bodies: comparison with centrifugation in repetitive biotransformations.

BACKGROUND: Physiological aggregation of a recombinant enzyme into enzymatically active inclusion bodies could be an excellent strategy to obtain immobilized enzymes for industrial biotransformation processes. However, it is not convenient to recycle "gelatinous masses" of protein inclusion bodies from one reaction cycle to another, as high centrifugation forces are needed in large volumes. The magnetization of inclusion bodies is a smart solution for large-scale applications, enabling an easier separation process using a magnetic field. RESULTS: Magnetically modified inclusion bodies of UDP-glucose pyrophosphorylase were recycled 50 times, in comparison, inclusion bodies of the same enzyme were inactivated during ten reaction cycles if they were recycled by centrifugation. Inclusion bodies of sialic acid aldolase also showed good performance and operational stability after the magnetization procedure. CONCLUSIONS: It is demonstrated here that inclusion bodies can be easily magnetically modified by magnetic iron oxide particles prepared by microwave-assisted synthesis from ferrous sulphate. The magnetic particles stabilize the repetitive use of the inclusion bodies .