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Microbial enzymes are increasingly finding applications as therapeutics due to their targeted activity and minimal side effects. Serratiopeptidase, also known as a miracle enzyme, has already proved its potential as an anti-inflammatory, mucolytic, fibrinolytic, analgesic in many studies. A cost effective, bioreactor level production process has been described here comprising of the fed-batch fermentation to produce recombinant serratiopeptidase protein expressed as a fusion construct. High yield of cell mass as well as protein was obtained by the optimization of bioreactor parameters. The downstream solubilization and purification processes were also optimized to achieve maximum yield of pure, active serratiopeptidase protein. A final yield of 2.5 ± 0.764 g L−1 of protein was obtained, having 8382 ± 291 U mg−1 of specific caseinolytic activity. Additionally, a novel, unexpected self-proteolytic activity of the enzyme that cleaves the N-terminal 6× His-SUMO fusion tag along with the enzyme propeptide, thus yielding a mature serratiopeptidase, was also found.

The fibronectin leucine rich transmembrane (FLRT) protein family consists in humans of 3 proteins, FLRT1, -2, and -3. The FLRT proteins contain two extracellular domains separated by an unstructured linker. The most membrane distal part is a leucine rich repeat (LRR) domain responsible for both cis- and trans -interactions, whereas the membrane proximal part is a fibronectin type III (FnIII) domain responsible for a cis -interaction with members of the fibroblast growth factor receptor 1 (FGFR1) family, which results in FGFR tyrosine kinase activation. Whereas the structures of FLRT LRR domains from various species have been determined, the expression and purification of recombinant FLRT FnIII domains, important steps for further structural and functional characterizations of the proteins, have not yet been described. Here we present a protocol for expressing recombinant FLRT-FnIII domains in inclusion bodies in Escherichia coli . His-tags permitted affinity purification of the domains, which subsequently were refolded on a Ni-NTA agarose column by reducing the concentration of urea. The refolding was confirmed by circular dichroism (CD) and 1 H-NMR. By thermal unfolding experiments we show that a strand-strand cystine bridge has significant effect on the stability of the FLRT FnIII fold. We further show by Surface Plasmon Resonance that all three FnIII domains bind to FGFR1, and roughly estimate a K d for each domain, all K d s being in the µM range.

A method for the production and purification of biologically active recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), expressed in Escherichia coli, has been developed. Washing of an inclusion body fraction which allows the removal of numerous ballast proteins and on-column refolding of rhGM-CSF are specific characteristics of the method. The refolding efficiency reached 70%; the purity of the preparation constituted 95%. The biological activity was similar to those of other recombinant hGM-CSF analogs.

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 M: guanidine hydrochloride (Gdm.HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm.HCl with a G25 column and simultaneously dissolved in 8 M: urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding.

Recombinant human interleukin-1 receptor antagonist (rHuIL-1ra) was produced in E. coli as an inclusion body. rHuIL-1ra was purified to Over 98% purity by anion exchange chromatography after on-column refolding. The optimized processes produced more than 2 g pure refolded rHuIL-1ra per 1 l culture, corresponding to a 44% recovery, without an intermediate dialysis step. Refolded rHuIL-1ra had full biological activity with the MTT assay. An intramolecular disulfide linkage in the oxidized recombinant protein was suggested by data from HPLC and non-reducing SDS-PAGE.

Halolysins are extracellular proteases secreted by halophilic archaea for nutritional purposes. They bear great application potentials in various industries. Yet the diversity of halolysins remains underexplored. In this study, a halolysin from the extremely halophilic archaeon Haladaptatus sp. DYF46 (Hly Hap ) was identified to be a novel type of halolysin without C-terminal extension (CTE). Addition of the CTE of a halolysin from Halococcus salifodinae to Hly Hap did not significantly affect its extracellular proteolytic activity. Mature Hly Hap was generated from recombinant Hly Hap precursor by high-affinity column refolding. Hly Hap displayed optimal activity at 0.25-0.50 M NaCl, 45 °C and pH 8.5-9.0. Interestingly, Hly Hap preferred a low salinity and was stable in a broad range of salinity, albeit from an extremely halophilic archaeon. Ca 2+ and Mg 2+ significantly promoted Hly Hap activity. Hly Hap activity was stable with organic solvents and detergents. The K m and V max values of Hly Hap against azocasein were 0.018 mM and 7,179 U/mg, and those against succinyl-Ala-Ala-Pro-Phe-pNA were 0.32 mM and 3×10 6 μmol/min/μg, respectively. The unusual traits of Hly Hap , a novel type of halolysin without CTE, may endow it with strong potential for various industrial uses, such as biocatalysis in fluctuating salinities and aqueous-organic solvent. Key points • This is the first report of a novel type of halolysin without C-terminal extension • Hly Hap was obtained by heterologous expression and high-affinity column refolding • Hly Hap exhibited good salinity tolerance

This chapter contains sections titled: Introduction Protein Folding In Vivo Molecular Simulation of Protein Refolding and Aggregation Refolding Methods Applications References