A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification. Materials and Methods: After PCR amplification using NcoI and HindIII-harboring primers, the gene fragment was cloned into pET-28a(+). Escherichia coli BL21 was used for expression of constructed vector and toxin expression was verified by SDS-PAGE. For one-step purification Ni-NTA sepharose affinity chromatography was employed. For examination of purified toxin function, RBC hemolytic test was conducted. Results: The results showed that the FraC-coding gene was successfully cloned between NcoI and HindIII restriction sites and purified with affinity chromatography. Densitometric analysis represented the purity of approximately 97%. Hemolytic test indicated the purified FraC had remarkable lytic activity on RBC and almost lysed 50% of cells at the concentration value of 6. 25 nM. Conclusion: The results indicated that not only purified toxin preserved its activity during expression and purification processes but also exerted its function at lower concentrations so that even the 0. 09 nM displayed hemolytic effect.

Background: In the field of recombinant protein production, downstream processing, especially protein purification, is critical and often the most expensive step. Carbohydrate binding module 64 (CBM64) was shown in 2011 to bind efficiently to a broad range of cellulose materials. Methods: In this study, we developed a protein purification method using nanocrystalline cellulose embedded in a polyacrylamide monolith cryogel and CBM64 affinity tag linked by intein to PD1 as a model protein. The CBM64-Intein-PD1 gene cassette was expressed in E. coli. Following cell lysis, CBM64-Intein-PD1 protein bound to the monolith PA-NCC cryogel. After washing and reducing the pH from 8. 0 to 6. 5, the intein underwent self-cleavage, resulting in the release and elution of pure PD1 protein. Results: The synthesized monolith column had a porous structure with an average pore size of 30 μ, m and a maximum binding capacity of 497 μ, g per gram of dried column. The yield of this purification method was 84%, while the yield of the His tag-acquired CBM64-Intein-PD1 method was 89%. Conclusions: We used cellulose as support for affinity chromatography, which can be used as a costeffective method for protein purification.

DapE is an enzyme which is highly essential in lysine biosynthetic pathway for the growth and survival of Mycobacterium tuberculosis (Mtb) and other bacterial species although absent in human host. However, the sequence identity of Mtb-DapE with other mycobacterium and bacterial species estimated between 93 to 75% and approximately 50%, respectively. DapE is a metallo-enzyme requires few of transition metals for activity and bacterial proliferation. The homology model based structural studies including published structures revealed an availability of zinc interacting conserved amino residues in Mtb-DapE. In this study, we purified full length recombinant Mtb-DapE as tag-free (Mtb-DapETagFree) protein from inclusion bodies using zinc-NTA column. The single step purified protein observed with 96-98% purity and high yield. The indirect ELISA had 40% sensitivity using Mtb-DapE as an antigen against bovine tuberculosis (bTB) serum samples. IFA analysis with clear fluorescent spots of Mtb native DapE antigen in (3+) human TB positive sputum samples and recombinant Mtb-DapE positive control against Mtb-DapE polyclonal antibody are highly encouraging. The ELISA and IFA results incite for the consideration of Mtb-DapE in future development of quick and ideal TB detection assay along with other mycobacterium antigens. The future advanced detailed structural and functional studies using this highly purified and tag-free Mtb-DapE may provide discovery of antitubercular drug(s) and promising inhibitory molecules.