The Optimizing Expression Of LZ-8 Protein And Primary Assaying Of Its Immunomodulatory Activity

Objective: To obtain sufficient recombinant LZ-8 protein (rLZ-8) for assaying its immunosuppressive activity. Methods: To optimize the experiment, we first analyze the Fip family using biofomatic tools and methods. 12 overlapping oligonucleotides optimized by the usage of the favorite codons in E. coli were synthesized. Then these fragments were connected to form a new LZ-8 gene by PCR method. The pET-28b_lz8 containing new LZ-8 gene was constructed by inserting the new LZ-8 fragment into the expression plasmid pET-28b. The rLZ-8 protein was purified with Ni-NTA matrices from the BL21(DE3) strain transformed with pET-28b_lz8 plasmid and then assayed by using skin transplantation model in mice. Results: The recombinant plasmid was verified with sequencing. High yield (about 70mg/liter of induced culture) and purity (about 90%) of rLZ-8 was achieved by one-step nickel-affinity chromatography. The immunological activity of the mice injected with the purified rLZ-8 was suppressed.Conclusion: The rLZ-8 protein can be highly expressed after codon usage optimization in E. coli.