Molecular characterization, expression analysis, recombinant expression and refolding of the chicken-type and goose-type Lysozymes from Totoaba (Totoaba macdonaldi)

Abstract

Lysozyme (EC 3.2.1.17) plays a critical role in the innate immune response against bacterial pathogens, hydrolyzing the peptidoglycan, which shapes their cell walls. C-type and g-type lysozymes exhibiting unique features are found in teleost fish species, suggesting lysozyme´s critical role in these organisms´ defense system. Totoaba (Totoaba macdonaldi) is an endemic and critically endangered species from de Gulf of California, Mexico. Totoaba´s aquaculture demands further research towards the development of immunoprophylactic strategies and improve this species large-scale farming production. In this study, the c-type (TmLyzc) and g-type (TmLyzg) lysozymes genes from totoaba were cloned and characterized. The TmLyzc and TmLyzg full-length cDNA sequences were of 432 bp and 582 bp, encoding to polypeptides of 143 and 193 amino acids, respectively. The amino acid sequences shared high identities (90-60%) and close phylogenetic relationships with other fish and higher vertebrate lysozymes, as well as structural and functional domains typical of the lysozyme superfamily. Expression analysis by qRT-PCR showed that TmLyzc and TmLyzg were mainly expressed in the stomach, pyloric caeca, and heart. The findings suggest that lysozymes have a significant role in defense of totoaba against bacterial infections and also may be involved in digestion. In the second leg of this research, TmLyzc and TmLyzg were recombinantly expressed in E. coli BL21 (DE3) at 25 °C with 0.5 mM IPTG, isolated from inclusion bodies and subjected to in vitro refolding experiments using the PierceÒ Protein Folding system. Refolding both lysozymes did not yield active enzymes, either because the proteins were partially folded or because fusion tags somehow hindered their active site. Although the tested refolding conditions were found promising, further optimization is required to recover active enzymes.