To establish a program to prevent diseases in artificially-reared shrimps in ponds, the understanding of the function and mechanism of antibacterial proteins is fundamental. There are some proteins reported to be involved in shrimp protection against microorganisms. One of these proteins is the enzyme lysozyme (E.C. 3.2.1.17). It hydrolyses the b-1,4glycosidic bond of bacterial cell wall peptidoglycans. The amino acid sequence of shrimp lysozyme has been deduced by cloning and sequencing its cDNA (Sotelo-Mundo et al. 2003). This protein presents unique features like two extra stretches of residues inserted at its carboxy-terminal extreme. The function of such extra amino acid residues remains to be elucidated and makes interesting its study. Efforts to purify this protein have been difficult probably due to the very low amounts synthesized in the cells.

In this work, we produced the marine lysozyme by a recombinant expression system, a biotechnological tool that allows synthesizing heterologous proteins in bacteria. The lysozyme coding region from the Pacific white shrimp (Penaeusvannamei) was subcloned into an expression vector. This plasmid DNA (pET5a) encodes the signal for a bacteria to synthesize the foreign protein. The bacterial system contained the promoter region for the T7 bacteriophage RNA polymerase and it is called the T7 expression system (Studier and Moffat, 1986). To perform the subcloning, the polymerase chain reaction (PCR) amplified lysozyme coding sequence and the pET5a vector were digested with restriction enzymes to produce complementary cohesive ends. To connect these DNA fragments, the T4 DNA ligase was used and the recombinant or heterologous plasmid was obtained. The DNA construct was then introduced into a special strain of Escherichia coli compatible with the T7 expression system.

Recombinant bacteria (strain that contains the recombinant plasmid) was grown in liquid media at a constant temperature of 37ºC and continuous shaking (220 rpm). Cell growth was monitored by turbidity or optical density: then, when it reached A₃₂₅=0.6, the inducer IPTG (isopropylthioglycoside) was added to the bacterial culture to induce expression of the shrimp lysozyme, and it was kept in the incubator overnight. The bacteria waspelleted from the media by centrifugation. Then, the bacterial cells were disrupted by sonication, and the shrimp lysozyme was located in inclusion bodies (insoluble fraction). The inclusion bodies are made of denatured proteins which are not folded by the bacteria, but they can be isolated and folded artificially by adding first and removing later denaturing or chaotropic agents such as guanidine hydrochloride or urea. We used such a protocol to recover the shrimp lysozyme from the inclusion bodies. Since the lysozyme was denatured, a refolding step was included. The enzyme solution was dialyzed against a buffered solution containing reagents that facilitated refolding.

To ensure that the refolding process was successful, we tested the lysozyme activity after the renaturation step in a solid phase assay (Shugar et al. 1952). In this assay, an agarose gel is prepared and  Micrococcusluteus is included in the agarose, the enzyme extract is added in small wells made in the gel. When lysozyme is properly folded, it produces a clear halo in the agarose gel due to the lysis of the M. luteus bacteria. This activity assay together with the SDS-PAGE electrophoretic analysis of the recombinant protein have led us to the conclusion that it is possible to obtain the recombinant shrimp lysozyme expressed in E. coli. These results also show that the expressed lysozyme is functional and retains its bacteriolytic activity against Gram (+) bacteria. Further studies of this protein are currently being done.

SHUGAR, D. The measurement of lysozyme activity and the ultraviolet inactivation of lysozyme.BiochimicaetBiophysicaActa, 1952, vol. 8, no. 2, p. 302-309.

SOTELO-MUNDO, R.R.; ISLAS-OSUNA, M.A.; DE-LA-RE-VEGA, E.; HERNANDEZ-LOPEZ, J.; VARGAS-ALBORES, F. and YEPIZ-PLASCENCIA, G. cDNA cloning of the lysozyme of the white shrimp Penaeusvannamei. Fish and Shellfish Immunology, October 2003, vol. 15, no. 4, p. 325-331.

STUDIER, F.W. and MOFFATT, B.A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology, 1986, vol. 189, no. 1, p. 113-30.

Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication.