A 61kDa antibacterial protein isolated and purified from the hemolymph of the American cockroach Periplaneta amreicana
2003
Seraj, U.M. | Hoq, M.I. | Anwar, M.N. | Chowdhury, S. (Chittagong Univ. (Bangladesh). Dept. of Microbiology)
Attempts were made to isolate, purify and characterize antibacterial peptides from the haemolymph of the American cockroach Periplaneta americana. Both non-induced and induced haemolymph were tested for their antibacterial activity against several Gram-positive and Gram-negative bacteria. Induction was done by injecting Escherichia coli into the abdominal cavity of the cockroach. A time-induction study showed that antibacterial peptides were induced as early as 1/2 an hour with a peak at 9 h which started to decline around 24 h. The non-induced haemolymph showed activity only against E. coli whereas induced haemolymph showed activity against several Gram-positive and Gram-negative bacteria as well as against one antibiotic resistant E. coli. The induced haemolymph was subjected to SDS-PAGE to estimate the number and molecular weight of proteins present in the crude haemolymph. Seven distinct protein bands were detected by SDS- PAGE. The haemolymph was then subjected to gel filtration chromatography to purify the proteins responsible for the antibacterial activity. Twenty fractions, one ml each, were collected and all the fractions were tested against those bacteria which previously showed sensitivity to the crude haemolymph. Only six fractions were found to be effective against the tested bacteria. The protein concentrations in the active fractions were determined by spectrophotometry. The active fractions were finally subjected to SDS-PAGE to determine the molecular weight of the protein(s) which were responsible for the antibacterial activity. There was one protein in all these six fractions except fraction F13 where an additional protein of 67 kDa was present. The approximate molecular weight of the isolated antibacterial protein present in all the fractions was 61 kDa. It was found that only 2.87 mug of the protein could inhibit bacterial growth whereas approx. 10 mug of conventional antibiotics was required to obtain similar result.
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