Rat ¦Á-amide Enzyme Secretion Expression In Streptomyces

Bioactive peptides from neural and endocrine tissues play a very important regulatory role in human and animals. Most of these peptides are amidated at their C-terminals, which is essential for their full activities. The a-amide comes from posttranslational modification that is catalyzed by α-amidase (α-AE or PAM). The amidation reaction is a rate-limiting step in the biosynthesis of regulatory peptides. Although we are now able to produce pharmacological peptides by recombinant DNA technology, yet we fail to produce amidated peptides in the same way because bacteria lack the a-amidase. In this study rat a-amidase was expressed secretively in S. lividans so that a-amidated peptides can be produced in the organism. The rat a-amidase, with molecular weight of 75kDa (α-AE₇₅), is a bifunctional and most potent one of the a-amidating enzymes .Total RNA was extracted from rat's atrium by acid guanidine isothiocyanate-phenol-chloroform. Total RNA was successfully retro-transcribed with Superscript II RNase H^- Reverse Transcriptase primedby random hexamers. PCR was carried out in a special buffer with 14mmol/L (NH₄)₂SO₄ substituted for commonly used 50mmol/L KCI and 1/20 dilution of RT products as templates. The cycling denaturation time was optimized to 20 sec at 94 ℃. Two cDNA fragments of about 2.1kband 2.4kb respectively were obtained as a result of different mRNAs produced by alternative splicing of transcripts, the former being deduced to encode α-AE₇₅. The 2.1kb cDNA fragment was cloned, subcloned and sequenced. The sequence of the cloned cDNA proved to be identical to natural α-AE₇₅ encoding sequence. The α-AE₇₅ cDNA was fused downstream of melC1 signal peptide encoding sequence in correct reading frame so that α-AE₇₅ will be expressed secretively. The fusion gene (mel/AE) was then inserted into plJ680 to...