A precursor form of the penicillinase from Bacillus licheniformis

Abstract

All but one of the secretory proteins studied so far are made initially with an amino-terminal extension of 15-30 amino acids. This signal sequence is thought to bind the ribosomal complex containing the nascent chain to the endoplasmic reticulum in eukaryotic cells [l-3] and to the cytoplasmic membrane in prokaryotic cells [4,5]. The vectorial transfer of the peptide chain across the membrane is coupled to translation and as sdon as the chain is finished the protein is usually released from the extra-cytoplasmic side of the membrane. We are studying the secretion of the penicillinase enzyme in Bacillus licheniformis. This penicillinase is unique among secretory proteins, since it remains membrane bound after completion of the polypeptide chain [6]. We have shown that the membrane-bound form is attached by an amino-terminal peptide which is hydrophobic [7]. This hydrophobic tail is then removed by a proteolytic cleavage, and the penicillinase is released from the cell [6]. To study the relationship of the amino-terminal extension of the membrane penicillinase to the signal peptides found’in other secretory proteins we have synthesized the protein in vitro. DNA containing the penicillinase gene from B. lichenifomis was transcribed and translated in a cell-free system from Escherichia coli. We found that the penicillin&e made in vitro was larger than the membrane penicillinase. This extension is located at the amino terminus of the protein and is probably equivalent to the signal sequence present in other secretory proteins. 2. Material and methods