Construction and use of chimeric α-amylases to study protein secretion in Bacillus subtilis
To investigate the influence of exoprotein charge on protein secretion, genes encoding a range of chimeric α-amylases with altered net charge were constructed by a PCR-based technique and expressed in B. subtilis. The chimeric α-amylases were based on AmyL and contained specific regions from two related Bacillus α-amylases, AmyQ and AmyS. The engineered changes were introduced into amyL to increase the net positive charge of the chimeric enzymes, when compared to wild type AmyL. Isoelectric focusing confirmed that chimeric α-amylases possessed considerable positive charge and the temperature and pH optima of the most basic chimeric protein, AmyLQS50.5, were largely unaffected by the engineered changes. However, the structural stability, thermostability and the specific activity of this chimeric α-amylase were adversely affected. In general, lower amounts of chimeric α-amylases were released into the culture supernatants. Pulse-chase experiments revealed that the rate of processing of AmyLQS50.5 was reduced when compared to AmyL and also that the mature forms of both α-amylases were subjected to degradation during or shortly after translocation, although the chimeric enzyme was affected most. When compared to AmyL, the rate of refolding of AmyLQS50.5 was reduced approximately 3-fold, maintaining this protein in a protease-sensitive conformation for an increased period of time. Therefore, it is proposed that the extensive co- or post-translocational degradation of the chimeric enzyme was a consequence of reduced folding kinetics on the outer surface of the cytoplasmic membrane since, when in its native conformation, AmyLQS50.5 is highly resistant to the activity of B. sublifis extracellular proteases. These observations have important implications for the use of B. subtilis as a host for the secretion of heterologous proteins. The most positively charged chimera, AmyLQS50.5, was shown to bind significantly to cell walls isolated from B. sublifis 168, whereas AmyL and human serum albumin did not. This suggests that protein charge can influence the degree to which exoproteins interact with, and bind to, the cell wall as a consequence of electrostatic interactions with the anionic polymers.