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PCR Cloning and Subcloning> | … The amplified gene fragments (pulA) and linearized plasmid pMA5 with the primers in Table S1 were ligated together by using seamless cloning kit (GenScript Co., Ltd., Nanjing, China) according to manufacturer's protocols. For pullulanase expression, the sequenced vectors … | Get A Quote |
Bacillus subtilis has been widely used as a prokaryotic host for the secretory expression of heterologous proteins. In this study, a pullulanase (PulA) from Anoxybacillus sp. LM18-11 was firstly identified to be expressed in Bacillus subtilis 1A751 through non-classical secretion pathway. Results showed that both the N- and C-terminal regions of PulA were essential for its soluble expression. To explore its specific structural basis of secretion in B. subtilis, we revealed a hydrophobic motif A501-H507 which is vital for the secretion of the whole protein of PulA. Through a series of site-specific mutagenesis, the triple-sites mutants R503E/I506E/H507E and R503E/I506Y/H507E showed the highest extracellular acti... More