Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system.
Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.
Długołecka
Anna
A
Department of Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
Cieśliński
Hubert
H
Turkiewicz
Marianna
M
Białkowska
Aneta M
AM
Kur
Józef
J
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D001483:Base Sequence; D003080:Cold Temperature; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism*; D004950:Esterases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D003470:Culture Media; D004950:Esterases