Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli.
Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ-proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ-proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5'-untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ-proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.
Mergulhão
Filipe J M
FJ
Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
Monteiro
Gabriel A
GA
Larsson
Gen
G
Bostrom
Maria
M
Farewell
Anne
A
Nyström
Thomas
T
Cabral
Joaquim M S
JM
Taipa
M Angela
MA
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011384:Proinsulin / Q000096:biosynthesis* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C116144:UspB protein, E coli; C078564:universal stress protein A, Bacteria; D011384:Proinsulin