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.
Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
Filipe J M
FJ
Mergulhão
Gabriel A
GA
Monteiro
Gen
G
Larsson
Maria
M
Bostrom
Anne
A
Farewell
Thomas
T
Nyström
Joaquim M S
JM
Cabral
M Angela
MA
Taipa
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