Analysis of factors affecting the periplasmic production of recombinant proteins in Escherichia coli.
Five fusion proteins between Z domains derived from Staphylococcal Protein A and Green Fluorescent Protein or Human Proinsulin were produced on the periplasm of Escherichia coli. The effects of the molecular weight and amino acid composition of the translocated peptide, culture medium composition, and growth phase of the bacterial culture were analyzed regarding the expression and periplasmic secretion of the recombinant proteins. It was found that secretion was not affected by the size of the translocated peptide (17-42 kDa) and that the highest periplasmic production values were obtained on the exponential phase of growth. Moreover, the highest periplasmic values were obtained in minimal medium, showing the relevance of the culture medium composition on secretion. In silico prediction analysis suggested that with respect to the five proteins used in this study, those that are prone to form alpha-helix structures are more translocated to the periplasm.
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal. filipem@fe.up.pt
Filipe J
FJ
Mergulhão
Gabriel A
GA
Monteiro
D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D033903:Periplasmic Proteins / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011384:Proinsulin