Selective purification of recombinant neuroactive peptides using the flagellar type III secretion system.
The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233-2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel Na(V)1.2 comparably to chemically synthesized SIIIA.


OBJECTIVE
Manipulation of the flagellar secretion system bypasses the problems of inclusion body formation and cellular degradation that occur during conventional recombinant protein expression. This work serves as a proof of principle for the use of engineered bacterial cells for rapid purification of recombinant neuroactive peptides and proteins by exploiting secretion via the well-characterized flagellator type III secretion system.
Dèpartement de Médecine, Université de Fribourg, Fribourg, Switzerland.
Hanna M
HM
Singer
Marc
M
Erhardt
Andrew M
AM
Steiner
Min-Min
MM
Zhang
Doju
D
Yoshikami
Grzegorz
G
Bulaj
Baldomero M
BM
Olivera
Kelly T
KT
Hughes
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020916:Conotoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005407:Flagella / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D012615:Sea Anemones; D012908:Snails; D012911:Snakes; D013112:Spiders
D001426:Bacterial Proteins; D020916:Conotoxins; D010455:Peptides; D011993:Recombinant Fusion Proteins