Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus.
We developed a modified flagellar type III secretion apparatus to secrete heterologous polypeptides into the growth medium of Escherichia coli. The secretion was facilitated by fusing the 173-bp untranslated DNA fragment upstream of the gene fliC (encoding flagellin) as well as a transcriptional terminator from fliC, into the gene encoding the polypeptide of interest. The polypeptides secreted into the growth medium at concentrations ranging from 1 to 15 mg/l were from Campylobacter jejuni (262 residues in length), Streptococcus pneumoniae (434 residues), Staphylococcus aureus (115 residues), and N-terminal FliC hybrid proteins, for example, the eukaryotic green fluorescent protein (238 residues). The expressed proteins represented >50% of total secreted protein. Previously reported protein yields from extracellular secretion of foreign proteins in E. coli have been low, approximately 100 microg/l. The strengths of our method are the concentration and purity of the secreted proteins and its versatility with regard to the proteins' length and origin.
Majander
Katariina
K
General Microbiology, Faculty of Biosciences, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland.
Anton
Lena
L
Antikainen
Jenni
J
Lång
Hannu
H
Brummer
Mirko
M
Korhonen
Timo K
TK
Westerlund-Wikström
Benita
B
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000737:chemistry; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005407:Flagella / Q000378:metabolism / Q000502:physiology*; D005408:Flagellin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005798:Genes, Bacterial; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry; D013296:Streptococcus pneumoniae / Q000737:chemistry
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D010455:Peptides; D011994:Recombinant Proteins; D005408:Flagellin; D049452:Green Fluorescent Proteins