Two distinct regions in the model protein Peb1 are critical for its heterologous transport out of Escherichia coli.
BACKGROUND
Escherichia coli is frequently the first-choice host organism in expression of heterologous recombinant proteins in basic research as well as in production of commercial, therapeutic polypeptides. Especially the secretion of proteins into the culture medium of E. coli is advantageous compared to intracellular production due to the ease in recovery of the recombinant protein. Since E. coli naturally is a poor secretor of proteins, a few strategies for optimization of extracellular secretion have been described. We have previously reported efficient secretion of the diagnostically interesting model protein Peb1 of Campylobacter jejuni into the growth medium of Escherichia coli strain MKS12 (ΔfliCfliD). To generate a more detailed understanding of the molecular mechanisms behind this interesting heterologous secretion system with biotechnological implications, we here analyzed further the transport of Peb1 in the E. coli host.


RESULTS
When mature Peb1 was expressed without its SecA-YEG-dependent signal sequence and without the putative signal peptidase II recognition sequence in E. coli MKS111ΔHBB lacking the flagellar secretion complex, the protein was found in the periplasm and growth medium which indicated a flagellum-independent translocation. We assessed the Peb1 secretion proficiency by an exhaustive search for transport-affecting regions using a transposition-based scanning mutagenesis strategy. Strikingly, insertion mutagenesis of only two segments, called TAR1 (residues 42 and 43) and TAR2 (residues 173 to 180), prevented Peb1 secretion individually. We confirmed the importance of TAR regions by subsequent site-specific mutagenesis and verified that the secretion deficiency of Peb1 mutants was not due to insolubility or aggregation of the proteins in the cytoplasm. We found by cell fractionation that the mutant proteins were present in the periplasm as well as in the cytoplasm of MKS12. Hence, mutagenesis of TAR regions did not affect export of Peb1 across the cytoplasmic membrane, whereas its export over the outer membrane was markedly impaired.


CONCLUSIONS
We propose that the localization of the model protein Peb1 in the growth medium of E. coli is due to active secretion by a still unknown pathway of E. coli. The secretion apparently is a two-step process involving a periplasmic step and the TAR regions.
Division of General Microbiology, Department of Biosciences, PO Box 56, FIN-00014 University of Helsinki, Helsinki, Finland.
Lena
L
Anton
Katariina
K
Majander
Harri
H
Savilahti
Liisa
L
Laakkonen
Benita
B
Westerlund-Wikström
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005407:Flagella / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019897:Periplasm / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000942:Antigens, Bacterial; C489977:PEB1 protein, Campylobacter; D011994:Recombinant Proteins