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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 μg/l1. 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.

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Figure 1: Schematic presentation of the plasmids, the gene constructs expressed in E. coli MKS12 and the secretion of the resulting polypeptides into culture medium.
Figure 2: Analysis of intracellular and secreted polypeptides from recombinant strains of E. coli: MKS12 expressing FliC fusion polypeptides and analysis of binding properties of the fusion polypeptides.
Figure 3: Analysis of intracellular and secreted polypeptides from recombinant strains of E. coli MKS12 expressing foreign polypeptides.

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We thank I. Blomfield for the E. coli strain MG1655 Δfim; H. Rautelin for the C. jejuni strain T-71431; H. Saarilahti for the plasmid psmGfp; and R. Lameranta, K. Alsti, L. Partanen and T. Vesa for technical assistance. This work was supported by the National Technology Agency (Tekes, grant number 40312/03), University of Helsinki (grant number 2105045) and the Academy of Finland (grant numbers 78141 and 202009, and the Microbes and Man Program).

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Correspondence to Benita Westerlund-Wikström.

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Majander, K., Anton, L., Antikainen, J. et al. Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol 23, 475–481 (2005).

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