Application of an E. coli signal sequence as a versatile inclusion body tag.
BACKGROUND
Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form.


RESULTS
When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins.


CONCLUSIONS
We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.
Abera Bioscience AB, 11145, Stockholm, Sweden. wouter.jong@aberabio.com.|Department of Molecular Cell Biology, Section Molecular Microbiology, Faculty of Earth and Life Sciences, VU University, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands. wouter.jong@aberabio.com.
Wouter S P
WS
Jong
Xbrane Biopharma AB, 11145, Stockholm, Sweden.
David
D
Vikström
Abera Bioscience AB, 11145, Stockholm, Sweden.
Diane
D
Houben
Abera Bioscience AB, 11145, Stockholm, Sweden.
H Bart
HB
van den Berg van Saparoea
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 10691, Stockholm, Sweden.
Jan-Willem
JW
de Gier
Abera Bioscience AB, 11145, Stockholm, Sweden. s.luirink@vu.nl.|Department of Molecular Cell Biology, Section Molecular Microbiology, Faculty of Earth and Life Sciences, VU University, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands. s.luirink@vu.nl.
Joen
J
Luirink
D002352:Carrier Proteins; D004815:Epidermal Growth Factor / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism*; D007377:Interleukin-3 / Q000096:biosynthesis* / Q000235:genetics; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012995:Solubility; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C508596:IL3 protein, human; D007377:Interleukin-3; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C517609:TrxA protein, E coli; D013879:Thioredoxins; D004815:Epidermal Growth Factor
Aggregation; E. coli; Fusion tag; Heterologous protein production; Inclusion bodies; Insolubility; Signal peptide; Twin-arginine translocation pathway