IGEM:Stanford/2009/Project Homeostasis/E.Coli Export

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Type 1 Secretion System – Recombinant Protein Secretion in E coli.

C-terminus signal peptide (post-translational) 46-50 (normally 218, can also use 38 or 113 residue with reduced transport efficiency) residue peptide recognized by two proteins HlyB and HlyD, a channel forms connecting the two membranes, translocation involves hydrolysis of ATP; protein is separated from the two linker proteins TolC


IL-6 secretion via hemolysin signal peptide pathway and secretion:


Drawback 1 – need to coexpress all the cofactors needed for transport; this can create a bottleneck in the number of proteins that require the use of the endogenous transport mechanism.

Drawback 2 – the signal peptide remains attached; is there cleavage step involved after? (See reference in paper).

Engineered protease cleavage sites (i.e. conserved sequences that are broadly recognized by proteases).

Recombinant Protein Secretion in E coli: Mergulhao et al


SecYEG + SecA ATPase system is cotranslational translocase:


Secretion via the Sec pathway generally requires the presence of an N-terminal signal peptide on the secreted protein(Note that Type II and type V secretion systems generally require the presence of an N-terminal signal peptide in order to utilise the Sec pathway for translocation from cytoplasm to periplasm [Wikipedia citation]):


signal peptide for translocase recognizion


"the net positively charged N-terminal n-region, the hydrophobic h-region, followed by a polar c-region"


examples of the signal peptide: KRR-LamB(sequence know)

Recombinant Protein Expression:



Vesicle secretion of stress products (i.e. accumulated misfolded proteins; would IL-6 be ):


Barley paper: gene III signal sequence (signal sequence is cleaved after transport; enzyme was active after secretion) tagged alpha amylase for export Article: http://www.springerlink.com/content/w8gj1t1j6v540083/fulltext.pdf

Review of Protein Secretion Systems in Bacteria: http://www.asm.org/ASM/files/ccLibraryFiles/Filename/000000002532/znw00906000414.pdf http://arjournals.annualreviews.org/doi/pdf/10.1146/annurev.mi.39.100185.003151

ATP-binding cassette (iii) Protein substrates are unfolded during secretion. Given the small size of a typical ABC transporter, it is unlikely that VOL. 72, 2008 BACTERIAL ATP-BINDING CASSETTE SYSTEMS 341 Downloaded from mmbr.asm.org at SERIALS CONTROL Lane Medical Library on June 24, 2009 folded proteins pass through ABC proteins in the cytoplasmic membrane. http://mmbr.asm.org/cgi/reprint/72/2/317 translocase (4 proteins in system): http://pdfserve.informaworld.com/460111_731200435_713736803.pdf

General secretory translocase (12 proteins in system): http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1T-479M5BX-1&_user=145269&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=938873558&_rerunOrigin=scholar.google&_acct=C000012078&_version=1&_urlVersion=0&_userid=145269&md5=1dfa6aeb703a318e82f5dfdd3c0581cf

Genetic Analysis of Protein Exporters Can be useful if we want to know the genes that code for export proteins and express them heterogenously or induce over-expression: http://arjournals.annualreviews.org/doi/pdf/10.1146/annurev.ge.24.120190.001243

Very fast overview of Type I throught Type VI Secretion systems: When it comes to type VI secretion (T6S), we are all largely beginners, given that this mechanism earned its place alongside the previous five categories of Gram-negative protein secretion as recently as 2006. All six types of secretion transport proteins from the cell's interior across the cell envelope to the external milieu. Before examining type VI secretion, it is worth pausing to outline what we know about secretion systems I–V (reviewed in [1 I.R. Henderson, F. Navarro-Garcia, M. Desvaux, R.C. Fernandez and D. Ala’Aldeen, Type V protein secretion pathway: the autotransporter story, Microbiol Mol Biol Rev 68 (2004), pp. 692–744. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (179)1]). Type II and type V secretion systems generally require the presence of an N-terminal signal peptide in order to utilise the Sec pathway for translocation from cytoplasm to periplasm. By contrast type I, type III and usually also type IV systems, can secrete a protein without any such signal. While type I and type V systems are relatively simple, consisting of only two or three proteins, secretion systems of types II–IV are large multi-protein complexes that span the entire cell envelope. Type III and type IV systems in particular have been implicated in interactions between bacterial and eukaryotic cells and in the translocation of bacterial effector molecules directly into the eukaryotic cytosol. These two secretion systems are commonly encoded in large clusters of contiguous genes, which, when they occur in pathogens, are often considered pathogenicity islands. The following review will briefly set the discovery of the T6SS in a historical context and then describe the current state of knowledge about these secretion systems.

Great paper about Bacterial Efflux Systems: Bacteria apparently get a lot of crap outside the cell, which is good for us I guess... harhar http://arjournals.annualreviews.org/doi/pdf/10.1146/annurev.biochem.73.011303.073626?cookieSet=1


GroEL belongs to the chaperonin family of molecular chaperones, and is found in a large number of bacteria[1]. It is required for the proper folding of many proteins. To function properly, GroEL requires the lid-like cochaperonin protein complex GroES. In eukaryotes the proteins Hsp60 and Hsp10 are structurally and functionally nearly identical to GroEL and GroES, respectively.

Wnt (just secretion) IL-6 (both folding and secretion)

Retinoic acid in bloodstream: "Also, it has been demonstrated that the bloodstream normally contains a low concentration of retinoic acid that can be taken up in significant amounts by many organs" http://endo.endojournals.org/cgi/content/full/138/7/3035