Export Proteins

Actinomycetes Large surface area to volume ratio should favour protein export Widely used in industrial microbiology Good expression systems being developed Promoters/gene regulation still poorly understood Rheology of fermentations important... 

PelZ is a hydrophilic protein of 420 amino acids with a short hydrophobic sequence at its N-terminal end which has Ae characteristics of the signal sequences of exported proteins. The signal peptide may be 24 amino acids long, which would corroborate wiA the usual length encountered in prokaryotes. The molecular cloning of the pelZ gene in an expression vector pT7-6 allowed for the specific 35S-cysteine-methionine raAo-labelling of PelZ in E. coli K38. We could detect, in crude extracts, the presence of a precursor and a mature form of PelZ. After cell fractionation, Ae mature form of PelZ could be localized in Ae periplasm of E. coli. So PelZ appears to be a protein exported by Ae Sec-dependent system of translocation. 

Koronakis, V., Sharff, A., Koronakis, E., Luisi, B. and Hughes, C. (2000). Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export, Nature, 405, 914—919. 

Economou, A. (1999). Following the leader bacterial protein export through the Sec pathway, Trends Microbiol., 7, 315-320. 

Bogsch, E., Sargent, F., Stanley, N., Berks, B., Robinson, C., and Palmer, T. (1998). An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. / Biol. Chem. 273, 18003-18008. 

Nunn, D. (1999). Bacterial type II protein export and pilus biogenesis more than just homologies Trends Cell Biol. 9, 402-408. 

Settles, A., and Martienssen, R. (1998). Old and new pathways of protein export in chloroplasts and bacteria. Trends Cell Biol. 8, 494-501. 

Y. Imai, S. Asada, S. Tsukahara, E. Ishikawa, T. Tsuruo, and Y. Sugimoto. Breast cancer resistance protein exports sulfated estrogens but not free estrogens. Mol Pharmacol. 64 610-618 (2003). 

Nickel W (2005) Unconventional secretory routes direct protein export across the plasma membrane of mammalian cells. Traffic 6 607-614... 

Bernhard, M., Eriedrich, B. and Siddiqui, R. A. (2000) Ralstonia eutropha TF93 is blocked in tat-mediated protein export./. BacterioL, 182, 581-8. 

Sargent F, Bogsch EG, Stanley NR, et al. 1998. Overlapping fnnctions of components of a bacterial Sec-independent protein export pathway. EMBO J 17 3640-50. 

Brodsky JL, Werner ED, Dubas ME, Goeckeler JL, Kruse KB, McCracken AA (1999) The requirement for molecular chaperones during endoplasmic reticulum-assodated protein degradation demonstrates that protein export and import are mechanistically distinct. J Biol Chem 274 3453-3460 Brown CR, Doxsey SJ, White E, Welch W (1994) Both viral (adenovirus ElB) and cellular (hsp 70, p53) components interact with centrosomes. J Cell Physiol 160 47-60... 

Figure 3.9. Generalized overview of the industrial-scale manufacture of recombinant E2 classical swine fever-based vaccine, using insect cell culture production systems. Clean (uninfected) cells are initially cultured in 500-1000 litre bioreactors for several days, followed by viral addition. Upon product recovery, viral inactivating agents such as /i-propiolactone or 2-bromoethyl-iminebromide are added in order to destroy any free viral particles in the product stream. No chromatographic purification is generally undertaken as the product is substantially pure the cell culture media is protein-free and the recombinant product is the only protein exported in any quantity by the producer cells. Excipients added can include liquid paraffin and polysorbate 80 (required to generate an emulsion). Thiomersal may also be added as a preservative. The final product generally displays a shelf-life of 18 months when stored refrigerated...

Ossareh-Nazari, B., Bachelerie, F. and Dargemont, C. (1997) Evidence for a role of CRM1 in signal-mediated nuclear protein export. Science, 278,141-144. 

GJ Phillips, TJ Silhavy. The E. coli fflt gene is necessary for viability and efficient protein export. Nature 359 744-746, 1992. 

The process of protein export involves a small, cytoplasmic ribonucleoprotein particle (the Signal Recognition Particle or SRP) with the signal coding mRNA sequence and/or the signal peptide itself. This interaction stops translation of the protein. Then, the stalled or arrested ribosome moves to the endoplasmic reticulum (ER). A receptor on the ER binds the SRP. 

Over several decades, multiple vector systems for recombinant gene expression in E. coli have been developed. Modem vectors suitable for recombinant protein production vary in the used promoter system in the presence or absence of coding sequences for affinity tags upstream or downstream of the multiple cloning site (MCS) and of sequences coding for leader peptides for the protein export. Moreover, different origins of replication (ori), antibiotic selection marker genes and MCS are used. 

Hell, K., Herrmann, J. M., Pratje, E., Neupert, W., and Stuart, R. A. (1998). Oxalp, an essential component of the N-tail protein export machinery in mitochondria. Proc. Natl. Acad. Sd. USA 95, 2250-2255. 

---