- Poster Presentation
- Open Access
DnaK-J are limiting for proper recombinant protein folding only at low production rates and when the physiological heat-shock stress response is not triggered
© Martínez-Alonso et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Recombinant Protein
- Heat Shock Response
- Recombinant Protein Production
- Specific Fluorescence
It is known that the bacterial production of recombinant, misfolding-prone proteins triggers the heat shock response, as observed through the monitoring of several marker genes . On the other hand, the co-production of selected chaperones along with the recombinant protein has been largely explored as a strategy to minimize aggregation of the product, with rather unpredictable and not always consistent results [2–4]. The reasons for the limited success of this approach could lie in suspected although not proven different folding requirements of specific protein species and the occurrence of differential bottlenecks in their in vivo folding pathways. Moreover, it has not been discarded that the protein-induced heat-shock response could eventually eclipse the rise of functional plasmid-encoded chaperones resulting from co-production. To explore this possibility and to offer more light on the functional mechanics of recombinant protein folding we have quantitatively determined the intracellular levels of DnaK under different conditions, during the production of a misfolding-prone GFP variant.
The production in E. coli MC4100 of an engineered GFP protein (VP1GFP), controlled by the Trc promoter (in pTVP1GFP) and triggered by 1 mM IPTG results in an important level of aggregation, with VP1GFP occurring in both the soluble and insoluble cell fractions at similar extents (42.2 % ± 1.6 and 57.8 % ± 1.6 respectively). When the producer strain carried pBB535  (containing both IPTG-inducible PA1/lac-O1 controlled dnaK-J chaperone genes) as a second plasmid, the addition of IPTG promotes the co-expression of both the chaperone gene set and the vp1gfp gene. However, this does not result in any detectable shift in the fractioning of VP1GFP, which still occurs in the insoluble cell fraction in an important level (48.2 % ± 3.6). In the same line, the specific fluorescence of VP1GFP is not modified by dnaK-J co-expression in both soluble (143.1 ± 31.5 fluorescence units/μg VP1GFP versus 125.4 ± 29.7) and insoluble (26.2 ± 6.7 versus 30.6 ± 5.4 fluorescence units/μg VP1GFP) cell fractions. Also, the total fluorescence determined by OD unit is not significantly affected by coexpression (378.0 ± 20.1 fluorescence units/ml of culture·OD550 versus 315.0 ± 8.5 fluorescence units/ml of culture·OD550).
DnaK-J are limiting for the proper folding of low amounts of VP1GFP, in absence of any endogenous heat-shock response. However, under strong production conditions, a physiological heat-shock response is triggered and an additional income of DnaK-J does not promote any detectable effect on VP1GFP protein quality. This indicates that the DnaK-J levels reached as a response to protein production are high enough to offer a sufficient supply of such chaperones. Therefore, the proper folding of VP1GFP under these conditions is probably restricted by a limiting factor other than DnaK-J, probably a heat-shock product or other cell element whose levels remain modest during recombinant protein production.
We appreciate the generous gift of pBB535 from Prof. B. Bukau. This work has been supported by grants BIO2004-0700 (MEC) and 2005SGR-00956 (AGAUR), Spain. AV, EGF and NGM are recipient of predoctoral fellowships from UAB, MEC and MEC respectively.
- Jurgen B, Lin HY, Riemschneider S, Scharf C, Neubauer P, Schmid R, Hecker M, Schweder T: Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations. Biotechnol Bioeng. 2000, 70: 217-224. 10.1002/1097-0290(20001020)70:2<217::AID-BIT11>3.0.CO;2-W.View ArticleGoogle Scholar
- Baneyx F, Palumbo JL: Improving heterologous protein folding via molecular chaperone and foldase co-expression. Methods Mol Biol. 2003, 205: 171-197.Google Scholar
- Thomas JG, Baneyx F: Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing Heat-shock proteins. J Biol Chem. 1996, 271: 11141-11147. 10.1074/jbc.271.19.11141.View ArticleGoogle Scholar
- Thomas JG, Baneyx F: Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli . Protein Expr Purif. 1997, 11: 289-296. 10.1006/prep.1997.0796.View ArticleGoogle Scholar
- Tomoyasu T, Mogk A, Langen H, Goloubinoff P, Bukau B: Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol. Mol Microbiol. 2001, 40: 397-413. 10.1046/j.1365-2958.2001.02383.x.View ArticleGoogle Scholar
- de Marco A, de Marco V: Bacteria co-transformed with recombinant proteins and chaperones cloned in independent plasmids are suitable for expression tuning. J Biotechnol. 2004, 109: 45-52. 10.1016/j.jbiotec.2003.10.025.View ArticleGoogle Scholar
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