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1 1 2 1Anne Stamm , Iliyana Pepelanova , Kerstin Reimers , Thomas Scheper 1Institute of Technical Chemistry, Leibniz University Hannover, Callinstr. 5, 30167 Hannover
2Department of Plastic, Aesthetic, Hand and Reconstructive Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover
Epidermal lipoxygenases as a new approach in wound healing: Heterologous production of human ALOXE3 in E. coli
IntroductionRecent studies reveal the key role of epidermal lipoxygenases (eLOX) from Homo sapiens and Ambystoma mexicanum (mexican axolotl) in wound healing and
[1] [2]epidermis regeneration . The epidermal lipoxygenase in mammals is involved in the regulation of keratinocyte differentiation. In the mexican salamander (axolotl) an epidermal lipoxygenase could be detected during limb development and in epidermal cells. Furthermore, it could be shown that human cells also respond to the epidermal lipoxygenases from the axolotl. Therefore, epidermal lipoxygenases offer a promising tool for clinical applications in wound healing within the multidisciplinary project “Biofabrication for NIFE”. Such alternative options are especially important in cases where conventional treatments fail.
AcknowledgementThis work has been carried out as an integral part of the BIOFABRICATION FOR NIFE Initiative, which is financially supported by the ministry of Lower Saxony and the VolkswagenStiftung. (NIFE is the Lower Saxony Center for Biomedical Engineering, Implant Research and Development, a joint translational research centre of the Hannover Medical School, the Leibniz University Hannover, the University of Veterinary Medicine Hannover and the Laser Center Hannover.)
References[1] Menger et al. (2011). Annals of Surgery, 253(2), 410–8 [2] Yu et al. (2005). Biochimica et Biophysica Acta,1686(3), 238–47[3] Vogel, R. (2012). Dissertation
[4] TaKaRa Chaperone Plasmide Set Product Manual
Figure 1. 3-dimensional structure
[3]of a LOX
Center for Applied ChemistryInstitute of Technical ChemistryCallinstr. 5 , 30167 Hanover
Experimental procedure
ConclusionThe human epidermal lipoxygenase ALOXE3 could be produced as a soluble protein in E. coli. In this case an approach using chaperone plasmids for coexperession did lead to the best results. Utilizing the polyhistidine-tag upstream of the gene, the produced protein can be purified and analyzed using different assays. Furthermore, a similar approach can be used for the heterologous production of the epidermal lipoxygenase AmbLOXe from the mexican axolotl.
Results
A. [bp]10.0008.0006.000
5.000
4.000
3.500
3.000
2.500
2.000
1.500
1.000
750
500
250
B.
A: ALOXE3 could be cloned sucessfully into a pET28b vector. A polyhistidine-tag and a TEV protease recognition site upstream of the cloned gene can be utilized for subsequent purification.B: Production of ALOXE3 in E. coli BL21 (DE3) led to insoluble protein fractions only. Different approaches for optimization of soluble protein production such as lower temperatures, different inducer concentrations and different media did not lead to a higher amount of soluble protein. Soluble protein production could be implemented by coexpression of certain chaperons using the commercial TaKaRa Chaperon Plasmid Set.
A side note to chaperones:
Ÿ Heterologous protein prodcution in E. coli often leads to formation of inclusion bodies and/or degradation of the protein of interest by proteases
Ÿ Coepxression of chaperons can decrease these problems and lead to a corretly folded protein
Plasmid Chaperones pG-KJE8 DnaK, DnaJ, GrpE, GroES, GroEL pGro7 GroES, GroEL pKJE7 DnaK, DnaJ, GrpE
Ÿ Genes of chaperones are downstream of a promotor which can be induced either by arabinose or tetracycline, therefore the system can be used with a plasmid containing a lactose sensitive promotor for the gene of interest
mRNA
SynthesizedProtein
DnaK
DnaJ
Proteolysis
GrpE
ATP
ADP
Aggregation
ProteolysisGroEL
GroES
ATP
ADP
ADP
ATP
GrpE
Tf
Native Form
Figure 2. Agarose gel electrophoresis after digestion with XhoI and NcoI of plasmid DNA from different E. coli colonies
Figure 3. SDS PAGE of samples before induction (BI), after induction (AI), pellet after cell lysis (P) and supernatant after cell lysis (S); coexpression of different chaperone plasmides
Figure 4. Possible model for chaperone-assisted protein [4]
folding in E. coli
General parameters found to work best in complex media:- OD for induction: 0,8 - Concentration of inducer (IPTG): 0,2 mM- Cultivation temperature during protein expression: 20 °C
Epidermal lipoxygenases Ÿ eLOX displays regenration capacity in wound healing Ÿ Size between 70 and 80 kDaŸ Small N-terminal PLAT domain (eight antiparallel ß-strands forming a ß-barral) and a larger
C-terminal catalytic domain (18-22 α-helices and 1-2 antiparallel ß-sheets)Codon optimized
sequence of gene of interest (ALOXE3)
pET28b_ALOXE3
•
Cloning ALOXE3
into suitable vector (e.g. pET28b) and verification
BL21 (DE3) E. coli strain with
pET28b_ALOXE3
•
Transformation and selection of E. coli production strain
•
Optimization of soluble protein expression (e.g. temperature, inducer concentration)
Soluble ALOXE3
Purified and characterized ALOXE3
•
Purification and enzyme assays
[kDa]
118
66
45
35
M BI AI P S BI AI P SBI AI P S
pG-KJE8 pGro7 pKJE7
Chaperone plasmid