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1 1 2 1 Anne Stamm , Iliyana Pepelanova , Kerstin Reimers , Thomas Scheper 1 Institute of Technical Chemistry, Leibniz University Hannover, Callinstr. 5, 30167 Hannover 2 Department 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 Introduction Recent 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. Acknowledgement This 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 Chemistry Institute of Technical Chemistry Callinstr. 5 , 30167 Hanover Experimental procedure Conclusion The 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] 10.000 8.000 6.000 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 Synthesized Protein DnaK DnaJ Proteolysis GrpE ATP ADP Aggregation Proteolysis GroEL 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 kDa Ÿ 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 S BI AI P S pG-KJE8 pGro7 pKJE7 Chaperone plasmid
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Epidermal lipoxygenases as a new approach in wound healing ... · Anne Stamm1, Iliyana Pepelanova1, Kerstin Reimers2, Thomas Scheper1 1Institute of Technical Chemistry, Leibniz University

Sep 29, 2020

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Page 1: Epidermal lipoxygenases as a new approach in wound healing ... · Anne Stamm1, Iliyana Pepelanova1, Kerstin Reimers2, Thomas Scheper1 1Institute of Technical Chemistry, Leibniz University

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