1. Vitiligo Vitiligo (leukoderma) is a skin disorder causing patches of the skin to turn white, resulting from the destruction of mel-anocytes, responsible for the pigment molecule melanin. Three major theories have been proposed: (1) a neural theory suggesting an accumulation of neurochemical sub-stances from nerve endings, (2) a biochemical theory sug-gesting an accumulation of toxic molecules such as reactive oxygen species, and (3) an autoimmune response theory suggesting an accumulation of immunoglobulin G (IgG) anti-self antibodies targeting the melanocytes [1]. There is no cure for vitiligo and current treatments are ine�cient, carrying many side e�ects [2].
In our �nal model, CPP-bound proteins are naturally secreted from bacteria, eliminating the need for pro-tein puri�cation. Our cloning chassis E. coli does not normally secrete proteins extracellularly. Although a number of treatments have been developed to pro-mote extracellular secretion, secretion tags are not su�ciently e�cient and convenient in E. coli. [16] However, they have been used with more success in gram-positive bacteria such as Lactobacillus [16]. In our model we propose application of a bacte-rial ointment directly onto the skin. This requires that the choice of bacterium be carefully taken into con-sideration. Lactobacilli have already been successfully used in bacteriotherapy [17]. This further suggests that Lactobacillus is a good choice as our future cellular chassis.
6. ModelingTo enable regulation of bacterial protein production on skin, a model for safe induction of pro-tein expression from our Ptac vector was developed from work by Ahmadzadeh et al. [15]. The proposed toy model is based on the well-studied E. coli lac operon with lactose as inducer.
SOD1 yCCS bFGF
(Tyrosinase)(MITF)
IgG Protease (IdeS)Protein A (PtA-Z)Autoimmune hypothesis
Biochemicalhypothesis
2. Therapeutic approach We propose a novel treatment for vitiligo, based on bacterial therapy. Bacteria secreting proteins bene�-cial for vitiligo re-pigmentation are applied onto af-fected skin. The proteins then relocate through the epidermal skin layers to the melanocytes, where they act towards skin re-pigmentation.
Team Stockholm 2010Andreas Constantinou1, Hassan Foroughi Asl2, Emmelie Lidh1, Johan Nordholm1, Nina Schiller1
1Stockholm University, 2Royal Institute of TechnologyCorrespondence: [email protected] More information: http://igem.se
Expression vector lac operon
Faculty of Science Swedish Vitiligo Association Thermo Fisher Scienti�c Biomatters Sigma-Aldrich
References[1] Autoantibody responses to melanocytes in the depigmenting skin disease vitiligo Anthony P. Weetman et al., 2007[2] Current remedies for vitiligo, Javed Ali et al., 2010[3] Interaction engineered three-helix bundle domains for protein recovery and detec-tion, Alm T., 2010[4] IdeS: A Bacterial Proteolytic Enzyme with Therapeutic Potential, Johansson B et al., 2008[5] Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library. Hallewell R et al., 1985
We want to thank Mentors Robert Daniels, Elisabeth Haggård, Gunnar von HeijneAdvisors Jaroslav Belotserkovsky, Sridhar Mandali, Richard Odegrip, Sergey SurkovSta� of department of Biochemistry & Biophysics, Stockholm UniversitySta� of department of Genetics, Microbiology and Toxicology, Stockholm University
mRNA dynamics :dM
dt= αM
K1 A2 + 1K1 A2 + K
+ Γ0 − M γM
Allolactose dynamics :dA
dt= M kb
(αA L
KL + L− βA A
K + A
)− AγA
Lactose dynamics :dL
dt= M kp
(αL Le
KLe + Le− βL L
KL1 + L
)−kb M
αA L
KL + L−L γL
M , Mp : mRNA L : lactose
A : allolactose p : vector protein
Vector mRNA dynamics :dMp
dt= αM p
K1 A2 + 1K1 A2 + K
+ Γ0p − Mp γM p
Vector protein dynamics :dp
dt= Mp αp − γp p
Degradation rate : γ = µ + γ
γ : degradation coefficientα , β : production coefficient
Γ0 , Γ0p : transcription leakage
3. Proteins For the autoimmune theory, proteins masking the autoimmune re-sponse by either binding or cleaving the Fc region of IgG antibodies were selected [3, 4]. A protein chimera of the Protein A Z-domain (PtA-Z) and a bacterial IgG protease (IdeS) was proposed. For the biochemical theory, a few key proteins important for the well-being of melanocytes were chosen. Superoxide dismutase (SOD1) is an important antioxidant converting superoxide into oxygen and hydrogen peroxide [5]; it requires a copper chaperone (yCCS) to gain full activity [6]. Basic �broblast growth factor (bFGF) promotes melanin synthesis and mitosis [7]. MITF is an important transcription factor for melanocyte development [8]. Tyrosinase catalyzes the production of melanin [9].
4. Cell-penetrating peptides Skin is an e�ective barrier for all macromolecules. A needle-free ap-proach for protein delivery into the skin can be achieved by coupling the proteins to cell-penetrating peptides (CPPs), small molecules shown to penetrate into cells and in some cases cross the skin barrier [10]. The CPPs Trans-Activating Transcriptional Activator (TAT), Low Molecular Weight Protamine (LMWP), and Transportan 10 (Tp10) were chosen.
5. Lab work CloningEscherichia coli was selected as our cloning chassis. Assembly Stand-ard 25 was used, as it enables protein fusions [11]. Each CPP was fused to both the N- and C-termini of all proteins due to the uncertainty of their e�ciency at each terminus; 6x His were fused at the opposite end to enable protein puri�cation. Also, to obtain full SOD1 activity, operons containing SOD1 and yCCS were assembled.
Protein expressionProtein overexpressions were performed in the expression vector pEX to verify protein production [12]. Resulting expression levels were in-su�cient for all tested constructs, inconsistent with levels from a simi-lar commercially available vector using the same promoter (Ptac) [13].
Puri�cationTo enable activity and penetration assays, e�orts were put into purify-ing our His-tagged proteins using nickel-a�nity columns. These at-tempts were unsuccessful, most likely due to low expression levels.
Activity assaysActivity of SOD1 was investigated using a SOD determination kit (Sigma-Aldrich) [14]. The kit measures the abundance of O2-, which is reversibly proportional to SOD1 levels. No elevated SOD1 activity was recorded. IgG protease activity was tested for IdeS by digesting peroxidase-conjugated IgG anti-bodies and recording the result-ing peroxidase activity. Although activity was recorded, this was at-tributed to unspeci�c E. coli proteases.
Cell- and skin penetrationImmunohistochemistry-based experiments were designed for testing cell and skin penetration of CPPs. Cultured melanocytes and keratino-cytes had been prepared, as well as human arti�cial skin. Also, skin would be harvested from dead mice.
Bacteria applied
Proteins secreted
Internalization
Re-pigmentation
[6] Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents. Ahl I. M et al., 2004[7] Basic Fibroblast Growth Factor from Human Keratinocytes Is a Natural Mitogen for Melanocytes, Halaban R et al., 1988[8] MITF: master regulator of melanocyte development and melanoma oncogene, Carmit Levy et al., 2006[9] Optimum melanin production using recombinant Escherichia coli, V.H. Lagunas-Muñoz et al., 2006[10] Synthetic Skin-Permeable Proteins Enabling Needleless Immunization, Yongzhuo Huang et al., 2010
[11] Fusion Protein (Freiburg) Biobrick assembly standard [12] Part:BBa_K243033 , Registry of Parts[13] pGEX Vectors (GST Gene Fusion System), GE Healthcare Life Sciences[14] 19160 SOD determination kit, Sigma-Aldrich[15] Analysis of the Lactose Metabolism in E Coli Using Sum of Squares Decomposition, A. Ah-madzadeh et al., 2005[16] Secretion of recombinant proteins by Gram-negative bacteria, Maria Sandkvist et al., 1996[17] Bacteriotherapy with Lactobacillus plantarum in burns, Maria C Peral et al., 2009
+
7. Transfer to Lactobacillus
