The physiological function of FGE encoded by SUMF1 is to perform the co-/post-translational modification in the active centre of the sulfatases. The function of SUMF2-encoded pFGE is still ill defined, however high similarity of the structure and the expression pattern to that of FGE indicates a cellular function in assisting or regulating FGE activity in the ER. The topological distribution of both proteins, FGE and pFGE, has indeed shown them to be localized in the lumen of the ER.
The mechanism of selective segregation of FGE and pFGE from other proteins that follow the secretory pathway is still unclear.
Most soluble proteins that reside in the lumen of the ER carry a specific tetrapeptide signal sequence like KDEL that prevents their secretion. However, human FGE and pFGE were found to lack the intrinsic ER retention signals like the prototype KDEL. Therefore, it remains unclear how these proteins are selectively segregated from other proteins that follow the secretory pathway and retained in the ER lumen. Consequently, we investigated the mechanism of FGE and pFGE retention in the ER, which could be based on one of the known general retention mechanism or by a new mechanism.
Chapter 2
Materials and Methods
2.1 Materials
2.1.1 Laboratory equipment
Analytic balances type 1602 MP and 1265 MP Sartorius, G¨ottingen Analytical HPLC:
SMART-system with the following columns: Amersham Biosciences Gel filtration columns:
Superdex 200 PC 3.2/30 (2.4 ml) Amersham Biosciences Anion exchange columns:
MonoQ PC 1.6/5 (0.1 ml) Amersham Biosciences
µPeak C2/C18 PC 3.2/3 (C2/C18, 2.1 x 30 mm) Amersham Biosciences UV-detectors for SMART-system:
µPeak Detector Amersham Biosciences
UV-Detector for Vision Workstation:
FLUOR-305 PerSeptive Biosystems
Intelligent Dark Box II, Las-1000+ Fuji, Japan
Ice machine Ziegra, Isernhagen
Centrifuges:
Eppendorf centrifuge Type 5415C and 5402 Eppendorf, Hamburg
Table ultracentrifuge TL-100 Beckmann, M¨unchen
Ulracentrifuge L8-70M Beckmann, M¨unchen
Cold Centrifuge J-21C and J2-MC Beckmann, M¨unchen
Labofuge GL Heraeus Sepatech
Rotors:
JA10, JA 20 Beckmann, M¨unchen
17
18 Chapter2. Materials and Methods
Ti 45, Ti 60, Ti 70 Beckmann, M¨unchen
TLA 45, Beckmann, M¨unchen
TLA-100.3 Beckmann, M¨unchen
Electrophoresis chambers for agarose gels Workshop of the Institute Electrophoresis chambers for polyacrylamide gels Workshop of the Institute Liquid scintillation counter 1900TR Packard, Frankfurt/Main
Gel dryer Bio-Rad, Hilden
Magnetic mixer IKA, Works, INC.
MALDI-TOF Mass Spectrometer, REFLEX III Bruker Daltonics, Bremen
Microwave oven Siemens, M¨unchen
pH-meter Beckmann, M¨unchen
Photometer, UV 160 A Shimadzu, Kioto/Japan
UV-hand lamp (365/254nm), Type 5415 and 5402 Eppendorf, Hamburg Vacuum concentrator model 100H Bachhofer, Reutlingen
Vortex-Genie Scientific Industries, USA.
DNA-Sequencer Type 310 ABI, PE Biosystems
Electroporator 1000 (used for bacteria) Stratagene, USA Confocal Laser Scanning Microscope Leica, Bensheim Leica TCS SP2 AOBS
(Ar: 488, 514 nm; He/Ne: 543 nm; 63x Oil Objective)
Incubators Innova 4230 and 4330 New Brunswick Scientific
Phosphoimager Fujix BAS1000 Fuji, Japan
Ultra turrax T8 IKA Labortechnik, Staufenv
Supersignal Chemiluminiscent Substrate Pierce, Illinois Thermocycler GeneAmp PCR system 9600 Perkin-Elmer Cetus
2.1.2 Chemicals, plasticware and membranes
Chemicals Boehringer/Roche, Mannheim,
Merck, Darmstadt, Roth, Karlsruhe, Serva, Heidelberg, Sigma, Deisenhofen Cell culture plasticware Greiner, Frickenhausen
Nalge Nunc International, Denmark Nitrocellulose membrane Schleich and Sch¨ull, Dassel
PVDF membrane, 0.2µM Schleich and Sch¨ull, Dassel Hybond-N Nylon membrane Amersham Biosciences, UK
2.1. Materials 19 Whatman GB002 paper Schleich and Sch¨ull, Dassel
Whatman GB003 paper extra thick Schleich and Sch¨ull, Dassel
2.1.3 Kits, spin columns and reagents
2.1.3.1 DNA
HiSpeed Plasmid Midi kit Qiagen Omniscript Reverse Transcription kit Qiagen
PCR purification kit Qiagen
QIAprep Spin Miniprep kit Qiagen QIAquick Gel Extraction kit Qiagen Effectene Transfection kit Qiagen Fugene Transfection reagent Roche
Lipofectamine 2000 Invitrogen
2.1.3.2 Protein
Bio-Rad Protein Assay Bio-Rad
DAKO fluorescent mounting medium DakoCytomation, USA
ECL Plus Amersham Biosciences
Ni-NTA agarose Qiagen
Protease Inhibitor Cocktail Sigma Supersignal Chemiluminescence Kit Pierce, USA Roti-blue Colloidal Coomassie Brilliant Blue Roth
PEFA Bloc Roth
Bovine Serum Albimin (BSA) Serva Iodoacetamide, Iodoacetic acid Sigma
PANSORBIN cells Calbiochem
Prestained Marker Biorad
2.1.4 Enzymes, nucleotides and standards
Restriction endonucleases New England Biolabs Klenow DNA polymerase New England Biolabs
DNA ligase New England Biolabs
TaqDNA polymerase Amersham Pharmacia Biotech Alkaline phosphatase Boehringer
Ultrapure dNTP Set Amersham Pharmacia Biotech
20 Chapter2. Materials and Methods Adenosine 5-triphosphate (ATP) Sigma
Oligonucleotide NAPS, G¨ottingen
Pfupolymerase Stratagene
1-kb DNA ladder Gibco BRL
2.1.5 Vectors
pBI BD CLONTECH
pSB 4.7 pA Transkaryotic Therapies Inc, Cambridge, MA pSV-pac gift from Prof. Stefan H¨oning
pUB/Bsd Invitrogen life technologies
pGBKT7-53 BD Bioscience
pGBKT7 This study
pGADT7 BD Bioscience
pLamC gift from Prof. Peter Schu pCMV2-Lys-cmyc-KDEL gift from Prof. H.D. S¨oling
2.1.6 Antibiotics and drugs
Ampicillin Serva
Neomycin (Gentamycin sulfate or G418) Gibco
Penicillin/Streptomycin Gibco
(100x =10,000 U/ml)
Kanamycin Serva
2.1.7 Radioactive substances
[35S]-Cysteine, 10 mCi/ml Amersham Pharmacia Biotech 6-Sulfo-GalNAc-β(1-4)-GlcUA-β(1-3)
-6-sulfo-N-Acetyl-galactosamine (1-3H) in H2O
2.1.8 Antibodies
2.1.8.1 Primary antibodies
antigen type Western blot reference
RGS-His6 tag mouse mAb 1:2000 Qiagen
HA tag mouse mAb 1:2000 Covance Inc., Princeton
2.1. Materials 21
pFGE rabbit pAb 1:2500 this study
pFGE mouse mAb 1:2500 this study
FGE rabbit pAb 1:2500 this study
FGE mouse mAb 1:2500 this study
Galactose-6-sulfatase mouse mAb 1:1000 Transkaryotic Therapies, Inc, USA
FKBP12 rabbit pAb
FKBP13 rabbit pAb
2.1.8.2 Secondary antibodies
Goat anti-rabbit Horseradish peroxidase conjugate Goat anti-mouse Horseradish peroxidase conjugate
2.1.9 Yeast and Bacterial strains
2.1.9.1 Yeast strains
Strain Genotype Reporters Transformation
markers AH109 MATa,trp1-901, leu2-3, 112, HIS3, trp1,leu2
ura3-52,his3-200, gal4∆, gal80∆, ADE2, LYS2::GAL1U AS-GAL1T AT A-HIS3, MEL1, GAL2U AS-GAL2T AT A-ADE2 lacZ URA3::MEL1U AS-MEL1T AT A-lacZ
MEL1
Y187 MATα,Ura3-52, His3-200, MEL1, trp1,leu2 ade2-101,trp1-901,leu2-3,112, lacZ
gal4∆, gal80∆,met
URA3::GAL1U AS-GAL1T AT A-lacZ MEL1
2.1.9.2 Bacterial strains DH5α, Gibco BRL, Eggenstein
22 Chapter2. Materials and Methods
2.1.10 Stock solutions and buffers
1 M Sodiumphosphate buffer
1M solution of sodium di-hydrogen phosphate was slowly added to 1 M di-sodium hydrogenphosphate solution with constant mixing on a magnetic stirrer till the pH reached 7.4.
10 x PBS
100 mM sodium phosphate pH 7.4 9 % sodium chloride
Dissolved in 800 ml water and pH was adjusted to 7.4 with HCl, volume was made up to 1000 ml and autoclaved. Stored at room temperature.
1 x TBS
10 mM Tris/ HCl pH 7.4 150 mM Sodium chloride 1 x TAE 0.04 M Tris-acetate 1mM EDTA (pH 8.0)
50x TAE 242 g Tris base
57.1 g glacial acetic acid
100 ml of 0.5 M EDTA (pH 8.0)
Dissolved in water and the final volume was made upto one litre.
TE Buffer
10 mM Tris/ HCl pH 7.5 1 mM EDTA
2.1.11 Media for E.coli
2.1.11.1 Luria Bertani (LB) medium
Ingredients Amount
Glucose 1 g
Bacto-yeast extract 5 g
NaCl 5 g
Bacto-Tryptone 10 g
The medium was autoclaved and stored at room temperature.
2.1. Materials 23 2.1.11.2 LB-Agar Plates
1.5% of Agar was added to the LB medium, autoclaved and the medium was let to cool down to around 50oC. 100 µg/ml Ampicillin or 50 µg/ml of Kanamycin was added to the medium and poured into Petri dishes.
2.1.12 Media for S.cerevisiae
2.1.12.1 YAPD medium
Ingredients For 1 liter
Amount or volume Yeast extract 10 g
Peptone 20 g
Adenine Sulfate 0.04 g
Glucose 2% (Stock 20%, Sterilized) Agar (for plates) 15 g
2.1.12.2 Synthetic minimal medium (SD)
Ingredients For 1 liter
Amount or volume Yeast Nitrogen base 1.7 g
Ammonium sulfate 5.0 g SC drop out mix (10 X) 10 ml
20% Glucose 100 ml
Agar (for plates) 15 g 2.1.12.3 SC drop mix (DO)
Supplements 10X Concentration
(mg/L) L-Adenine hemi sulfate 200
L-Arginine HCl 200
L-Histidine HCl monohydrate 200
L-Isoleucine 300
L-Leucine 1000
L-Lysine HCl 300
L-Methionine 200
24 Chapter2. Materials and Methods
L-Phenylalanine 500
L-Threonine 2000
L-Tryptophan 200
L-Tyrosine 300
L-Uracil 200
L-Valine 1500
10X dropout supplements may be autoclaved and stored at 4oC for up to 1 year.
For making selection medium, made dropout mix lacking the appropriate amino acids. Therefore, a combination of a SD base and a DO supplement will produce a synthetic, defined minimal medium lacking one or more specific nutrients. The specific nutrients omitted depends on the selection medium desired.