klonierung, expression und produktion einer marinen ... · klonierung, expression und produktion...
TRANSCRIPT
Klonierung, Expression und Produktion einer
marinen Chitindeacetylase
Hayssam Zakaria, Christian Schmalz, Arno Cordes & Bernd Otto
CHITOSAN- what it is
Deacetylated derivative of chitin
Deacetylation grade > 50%
Chain length variable
Secondary structure still remaining
Proteins and Calcium carbonat present or not
Chitosan Chemical Properties
Polymer of D-glucosamine
Reactive amino groups
Reactive hydroxyl groups available
Chelates many metal ions
Forms films
Chitosan Biological Properties
• Biocompatible• Natural polymer• Biodegradable• Non-toxic• Binds to mammalian cells• Regenerative effect on conective tissue• Accelarates the formation of osteoblasts• Fungistatic• Spermicidal
CHITOSAN- chemical production
Decalcification in dilute HCl solution
Deproteination in dilute NaOH solution
Decolorization in 0.5% KMnO4 and Oxalic acid
Chitin
Deacetylation in hot (80°C) concentrated (40-50%) NaOH solution
Neutralization
Chitosan
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CHITOSAN- Anwendungen
Biotechnology
Wound healingSlow release of drugs
Burn treatmentEnzyme immobilization
Contac lens
Agriculture
Seed coatingFertilizer
Animal feed additive
Membranes and paper
Permeability controlReverse osmosis
Photographic paperSurface treatment
Cosmetics
MoisturizerAntimicrobial agent
Fungistatic
Wastewater Treatment
Removal of metal ionsFlocculant/ Coagulant
Basics
„High tech“ Chitosan für medizinische Anwendungen
Neue Chitindeacetylase (CDa) zur enzymatischen Konvertierung von Chitin zu Chitosan
Enzymatische Prozesse sind leichter zu kontrollieren / zu reproduzieren (uniforme Qualität)
Bisherige Enzyme sind dafür unzureichend
Der Plan
Alignment bekannter Enzyme mit ähnlicher Aktivität
Design degenerierter Oligonukleotide
Degenerierte PCR von DNA aus isolierten marinen Mikroorganismen
Direkte genomische Sequenzierung für eine full length Sequenz
Heterologe Expression des klonierten Gens in E. coli
Aufreinigung (Affinitäts-Chromatographie)
PCR-products
lane 1: Marker λ-DNA-Bst EII-Digest
lane 2 + 20: Marker φX174 DNA-HaeIII-Digest
lane 3-19: PCR-products with several primer-pairs
2% TBE agarose-gel
Characteristics
Original-Stamm
• grampositive bacteria
• promicromonospora/ actinobacteria
Biologische Daten
• keine Sekretions-Sequenz
• möglicherweise Teil eines
Chitindegradations-Operons
open reading frame
gene length [bp]
molecular weight [kD]
theoretical IP startcodon
1 1068 38,65 11,86 CTG
2 915 33,05 10,64 CTG
3 789 28,69 8,52 GTG
4 759 27,50 6,84 ATC
5 747 27,01 8,52 GTG
6 633 23,09 6,83 CTG
• spezifische Aktivität (ORF 5): 5 nkat/mg
Klonierung der neuen Chitindeacetylase!"#$%&'(($) ) ) **))
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?8&($"@'$"/$)A$"$'45)+$")B83C(74457"'+,>$74$/C37($,>8.D&$)'(/).7"?'$"/=)
?B) 6/7"/48+8&) :) .'/) EcoEF,E$(/"'?/'8&((45&'//(/$33$&) &745) F&($"/'8&(,BGE) H&+)
I38&'$"H&#(@$?/8")JK6I,FAKL=))
CB) M$?/8") JK6I,FAKL) &745) F&($"/'8&) +$") cda) .'/) 6/7"/48+8&) :) 1%$") EcoEF,
E$(/"'?/'8&((45&'//(/$33$&=))
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• expression in E. coli strain BL21 (D3)
• vector: pASK-IBA 7
• induction with 200 µg/l Anhydro-Tetracycline
• expression for 4 h at 25°C
• expression levels:
- soluble: 100 µg/g wet-weight
- Inclusion bodies: > 10 mg/g wet- weight
Klonierung der neuen Chitindeacetylase
• expression in E. coli strain BL21 (D3)
• vector: pASK-IBA 7
• induction with 200 µg/l Anhydro-Tetracycline
• expression for 4 h at 25°C
• expression levels:
- soluble: 100 µg/g wet-weight
- Inclusion bodies: > 10 mg/g wet- weight
Aufreinigung - lösliche Produktion (ORF 5)
lane1: total lysat; lane 2-4 and 6-8: fractions of the Strep-tag II purification; lane 5: prestained low-range marker, Biorad
westernblotcoomassie stained sds-gel
Purification - inclusion bodies (ORF 5)
coomassie stainedsds-gel
lane1: prestained low-range marker, Biorad; lane 2: total lysat;lane 3: purification from inclusion bodies
pro:
• high total yield
• high purity
contra:
• protein has to be denaturated and refolded
• specific activity is lower compared to soluble
preparation
Temperatur Optimum
0
5
10
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4°C 15°C 25°C 30°C 37°C 42°C
acet
ic a
cid
[mM
]
Temperature Optimum
pH-Optimum
0
5
10
15
20
pH 6.0 pH 7.0 pH 8.0
Deacetylation of 45 mM N-Acetyl-Chitosan-Oligomers by 1 µM enzyme at 25°C for 48h in 100 mM PBS buffer
acet
ic a
cid
[mM
]
Kinetik
Deacetylation of 45 mM N-Acetyl-Chitosan-Oligomers by 1 µM enzyme at 25°C in PBS buffer, pH 7.0
0
5
10
15
20
25
30
35
40
45
0 h 24 h 48 h 72 h 96 h 120 h
time
acet
ic a
cid
[m
M]
0
10
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40
50
60
70
80
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100
dea
cety
lati
on
[%
]
Ausblick
Austesten weiterer Chitin-Substrate
Expression in patentfreiem System
Erhöhung der Proteinausbeute
Upscaling zum industriellen Maßstab
Optimierung des enzymatischen Prozesses
Die Hände & Köpfe
Tierärztliche Hochschule Hannover Prof. Bernd Otto
(ehemals FhG-IGB) Dr. Hayssam Zakaria
Dr. Christian Schmalz (Hugo-Geiger-Preis)
FH Oldenburg/ Ostfriesland/ Wilhelmshaven Prof. Eike Siefert
Uta Bünger
Bernd Schmietenknop
Universität Hannover Dr. Jochen Meens
Dipl. Biochem. Christine Schreiber
Dr. Irene Wagner-Döbler
ASA Spezialenzyme GmbH Dr. Arno Cordes