Farmaceutische biotechnologie
Inhoud
BIOTECHNOLOGIE..............................................................................................................................8
GESCHIEDENIS BIOTECHNOLOGIE..............................................................................................................8
BIOGENE VS. SYNTHETISCHE GENEESMIDDELEN...........................................................................................8
HOE KAN JE WERKEN MET NIET-GENETISCH GEMANIPULEERDE ORGANISMEN? (ONGEWIJZIGD)...............................9
3 TYPES FERMENTATIEPROCESSEN.............................................................................................................9
GEBRUIKTE MICRO-ORGANISMEN.............................................................................................................9
RECOMBINANT DNA TECHNOLOGIE (GEWIJZIGD, GENETISCH GEMANIPULEERD)............................10
INLEIDING........................................................................................................................................10
DNA ISOLATIE...................................................................................................................................11
RNA ISOLEREN..................................................................................................................................12
KWANTITATIEVE ANALYSE VAN NUCLEÏNEZUREN (3)........................................................................13
RECOMBINANT DNA TECHNOLOGIE : POLYMERASE KETTINGREACTIE.............................................14
HOE WERKT PCR?.............................................................................................................................14
VERSCHILLENDE VARIANTEN VAN PCR TESTEN (EX: UITLEGGEN)....................................................................15
RECOMBINANT DNA TECHNOLOGIE : RESTRICTIE-ENZYMEN...........................................................19
RESTRICTIE ENZYMEN..........................................................................................................................19
INHIBITIE VAN RESTRICTIE ENZYMEN DOOR DNA METHYLATIE.......................................................................20
INVLOED VAN OMGEVENDE SEQUENTIE OP KNIP EFFICIËNTIE.........................................................................20
RECOMBINANT DNA TECHNOLOGIE : HULPENZYMEN (MODIFYING ENZYMES)...............................21
ENZYMEN DIE HET DNA KUNNEN WIJZIGEN (7 GROEPEN)............................................................................21
1. METHYLASEN........................................................................................................................................21
2. LIGASEN..............................................................................................................................................21
3. POLYMERASEN......................................................................................................................................21
TAQ-DNA POLYMERASE.............................................................................................................................22
REVERSED TRANSCRIPTASE (RT)...................................................................................................................22
TERMINAAL DEOXYNUCLEOTIDYL TRANSFERASE (TDT)......................................................................................23
RNA POLYMERASE.....................................................................................................................................23
4. GUANILYLTRANSFERASE...........................................................................................................................23
5. T4 POLYNUCLEOTIDE KINASE....................................................................................................................23
6. FOSFATASEN.........................................................................................................................................23
,7. NUCLEASEN..........................................................................................................................................23
DNASE I..................................................................................................................................................23
S1 NUCLEASE............................................................................................................................................23
MUNG BEAN NUCLEASE..............................................................................................................................23
PLASMIDEN.....................................................................................................................................24
VERSCHILLENDE MANIEREN OM PLASMIDEN TE CLASSIFICEREN......................................................................24
SYNTHETISCHE PLASMIDEN...................................................................................................................24
RECOMBINANT DNA TECHNOLOGIE : LIGATIE - PCR PRODUCT CLONEREN IN PLASMIDE .................26
LIGATIE IN TOPO PLASMIDEN...............................................................................................................26
LIGASE INDEPENDENT CLONING (LIC) – T4 DNA POLYMERASE.....................................................................27
RECOMBINANT DNA TECHNOLOGIE : TRANSFORMATIE VAN E. COLI...............................................28
RECOMBINANT DNA TECHNOLOGIE : SELECTIE VAN E. COLI TRANSFORMANTEN............................30
WELKE PLASMIDEN ZIJN DIT DAT LAC Z KUNNEN VORMEN? PUC........................................................................31
RECOMBINANT DNA TECHNOLOGIE : IDEAAL PLASMIDE VOLDOET AAN….......................................32
RECOMBINANT DNA TECHNOLOGIE : ISOLATIE PLASMIDE DNA UIT E.COLI......................................32
METHODE VAN BIRNBOIM EN DOLY (ALKALISCHE LYSE)...............................................................................32
ZUIVERING PLASMIDE DNA OVER EEN CSCL GRADIËNT...............................................................................32
ZUIVERING PLASMIDE DNA MET COMMERCIEEL VERKRIJGBARE MATRICES.......................................................32
RECOMBINANT DNA TECHNOLOGIE : MUTAGENESE........................................................................33
IN VIVO METHODE..............................................................................................................................33
IN VITRO METHODE.............................................................................................................................33
PLAATSGERICHTE MUTATIE...................................................................................................................33
MUTAGENESE-EFFICIENTIE MET MUTATOR OLIGONUCLEOTIDE........................................................................34
METHODEN VOOR HOGERE FREQUENTIE MUTANTEN...................................................................................34
MUTATIES MET BEHULP VAN PCR INBOUWEN...........................................................................................34
DNA SEQUENTIEBEPALING...............................................................................................................34
ENZYMATISCHE METHODE (METHODE VAN SANGER)...................................................................................34
VERBETERINGEN OP DE SANGER METHODE...............................................................................................35
NEXT GENERATION SEQUENCING : GS-FLX TECHNIEK OF METHODE VAN ROCHE...............................................35
NEXT GENERATION SEQUENCING : ILLUMINA TECHNIEK................................................................................36
,DERDE GENERATIE SEQUENCING.............................................................................................................36
SMRT SEQUENCING..................................................................................................................................36
OXFORD NANOPORE SEQUENCING................................................................................................................37
RECOMBINANTE DNA TECHNOLOGIE : GEBRUIK MAKEN VAN SYNTHETISCHE GENEN...........................................37
DE NOVO GEN SYNTHESE.............................................................................................................................37
ANALYTISCHE METHODEN : GELELECTROFORESE.............................................................................38
SCHEIDING DNA(-FRAGMENTEN) OVER AGAROSE GELS...............................................................................38
SCHEIDING DNA FRAGMENTEN OVER POLYACRYLAMIDE GELS.......................................................................38
WERKWIJZE AGAROSE GEL..........................................................................................................................38
ZICHTBAAR MAKEN VAN DNA FRAGMENTEN..................................................................................................38
NIEUWE ONTWIKKELINGEN : REEDS GEMAAKTE GELS (FLASHGEL)..................................................................39
HETEROLOGE GENEXPRESSIE : BELANGRIJKE DNA/RNA ELEMENTEN...............................................39
ALGEMEEN.......................................................................................................................................39
WELKE FACTOREN ZIJN VAN BELANG OM ZO EFFICIËNT MOGELIJK EW TE VORMEN? (EX).........................................39
PROKARYOTISCHE PROMOTOR......................................................................................................................39
EUKARYOTISCHE PROMOTOR........................................................................................................................40
TWEE ZAKEN DIE VAN GROOT BELANG ZIJN VOOR DE PROMOTOR........................................................................40
STABILITEIT VAN HET MRNA........................................................................................................................40
EFFICIËNTIE VAN TRANSLATIE.................................................................................................................40
HOE GEEFT MRNA AANLEIDING TOT EW EN HOE GEBEURT DE TRANSLATIE: TWEE ZAKEN VAN BELANG......................40
HETEROLOGE GENEXPRESSIE : E.COLI..............................................................................................41
IN E. COLI WORDEN HEEL VEEL EW GEVORMD, MAAR HOE?..............................................................................41
GENEXPRESSIE IS VAAK GEBASEERD OP LACTOSE OPERON..................................................................................41
TRYPTOFAAN OPERON................................................................................................................................41
HYBRIDE PROMOTOREN..............................................................................................................................42
MOGELIJKE PROBLEMEN IN E.COLI OM EW TE VORMEN (5)............................................................42
BEVORDEREN VAN SECRETIE VAN HETEROLOGE EW IN E.COLI.........................................................42
SEC- EIWIT TRANSLOCATIE SYSTEEM :......................................................................................................43
NIET OPGEVOUWEN EIWITTEN VAN CYTOPLASMA NAAR PERIPLASMA BRENGEN......................................................43
TAT-SYSTEEM: TWIN ARGININE TRANSLOCATIE..........................................................................................44
TRANSLOCATIE OPGEVOUWDE EW VAN CYTOPLASMA NAAR PERIPLASMA.............................................................44
THIOREDOXINE MET HIS PATCH.............................................................................................................44
EXTRACELLULAIRE PRODUCTIE VAN RECOMBINANTE EW IN E.COLI.................................................44
HOE EW VAN PERIPLASMA NAAR BUITEN BRENGEN?..................................................................................44
, VB DIE IN E.COLI WORDEN AANGEMAAKT EN VAN THERAPEUTISCHE BELANG ZIJN........................45
INSULINE..........................................................................................................................................45
VROEGER.................................................................................................................................................45
TEGENWOORDIG.......................................................................................................................................46
VOORBEELDEN VAN INSULINE PREPARATEN BEKOMEN MET DNA RECOMBINANT....................................................46
GROEIHORMOON...............................................................................................................................46
HETEROLOGE GENEXPRESSIE : SACCHAROMYCES CEREVISIAE.........................................................47
ALGEMEEN.......................................................................................................................................47
SECRETIEWEG IN GIST..........................................................................................................................47
EXPRESSIEVECTOREN IN DEZE GIST..........................................................................................................48
YEP VECTOR.............................................................................................................................................48
ARS BEVATTENDE VECTOREN.......................................................................................................................49
YAC........................................................................................................................................................49
PROMOTOREN IN GIST.........................................................................................................................49
GAL-REGULATIEMODEL..............................................................................................................................49
TRANSFORMATIE GISTCELLEN (2 WIJZEN).................................................................................................50
ELECTROPORATIE.......................................................................................................................................50
CHEMISCHE TRANSFORMATIE : LITHIUM EN PEG.............................................................................................50
PROBLEMEN BIJ PRODUCTIE VAN HETEROLOGE EW IN GISTEN.......................................................................50
2 PROBLEMEN DOOR DIT TYPE GLYCOSYLATIE...................................................................................................50
SUIKERPROBLEEM OPLOSSEN.......................................................................................................................51
SUIKERSTRUCTUUR....................................................................................................................................51
OPLOSSING..............................................................................................................................................51
SECTRECIEPROBLEEM OPLOSSEN (5)..............................................................................................................51
HBV VACCIN GEMAAKT IN SACCHAROMYCES CEREVISIAE...................................................................................52
HETEROLOGE GENEXPRESSIE : PICHIA PASTORIS..............................................................................52
PICHIA VECTOREN (PLASMIDEN).............................................................................................................52
2 MOGELIJKHEDEN VAN CLONEREN IN GIST.....................................................................................................53
TRANSFORMANTIE P PASTORIS (3 METHODEN).........................................................................................54
BELANGRIJKE PARAMETERS BIJ EXPRESSIE IN PICHIA..........................................................................................55
OCRI OF MICROPLASMINE ; THERAPEUTISCH EW DAT IN PICHIA WORDT AANGEMAAKT...........................................55
HETEROLOGE GENEXPRESSIE: KLUYVEROMYCES LACTIS EXPRESSIESYSTEEM...................................55
HETEROLOGE GENEXPRESSIE: FILAMENTEUZE SCHIMMELS.............................................................55
PRODUCTIE VAN RECOMBINANTE EW IN ASPERGILLUS DOOR FUSIE MET GLUCOAMYLASE....................................56
PRODUCTIE VAN RECOMBINANTE EW IN TRICHODERMA.............................................................................56
HETEROLOGE GENEXPRESSIE: PLANTEN...........................................................................................56
Inhoud
BIOTECHNOLOGIE..............................................................................................................................8
GESCHIEDENIS BIOTECHNOLOGIE..............................................................................................................8
BIOGENE VS. SYNTHETISCHE GENEESMIDDELEN...........................................................................................8
HOE KAN JE WERKEN MET NIET-GENETISCH GEMANIPULEERDE ORGANISMEN? (ONGEWIJZIGD)...............................9
3 TYPES FERMENTATIEPROCESSEN.............................................................................................................9
GEBRUIKTE MICRO-ORGANISMEN.............................................................................................................9
RECOMBINANT DNA TECHNOLOGIE (GEWIJZIGD, GENETISCH GEMANIPULEERD)............................10
INLEIDING........................................................................................................................................10
DNA ISOLATIE...................................................................................................................................11
RNA ISOLEREN..................................................................................................................................12
KWANTITATIEVE ANALYSE VAN NUCLEÏNEZUREN (3)........................................................................13
RECOMBINANT DNA TECHNOLOGIE : POLYMERASE KETTINGREACTIE.............................................14
HOE WERKT PCR?.............................................................................................................................14
VERSCHILLENDE VARIANTEN VAN PCR TESTEN (EX: UITLEGGEN)....................................................................15
RECOMBINANT DNA TECHNOLOGIE : RESTRICTIE-ENZYMEN...........................................................19
RESTRICTIE ENZYMEN..........................................................................................................................19
INHIBITIE VAN RESTRICTIE ENZYMEN DOOR DNA METHYLATIE.......................................................................20
INVLOED VAN OMGEVENDE SEQUENTIE OP KNIP EFFICIËNTIE.........................................................................20
RECOMBINANT DNA TECHNOLOGIE : HULPENZYMEN (MODIFYING ENZYMES)...............................21
ENZYMEN DIE HET DNA KUNNEN WIJZIGEN (7 GROEPEN)............................................................................21
1. METHYLASEN........................................................................................................................................21
2. LIGASEN..............................................................................................................................................21
3. POLYMERASEN......................................................................................................................................21
TAQ-DNA POLYMERASE.............................................................................................................................22
REVERSED TRANSCRIPTASE (RT)...................................................................................................................22
TERMINAAL DEOXYNUCLEOTIDYL TRANSFERASE (TDT)......................................................................................23
RNA POLYMERASE.....................................................................................................................................23
4. GUANILYLTRANSFERASE...........................................................................................................................23
5. T4 POLYNUCLEOTIDE KINASE....................................................................................................................23
6. FOSFATASEN.........................................................................................................................................23
,7. NUCLEASEN..........................................................................................................................................23
DNASE I..................................................................................................................................................23
S1 NUCLEASE............................................................................................................................................23
MUNG BEAN NUCLEASE..............................................................................................................................23
PLASMIDEN.....................................................................................................................................24
VERSCHILLENDE MANIEREN OM PLASMIDEN TE CLASSIFICEREN......................................................................24
SYNTHETISCHE PLASMIDEN...................................................................................................................24
RECOMBINANT DNA TECHNOLOGIE : LIGATIE - PCR PRODUCT CLONEREN IN PLASMIDE .................26
LIGATIE IN TOPO PLASMIDEN...............................................................................................................26
LIGASE INDEPENDENT CLONING (LIC) – T4 DNA POLYMERASE.....................................................................27
RECOMBINANT DNA TECHNOLOGIE : TRANSFORMATIE VAN E. COLI...............................................28
RECOMBINANT DNA TECHNOLOGIE : SELECTIE VAN E. COLI TRANSFORMANTEN............................30
WELKE PLASMIDEN ZIJN DIT DAT LAC Z KUNNEN VORMEN? PUC........................................................................31
RECOMBINANT DNA TECHNOLOGIE : IDEAAL PLASMIDE VOLDOET AAN….......................................32
RECOMBINANT DNA TECHNOLOGIE : ISOLATIE PLASMIDE DNA UIT E.COLI......................................32
METHODE VAN BIRNBOIM EN DOLY (ALKALISCHE LYSE)...............................................................................32
ZUIVERING PLASMIDE DNA OVER EEN CSCL GRADIËNT...............................................................................32
ZUIVERING PLASMIDE DNA MET COMMERCIEEL VERKRIJGBARE MATRICES.......................................................32
RECOMBINANT DNA TECHNOLOGIE : MUTAGENESE........................................................................33
IN VIVO METHODE..............................................................................................................................33
IN VITRO METHODE.............................................................................................................................33
PLAATSGERICHTE MUTATIE...................................................................................................................33
MUTAGENESE-EFFICIENTIE MET MUTATOR OLIGONUCLEOTIDE........................................................................34
METHODEN VOOR HOGERE FREQUENTIE MUTANTEN...................................................................................34
MUTATIES MET BEHULP VAN PCR INBOUWEN...........................................................................................34
DNA SEQUENTIEBEPALING...............................................................................................................34
ENZYMATISCHE METHODE (METHODE VAN SANGER)...................................................................................34
VERBETERINGEN OP DE SANGER METHODE...............................................................................................35
NEXT GENERATION SEQUENCING : GS-FLX TECHNIEK OF METHODE VAN ROCHE...............................................35
NEXT GENERATION SEQUENCING : ILLUMINA TECHNIEK................................................................................36
,DERDE GENERATIE SEQUENCING.............................................................................................................36
SMRT SEQUENCING..................................................................................................................................36
OXFORD NANOPORE SEQUENCING................................................................................................................37
RECOMBINANTE DNA TECHNOLOGIE : GEBRUIK MAKEN VAN SYNTHETISCHE GENEN...........................................37
DE NOVO GEN SYNTHESE.............................................................................................................................37
ANALYTISCHE METHODEN : GELELECTROFORESE.............................................................................38
SCHEIDING DNA(-FRAGMENTEN) OVER AGAROSE GELS...............................................................................38
SCHEIDING DNA FRAGMENTEN OVER POLYACRYLAMIDE GELS.......................................................................38
WERKWIJZE AGAROSE GEL..........................................................................................................................38
ZICHTBAAR MAKEN VAN DNA FRAGMENTEN..................................................................................................38
NIEUWE ONTWIKKELINGEN : REEDS GEMAAKTE GELS (FLASHGEL)..................................................................39
HETEROLOGE GENEXPRESSIE : BELANGRIJKE DNA/RNA ELEMENTEN...............................................39
ALGEMEEN.......................................................................................................................................39
WELKE FACTOREN ZIJN VAN BELANG OM ZO EFFICIËNT MOGELIJK EW TE VORMEN? (EX).........................................39
PROKARYOTISCHE PROMOTOR......................................................................................................................39
EUKARYOTISCHE PROMOTOR........................................................................................................................40
TWEE ZAKEN DIE VAN GROOT BELANG ZIJN VOOR DE PROMOTOR........................................................................40
STABILITEIT VAN HET MRNA........................................................................................................................40
EFFICIËNTIE VAN TRANSLATIE.................................................................................................................40
HOE GEEFT MRNA AANLEIDING TOT EW EN HOE GEBEURT DE TRANSLATIE: TWEE ZAKEN VAN BELANG......................40
HETEROLOGE GENEXPRESSIE : E.COLI..............................................................................................41
IN E. COLI WORDEN HEEL VEEL EW GEVORMD, MAAR HOE?..............................................................................41
GENEXPRESSIE IS VAAK GEBASEERD OP LACTOSE OPERON..................................................................................41
TRYPTOFAAN OPERON................................................................................................................................41
HYBRIDE PROMOTOREN..............................................................................................................................42
MOGELIJKE PROBLEMEN IN E.COLI OM EW TE VORMEN (5)............................................................42
BEVORDEREN VAN SECRETIE VAN HETEROLOGE EW IN E.COLI.........................................................42
SEC- EIWIT TRANSLOCATIE SYSTEEM :......................................................................................................43
NIET OPGEVOUWEN EIWITTEN VAN CYTOPLASMA NAAR PERIPLASMA BRENGEN......................................................43
TAT-SYSTEEM: TWIN ARGININE TRANSLOCATIE..........................................................................................44
TRANSLOCATIE OPGEVOUWDE EW VAN CYTOPLASMA NAAR PERIPLASMA.............................................................44
THIOREDOXINE MET HIS PATCH.............................................................................................................44
EXTRACELLULAIRE PRODUCTIE VAN RECOMBINANTE EW IN E.COLI.................................................44
HOE EW VAN PERIPLASMA NAAR BUITEN BRENGEN?..................................................................................44
, VB DIE IN E.COLI WORDEN AANGEMAAKT EN VAN THERAPEUTISCHE BELANG ZIJN........................45
INSULINE..........................................................................................................................................45
VROEGER.................................................................................................................................................45
TEGENWOORDIG.......................................................................................................................................46
VOORBEELDEN VAN INSULINE PREPARATEN BEKOMEN MET DNA RECOMBINANT....................................................46
GROEIHORMOON...............................................................................................................................46
HETEROLOGE GENEXPRESSIE : SACCHAROMYCES CEREVISIAE.........................................................47
ALGEMEEN.......................................................................................................................................47
SECRETIEWEG IN GIST..........................................................................................................................47
EXPRESSIEVECTOREN IN DEZE GIST..........................................................................................................48
YEP VECTOR.............................................................................................................................................48
ARS BEVATTENDE VECTOREN.......................................................................................................................49
YAC........................................................................................................................................................49
PROMOTOREN IN GIST.........................................................................................................................49
GAL-REGULATIEMODEL..............................................................................................................................49
TRANSFORMATIE GISTCELLEN (2 WIJZEN).................................................................................................50
ELECTROPORATIE.......................................................................................................................................50
CHEMISCHE TRANSFORMATIE : LITHIUM EN PEG.............................................................................................50
PROBLEMEN BIJ PRODUCTIE VAN HETEROLOGE EW IN GISTEN.......................................................................50
2 PROBLEMEN DOOR DIT TYPE GLYCOSYLATIE...................................................................................................50
SUIKERPROBLEEM OPLOSSEN.......................................................................................................................51
SUIKERSTRUCTUUR....................................................................................................................................51
OPLOSSING..............................................................................................................................................51
SECTRECIEPROBLEEM OPLOSSEN (5)..............................................................................................................51
HBV VACCIN GEMAAKT IN SACCHAROMYCES CEREVISIAE...................................................................................52
HETEROLOGE GENEXPRESSIE : PICHIA PASTORIS..............................................................................52
PICHIA VECTOREN (PLASMIDEN).............................................................................................................52
2 MOGELIJKHEDEN VAN CLONEREN IN GIST.....................................................................................................53
TRANSFORMANTIE P PASTORIS (3 METHODEN).........................................................................................54
BELANGRIJKE PARAMETERS BIJ EXPRESSIE IN PICHIA..........................................................................................55
OCRI OF MICROPLASMINE ; THERAPEUTISCH EW DAT IN PICHIA WORDT AANGEMAAKT...........................................55
HETEROLOGE GENEXPRESSIE: KLUYVEROMYCES LACTIS EXPRESSIESYSTEEM...................................55
HETEROLOGE GENEXPRESSIE: FILAMENTEUZE SCHIMMELS.............................................................55
PRODUCTIE VAN RECOMBINANTE EW IN ASPERGILLUS DOOR FUSIE MET GLUCOAMYLASE....................................56
PRODUCTIE VAN RECOMBINANTE EW IN TRICHODERMA.............................................................................56
HETEROLOGE GENEXPRESSIE: PLANTEN...........................................................................................56
