High Yield Ekspression af rekombinant human Proteiner med transient transfektion af HEK293-celler i suspension
Summary
Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.
Abstract
Kunsten at producere rekombinante proteiner med komplekse post-translationelle modifikationer er en stor udfordring for studier af struktur og funktion. Den hurtige etablering og høj nyttiggørelse fra kortvarigt transficeret pattedyrcellelinier adresser denne barriere, og er et effektivt middel til at udtrykke proteiner, som er naturligt kanaliseres gennem ER og Golgi-medieret sekretorisk vej. Her er en protokol for proteinekspression anvendelse af den humane HEK293F og HEK293S cellelinier transficeret med en mammal ekspressionsvektor konstrueret til høje proteinudbytter. Anvendeligheden af dette system demonstreres ved hjælp af tre repræsentative glycoproteiner, der har givet udtryk for med udbyttet mellem 95-120 mg renset protein udvundet per liter kultur. Disse proteiner er den humane FcγRIIIa og rotte α2-6 sialyltransferase ST6GalI, begge udtrykt med en N-terminal GFP fusion, samt umodificeret humant immunoglobulin G1 Fc. Denne robust system UTIlizes et serum-frit medium, der er passende til ekspression af isotopisk beriget proteiner og kulhydrater til strukturelle studier ved hjælp af massespektrometri og kernemagnetisk resonansspektroskopi. Desuden kan sammensætningen af N-glycan indstilles ved tilsætning af et lille molekyle for at undgå visse glycan ændringer på en måde, der ikke mindsker udbyttet.
Introduction
Producerer høje udbytter af passende foldede og posttranslationelt modificerede humane proteiner til detaljeret analyse af struktur og funktion er fortsat en stor udfordring. Et stort antal ekspressionssystemer er tilgængelige, der producerer rekombinante proteiner med nativ-lignende funktion og opførsel. Bakterielle ekspressionssystemer, hovedsagelig Escherichia coli stammer, repræsenterer de mest tilgængelige og almindeligt anvendte værktøjer i forskning arena, på grund af enkelheden af disse ekspressionssystemer, selvom gær-, plante-, insekt- og mammale systemer er også beskrevet 1-4. Men de fleste af disse systemer er i stand til passende post-translationel modifikation af målproteiner. En grundlæggende interesse for Barb og Moremen laboratorier producerer eukaryote proteiner med passende glycosylering. Mange humane proteiner kræver passende glykosylering for korrekt funktion (se 5).
Den eukaryoteglycosylerings maskiner er omfattende og i stand til at gøre en bred vifte af ændringer, herunder både asparagin (N) – og serin / threonin (O) -bundet komplekse glykaner 6. Det anslås, at> 50% af humane proteiner er N-glycosyleret 7. Glykaner er væsentlige elementer i mange proteiner, herunder terapeutiske monoklonale antistoffer, erythropoietin, og blod koagulationsfaktorer som faktor IX, for at nævne et par stykker. Selvom flere metoder eksisterer for at forberede passende N-glycosylerede proteiner og spænder fra rent syntetisk 8-10, at chemoenzymatic 11-14 eller nyttiggørelse fra manipuleret rekombinante systemer 15-20, ikke overraskende, har menneskelige ekspressionssystemer hidtil vist sig at være den mest robuste metoder til at generere humane proteiner.
Mange terapeutiske humane glycoproteiner fremstillet i rekombinante systemer, der anvender pattedyrceller. Systemer af note er den kinesiske hamster ovarie (CHO), musemyelom (NS0), Baby Hamster Kidney (BHK), humane embryonale Nyre (HEK-293) og human retinale cellelinier, der er ansat i vedhæftning eller suspension kultur til proteinproduktion 4,21,22. Imidlertid har pattedyrprotein ekspressionssystemer kræves dannelse af stabile cellelinier, dyre vækstmedier og substrat assisteret transfektion procedurer 23.
Pattedyrcelle transfektion opnås ved hjælp af talrige midler, herunder calciumphosphater 24,25, kationiske polymerer (DEAE-dextran, polybren, polylysin, polyethylimin (PEI)) eller positivt ladede kationiske liposomer 26-29. PEI er en polykationisk, opladet, lineær eller forgrenet polymer (25 kDa) 26, der danner et stabilt kompleks med DNA og endocytose. Efter syrning af endosomet, er PEI syntes at svulme op, hvilket fører til sprængning af endosomer og frigivelse af DNA'et i cytoplasmaet 26,30.
Indtil for nylig, forbigående transfektion i suspensiom kultur blev udført ved forudgående DNA / PEI kompleksdannelse efterfulgt af tilsætning til cellekulturen 29. Men Würm og medarbejdere rapporterede en yderst effektiv protokol optimeret til rekombinant proteinproduktion i HEK293-celler, der dannede et DNA / PEI-kompleks in situ 31,32. Dette undgås fremstilling, sterilisering af komplekset, og bufferudskiftning i et dyrkningsmedium. Yderligere optimering ved at inkludere udtryk-styrke plasmider førte til betydelige rentestigninger 33. Heri er en fremgangsmåde, der bygger på disse fremskridt og er bredt anvendelig. Ekspressionsbetingelser kan også ændres for at påvirke den N-glycan sammensætning.
Den HEK293S cellelinje, med en gendeletion der stopper N-glycan behandling på et mellemstadium, fører til ekspression af proteiner med ensartede N-glycaner bestående af 2 N-acetylglucosamin rester plus fem mannoserester (Man 5 GIcNAc 2) 34, 35. Disse cellermangler den N-acetylglucosaminyltransferase I (GNTI) gen, som er nødvendig for downstream N-glycan behandling 36,37. Anvendelsen af glycosyltransferase inhibitorer herunder kifunensine, sialinsyrereceptorer analoger og fucose analoge og 2-deoxy-2-fluor-fucose har lignende virkninger, og grænser N-glycan forarbejdning 38-41.
Protokollen rapporterede her anvender pGEN2 vektoren som vist i figur 1 42,43, PEI assisteret transient transfektion i mammale cellelinjer (HEK293F eller HEK293S celler), og genvinding af høje udbytter af passende glycosyleret protein. Dette system er robust og kan rumme forskellige faktorer, herunder isotop mærkning og glycan teknik til produktion af store titere af rekombinante proteiner.
Protocol
Representative Results
Discussion
Denne protokol illustrerer proteinekspression via transient transfektion af HEK293F eller S-celler. De optimale transfektion betingelserne i Barb og Moremen labs ansætte en kritisk kombination af celle tæthed og reagenskoncentrationer at opnå høj effektivitet transfektion. Kritiske overvejelser, når anvendelsen af denne protokol omfatter: opretholde en stabil kultur forud for transfektion (med konsekvent kultur fordobling gange); transfektion af aktivt voksende celler (opnået ved at fortynde celler til 1 x 1…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af de tilskud K22AI099165 (AWB), P41GM103390 (KWM) og P01GM107012 (KWM) fra National Institutes of Health, og ved midler fra Roy J. Carver Institut for Biokemi, Biofysik & Molekylær Biologi på Iowa State University . Indholdet af dette arbejde er alene forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter NIH.
Materials
| Biosafety cabinet | NuAire, Inc. | CellGard ES NU-S475-400 | Class II, Type A2 Biological Safety Cabinet |
| Incubation shaker | INFORS HT | Multitron Cell | |
| Medium A: FreeStyle Expression Medium | Life Technologies | 12338-018 | |
| Medium B: ExCell 293 Serum-Free Medium | SIGMA | 14571C | |
| 125 Erlenmeyer Flask with Vented Cap | Corning Incorporated/Life Sciences | 431143 | |
| 250 Erlenmeyer Flask with Vented Cap | Corning Incorporated/Life Sciences | 431144 | |
| FreeStyle HEK 293F Cells | Life Technologies | R790-07 | |
| 1 ml Disposable serological pipette | Fisher Scietific | 13-676-10B | |
| 10 ml Disposable serological pipette | Fisher Scietific | 13-676-10J | |
| 25 ml Disposable serological pipette | Fisher Scietific | 13-676-10K | |
| Pipettor (Pipet-Aid XP) | Drummond Scientific | 161263 | |
| Trypan Blue Solution | Thermo Scientific | SV30084.01 | |
| Counting slides | Bio-Rad | 145-0011 | |
| TC20 Automated Cell Counter | Bio-Rad | 145-0102 | |
| Polyethylenimine (PEI) | Polysciences Inc. | 23966 | Prepare stock solution at a concentration of 1 mg/ml in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 150 mM NaCl (pH 7.5). Dissolve PEI completely; sterilize through 0.22 μM syringe filter and store at -20 °C. |
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | EMD Chemicals Inc. | 7365-45-9 | |
| 25 mm Syringe Filter, 0.22 μM | Fisher Scietific | 09-719A | |
| Trisaminomethane (Tris base) | Fisher Scietific | BP152-1 | |
| XL1-Blue | Stratagene | 222249 | |
| Trypton | Fisher Scietific | BP1421-2 | |
| Yeast extract | Fluka Analytical | 92144 | |
| Sodium chloride | BDH chemicals | BDH8014 | |
| Plasmid Purification Kit | QIAGEN | 12145 | |
| Valproic (VPA) | SIGMA | P4543 | Prepare stock solution of 220 mM in water, sterilize by passage through a sterile 0.22 mm filter and store at -20 °C |
| Corning 250 mL Centrifuge Tube | Corning Incorporated/Life Sciences | 430776 | |
| Centrifuge | Thermo Scientific | EW-17707-65 | |
| Protein A-Sepharose column | SIGMA | P9424 | |
| Ni-NTA superflow | QIAGEN | 30430 | |
| 3-(N-morpholino)propanesulfonic acid (MOPS) | Fisher Scietific | BP308-500 | |
| Glycine | Fisher Scietific | BP381-500 | |
| 10 kDa molecular weight cut-off Amicon® Ultra centrifugal filters | Millipore | UFC901096 | |
| Sodium dodecyl sulfate | ALDRICH | L3771 | |
| Beta-mercaptoethanol | ALDRICH | M6250 | |
| Glycerol | SIGMA | G5516 | |
| Precision Plus Protein All Blue Standards | Bio-Rad | 161-0373 | |
| Acetic Acid, Glacial | Fisher Scietific | 64-19-7 | |
| coomassie brilliant blue | Bio-Rad | 161-0406 | |
| MALDI-TOFMS Voyager-DE PRO | Applied Biosystems | ||
| 15N labeled L-Tyrosine | ALDRICH | 332151 | |
| 15N labeled L-Lysine | ALDRICH | 592900 | |
| Unlabeled L-Phenylalanine | SIGMA-ALDRICH | P2126 | |
| 13C6-Glucose | ALDRICH | 389374 | |
| 2-deoxy-2-fluoro-l-fucose | SANTA CRUZ Biotechnology | sc-283123 |
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