맞춤형 HPLC 정화 프로토콜 즉, 올리고머 형성 할 수있는 수율 고순도 아밀로이드 베타 42과 아밀로이드 베타 펩티드 (40),
Özet
Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer's disease. The amyloidogenic nature of the peptide makes its purification a challenge.
Abstract
Amyloidogenic peptides such as the Alzheimer’s disease-implicated Amyloid beta (Aβ), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase – styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (>95%) Aβ42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the Aβ40 peptide with identical success and without the need to alter the method.
Introduction
알츠하이머 병은 신경 퇴행성 질환 효과 만 35 명 이상의 세계이다. 질병의 발병 및 발달에 크게 관여 1은 매우 응집하기 쉬운 소수성 펩타이드 베타 아밀로이드 (Aβ)이다. 이 Aβ가 그러나, 아밀로이드 베타 42 (Aβ42)는 단백질의 가장 독성 형태 42 아미노산 변이체 인 것으로 생각하고, 길이 36 내지 43 개의 아미노산의 범위이다. (3)이 용이 특히 신경 독성 엔티티 것으로 여겨진다 확산 성, 올리고머 성 종을 형성하는 Aβ42의 능력에 대부분 기인한다. 도 4는 Aβ 펩티드에 대한 이해를 증진하기 위하여, 일상적으로 고순도 재료를 얻는 것이 필수적이다. 미량 불순물의 존재는 크게 펩티드 응집 성향의 특성을 변화하는 것으로 나타났다. (5)
티raditionally 같은 소수성 Aβ 펩티드의 고속 액체 크로마토 그래피 (HPLC) 분리 C 사 또는 C 8 실리카 계 정지상과 이동상 산성의 조합을 사용하여 수행되었다. (6) 그러나, 이러한 조건은 펩티드의 정제에 도전을 제공 할 수있다. Aβ 펩티드의 낮은 등전점은 PI (약 5.5) (7)는 산성 조건 하에서, 펩티드의 응집이 증가되고, 그 결과 종종 분리하기 어려운 넓은 비 – 분해 HPLC 피크는 (도 2A)을 생산하는 것을 의미한다. 또한, 이러한 넓은 피크들은 펩티드의 응집 프로필 영향 및 일반적 극적 제조 펩타이드의 양에 영향을 미칠 수있는 정제의 후속 발사가 필요할 수 불순물을 함유한다.
폴리 (스티렌 / 디 비닐 벤젠) 정지상 PLRP-S는을 정제의 대안적인 수단을 나타낸다잉 소수성 펩타이드. 정지상 다른 단백질과 메신저 리보 핵산 (mRNA의) 다수의 정제에 사용되어왔다. 8, 9 PLRP-S 정지상은 역상 분리 추가적인 알킬 리간드를 필요로하지 않고,보다 중요한 펩티드 deaggregation 리드 높은 pH에서 화학적으로 안정하다. 7 여기서, 우리는 고순도 아밀로이드 베타 42 (Aβ42)과 아밀로이드 베타 40 (Aβ40) 펩타이드를 산출 맞춤형 HPLC 정화 프로토콜을보고합니다.
Protocol
Representative Results
Discussion
The HPLC purification of the Aβ peptide is highly dependent upon the choice of both the stationary phase employed in the purification and the mobile phase chosen to elute the peptide. The low isoelectric point of the peptide and high propensity for aggregation render traditional chromatographic conditions for the separation of hydrophobic proteins (C4 or C8 stationary phase coupled with an acidic mobile eluent) challenging, with the Aβ peptide eluting as a prolonged broad, non-resolved peak (Figure 2A</…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Agilent for their technical assistance. Kate Markham and Rafael Palomino are credited for their initial help in the synthesis and purification of the Aβ peptide and Dr Hsiau-Wei Lee is thanked for his help in preparing Figure 1 of the manuscript.
Materials
| Agilent 1260 Infinity II quarternary pump | Agilent | G7111B | http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1260-infinity-ii-quaternary-pump |
| Agilent 1260 Infinity II Dual variable wavelength detector | Agilent | G7114A | http://www.agilent.com/en-us/products/liquid-chromatography/lc-detectors/1260-infinity-ii-variable-wavelength-detector |
| Agilent 1260 Infinity II Manual Injector fitted with 10 mL stainless steel sample loop | Agilent | 0101-1232 | http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector |
| Agilent 1260 Infinity II Manual Injector fitted with 20 µL stainless steel sample loop | Agilent | G1328C | http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector |
| Ring Stand Mounting Bracket | Agilent | 1400-3166 | |
| PEEK Tubing Blue (1/32" outer diameter х 0.010" internal diameter) | Thermo Scientific | 03-050-399 | |
| Agilent PLRP-S 300Å 8µm 25 х 300 mm column (Preparative) | Agilent | PL1212-6801 | http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features |
| Agilent PLRP-S 300Å 8µm 7.5 х 300 mm (Semi-Preparative) | Agilent | PL1112-6801 | http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features |
| Agilent PLRP-S 300Å 5 µm 4.6 x 250 mm (Analytical) | Agilent | PL1512-5501 | http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features |
| Aβ42 or Aβ40 peptide | Synthesized in-house using a CEM liberty automated peptide synthesizer. | ||
| Ammonium Hydroxide (NH4OH, 28% solution) | Fisher Scientific | A669-500 | |
| Acetonitrile | Fisher Scientific | A998-4 | |
| HPLC grade water | Fisher Scientific | W5-4 | |
| Falcon 50 ml conical centrifuge tube | Fisher Scientific | 14-954-49A | |
| Supelco PEEK Fitting One-piece fingertight, pkg of 5 ea | Sigma-Aldrich | Z227250 | |
| Normject 5cc sterile syringe | Fisher Scientific | 1481729 | |
| 16 Gauge SS Needle | Rheodyne | 3725-086 |
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