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使用类弹性蛋白多肽融合蛋白检测乙型肝炎病毒X蛋白的非色谱方法

Non-chromatographic Method for the Hepatitis B Virus X Protein Using Elastin-Like Polypeptide Fusion Protein.

作者信息

Kwon Soon-Hwan, Cho Hyeseong

机构信息

Division of High-Risk Pathogen Research, Korea National Institute of Health, Osong, Korea ; Department of Biochemistry, School of Medicine, Ajou University, Suwon, Korea.

出版信息

Osong Public Health Res Perspect. 2012 Jun;3(2):79-84. doi: 10.1016/j.phrp.2012.04.003.

DOI:10.1016/j.phrp.2012.04.003
PMID:24159495
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3747649/
Abstract

OBJECTIVES

Hepatitis B virus (HBV) is a member of the hepadnavirus family. The HBV genome contains four genes designated as S, C, P, and X. The HBV X (HBx) gene encodes for a 16.5-kDa regulatory protein that enhances HBV replication and exerts multifunctional activities. The aim of this study is to describe the rapid and easy purification of HBx using ELP (elastin-like polypeptide) fusion protein.

METHODS

The ELP-HBx fusion protein was overexpressed in Escherichia coli. Environmental sensitivity was demonstrated via turbidity and dynamic light scattering as a function of temperature. HBx was purified as an ELP fusion protein. ELPs are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. ELP follows in temperature and salt consistency, precipitation, and solution repetition (inverse transition cycling) with polypeptide, where it purifies the protein in a simple manner.

RESULTS

Fusion proteins underwent supramolecular aggregation at 40 ℃ in 1 M NaCl and slowly resolubilized at subphysiologic temperatures. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity to the commercial HBx.

CONCLUSION

This study suggests that HBx can be purified rapidly and easily using inverse transition cycling, and that this method can be applied in determination of HBx 3D structures and HBx stability study.

摘要

目的

乙型肝炎病毒（HBV）是嗜肝DNA病毒科的成员。HBV基因组包含四个基因，分别命名为S、C、P和X。HBV X（HBx）基因编码一种16.5 kDa的调节蛋白，该蛋白可增强HBV复制并发挥多种功能。本研究的目的是描述使用弹性蛋白样多肽（ELP）融合蛋白快速简便地纯化HBx的方法。

方法

ELP-HBx融合蛋白在大肠杆菌中过表达。通过浊度和动态光散射作为温度的函数来证明环境敏感性。HBx作为ELP融合蛋白进行纯化。ELP是五肽重复序列Val-Pro-Gly-Xaa-Gly的生物聚合物，会发生逆温度相变。ELP在温度和盐浓度、沉淀以及与多肽的溶液重复（逆转变循环）方面表现一致，它以简单的方式纯化蛋白质。

结果

融合蛋白在40℃、1 M NaCl中发生超分子聚集，并在亚生理温度下缓慢重新溶解。ELP结构域蛋白水解产生了一种与市售HBx大小和免疫反应性相当的肽。

结论

本研究表明，使用逆转变循环可以快速简便地纯化HBx，并且该方法可应用于HBx三维结构的测定和HBx稳定性研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/629f0952bccf/EPHRP1-03-79-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/fa19a9bca0d4/EPHRP1-03-79-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/d4d00424b9a2/EPHRP1-03-79-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/6a97997a6d36/EPHRP1-03-79-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/629f0952bccf/EPHRP1-03-79-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/fa19a9bca0d4/EPHRP1-03-79-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/d4d00424b9a2/EPHRP1-03-79-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/6a97997a6d36/EPHRP1-03-79-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef0c/3747649/629f0952bccf/EPHRP1-03-79-g004.jpg

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