昆虫杆状病毒细胞表达系统 课件

9) Ease of purification
Some 6xHis and glutathione S-transferase (GSST) Baculoviirruuss EExxpprreessssiioonn aanndd Purification Kits has been developed, designed for easy and reliable singlestep ppuurriiffiiccaattiioonn ooff rreeccoommbbiinnaanntt pprrootteeiinnss.. TThhee 66xxHHiiss ppuurriiffiiccaattiioonn ssyysstteemm relies on the high specificity of the 6xHis tag for Ni-NTA Agarose. The GST purification system takes advantage of the high affinity of glutathione agarose beads for reduced glutathione. These kits combine the advantages of expressing functional and soluble recombinant proteins using BEVS technology with the convenience of a GST or 6xHis affinity purification system. Even under the highest expression levels, most GST and 6xHis fusion proteins expressed in insect cells remain predominantly soluble.
7) Simultaneous expression of multiple genes
BEVS has the capability to express two or more genes simultaneously within single infected insect cells. Protein complexes that depend on dimer or multidimer formation for activity can be assembled. A well known example is the formation of complete virus capsids from a variety of viruses which have been assembled in vitro, using BEVS, by coexpressing the capsid subunits simultaneously.
The highest expression level reported is 50% of the total cellular protein of an infected insect cell corresponding to approximately 1g of recombinant protein per 1 × 1099 cells. However, many recombinant pprrootteeiinnss aarree nnoott pprroodduucceedd at such high amounts and it is usually difficult to predict the amount of protein expression. There are some guidelines one can follow to optimize protein production. Of primary importance is optimizing the design of the recombinant Baculovirus Transfer Vector.
2) Post-translational modifications
Several post-translational modifications have been reported to occur in the BEVS, including N- and O-linked glycosylation, phosphorylation, acylation, amidation, carboxymethylation, isoprenylation, signal peptide cleavage and proteolytic cleavage. The sites where these modifications occur are often identical to those of the authentic protein in its native cellular environment.
The BEVS typically produces overexpressed recombinant proteins containing properfolding, disulfide bond formation and oligomerization. Additionally, this system is capable of performing several post-translational modifications. This leads to a protein that is similar to its native counterpart, both structurally and functionally. However, in cases where the authentic protein functions as a heterodimer or relies on tissue- or species-specific modifications, the recombinant Baculovirus-expressed protein may not be functionally active, unless its binding partner or modifying enzyme is coexpressed.
4) Capacity for large inserts
The expandability of the capsid structure of Baculoviruses allows the packaging and expression of very large genes. There is no known upper size limit for the insertion of foreign sequences into the BV genome.
6) Simplicity of technology
This technology has made expression of full-length proteins fast, easy and reliable. Recombinant Baculovirus can be obtained in two simple steps–cloning and co-transfection–in as little as 5 days. The ease of use now rivals that of bacterial expression systems and BEVS technology does not require that the recombinant protein be expressed as a fusion protein.
昆虫杆状病毒表达载体系统（Baculovirus Expression Vector System，简称BEVS），基于昆虫杆状病毒及其宿主细胞建立起来 的表达体系；是继大肠杆菌、酵母和哺乳动物细胞表达系统之后 建立起来的，始于本世纪80年代。
昆虫杆状病毒表达载体系统
过去20多年来，昆虫杆状病毒表达系统已经成为重组蛋白常 规表达最广泛使用的系统之一。
Βιβλιοθήκη Baidu
5) Capacity to express unspliced genes
Insect cells have the capability to perform intron/exon splicing. However, certain virus-, tissue- or species-specific splicing patterns will not be obtained if they require the presence of particular splicing factors which are not available in the infected insect cell environment. In general, for high protein expression levels, a cDNA insert rather than a genomic DNA fragment is recommended.
3) High expression levels
Compared to other higher eukaryotic expression systems, the most dissttiinngguuiisshhiinngg ffeeaattuurree oof the BEVS is iittss ppootteennttiiaall to achieve high levels of expressionn ooff aa cclloonneedd ggeennee. The BEVS ssyysstteemm hhaass pprroovveenn ppaarrttiiccuullaarrllyy uusseeffuull in the generation of large quantities of proteinss ffoorr ssttrruuccttuurraall aannaallyyssiiss.
8) Localization of recombinant proteins
Baculovirus-expressed recombinant proteins are usually localized in the same subcellular compartment as the authentic protein. Nuclear proteins will be transported to the insect nucleus, membrane proteins will be anchored into the cell membrane, and secreted protein will be secreted by infected insect cells.
今天，已经有成千上万种重组蛋白成功利用昆虫杆状病毒表 达系统得到表达，其范围涵盖从细胞器酶到膜结合蛋白。
这种表达系统得到如此广泛的应用主要是因为其独特的优点
。
优势
简单易用 蛋白大小
多重表达 信号肽切除 内含子剪切 核转运 活性蛋白 磷酸化 糖基化 乙酰化
1) Functional activity of the recombinant protein
5.2.2 真核细胞表达系统 —昆虫杆状病毒细胞表达系统
传统基因工程技术的四个 要素
•工 具 酶：内切酶、连接酶等 •载 体：pUC、pET系列等 •目的基因：X or Y •宿 主：
原核细胞 ：大肠杆菌等
真核细胞 ：酵母、昆虫细胞、哺乳动物细胞
等
昆虫杆状病毒表达载体系统（BEVS）
世界上的昆虫种类约有150多万种，可以感染昆虫的病毒大约有 1200多种，分属于7个不同的科，杆状病毒科是其中最普遍的一 群。杆状病毒科又被分为3个亚群：核型多角体病毒（Nuclear Polyhedrosis Virus, NPV）、颗粒状病毒（Granulosis virus, GV）与非包涵型杆状病毒（Nonoccluded Virus, NOV）。