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利用杆状病毒载体在昆虫细胞中表达具有配体刺激激酶活性的人表皮生长因子受体。

Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.

作者信息

Greenfield C, Patel G, Clark S, Jones N, Waterfield M D

机构信息

Ludwig Institute for Cancer Research, London, UK.

出版信息

EMBO J. 1988 Jan;7(1):139-46. doi: 10.1002/j.1460-2075.1988.tb02793.x.

DOI:10.1002/j.1460-2075.1988.tb02793.x
PMID:2834199
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC454229/
Abstract

The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production.

摘要

表皮生长因子（EGF）与特定细胞表面受体结合从而引发一系列生物学反应的机制仍未被充分理解。该系统中信号转导研究的一个重要部分涉及生产足够数量的天然和突变型EGF受体，用于X射线晶体学和光谱分析。此处利用含有编码人EGF受体蛋白cDNA的杆状病毒载体感染昆虫细胞。这导致一种155-kb跨膜蛋白的表达，该蛋白可被四种针对人EGF受体不同区域的抗体识别。用衣霉素、莫能菌素和内切糖苷酶H进行的研究表明，重组受体与天然受体在大小上的差异是由于糖基化的改变。对[125I] EGF结合的研究表明，在完整的受感染昆虫细胞中，Kd为2×10^(-9) M，经去污剂溶解后降至2×10^(-7) M。重组蛋白表现出EGF刺激的酪氨酸蛋白激酶活性，对胰蛋白酶肽段的分析表明，磷酸化受体位点与从A431细胞分离的EGF受体相似。这些观察结果表明，功能性EGF受体可以在昆虫细胞中表达，此外，该系统可用于大规模生产。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/ef79fea3998d/emboj00138-0143-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/0b326e454e3e/emboj00138-0140-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/ed34a3828ccd/emboj00138-0140-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/3e0c8265d8d2/emboj00138-0141-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/11191970bdbc/emboj00138-0141-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/d6c66e7cfc3a/emboj00138-0142-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/ef79fea3998d/emboj00138-0143-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/0b326e454e3e/emboj00138-0140-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/ed34a3828ccd/emboj00138-0140-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/3e0c8265d8d2/emboj00138-0141-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/11191970bdbc/emboj00138-0141-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/d6c66e7cfc3a/emboj00138-0142-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3e36/454229/ef79fea3998d/emboj00138-0143-a.jpg

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Synthesis and processing of asparagine-linked oligosaccharides.天冬酰胺连接寡糖的合成与加工
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