Rapid Protein Anchoring into the Membranes of Mammalian Cells Using Oleyl Chain and Poly(ethylene glycol) Derivatives

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• 作者：Koichi Kato ; Chika Itoh ; Tohru Yasukouchi ; and Teruyuki Nagamune
• 刊名：Biotechnology Progress
• 出版年：2004
• 出版时间：June 2004
• 年：2004
• 卷：20
• 期：3
• 页码：897 - 904
• 全文大小：191K
• 年卷期：v.20,no.3(June 2004)
• ISSN：1520-6033

文摘

The cell membrane is an important interface for communication with extracellularevents, and incorporation of bioactive substances, such as antibodies and receptors,into the cell membrane may enhance the potential abilities of cells. Gene manipulation,chemical modification of membrane proteins, and cell surface painting using a GPIanchor have been performed to introduce substances into cell membranes. Furthermore, many lipid anchors have also been used to modify lipid membranes such asliposomes. In this study, we have focused on developing an easy and rapid method foranchoring of substances including macromolecular proteins into the membranes ofliving mammalian cells. We employed a single oleyl chain derivative coupled withhydrophilic poly(ethylene glycol) (PEG90, the ethyleneoxide (EO) unit is 90) to facilitatesolubilization in water. This water-soluble derivative was designated BiocompatibleAnchor for Membrane (BAM). Some proteins (streptavidin, EGFP and an antibody)were coupled with BAM90 at the distal terminal of PEG and rapidly (within a fewminutes) anchored into the membranes of various cells (NIH3T3, 32D, Ba/F3,hybridoma 9E10). However, the anchored BAM90 disappeared from the cell membranes within 4-5 h in serum-free culture media, and moreover, the retention timeof anchoring was shortened (1-2 h) in culture medium containing 10% FBS. We furtherprepared a dioleylphosphatidylethanolamine (DOPE)-PEG derivative (DOPE-BAM80,the EO unit is 80) as a double oleyl chain derivative for comparison with the singleoleyl chain derivative, BAM90. The retention time of anchored DOPE-BAM80 waslonger than that of BAM90 and more than 8 h in culture media with and without10% serum. Furthermore, the treatment time of DOPE-BAM80 for anchoring wasnearly as short (within a few minutes) as that of BAM90. In addition, both types ofBAMs, BAM90 and DOPE-BAM80, showed no cytotoxicity. Therefore, DOPE-BAM80is useful for protein anchoring to cells. Although the utilization of BAM90 is consideredto be limited, it is expected to useful in restricted environments, for example, in tissuessuch as the cornea, peritoneum, bladder, and various mucosae, which are less exposedto serum. Thus, we suggest the possibility that both types of BAM can be applied tocell surface engineering.