Expression and chain assembly of human laminin-332 in an insect cell-free translation system |
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Authors: | Phan Hoang-Phuong Ezure Toru Ito Masaaki Kadowaki Tatsuhiko Kitagawa Yasuo Niimi Tomoaki |
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Institution: | Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, Japan. |
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Abstract: | Laminins are a family of large heterotrimeric glycoproteins comprising alpha, beta, and gamma chains. To determine the molecular mechanisms underlying chain assembly in vitro, we expressed human laminin-332 subunits in an insect cell-free translation system. We successfully produced the beta3-gamma2 heterodimer and the alpha3-beta3-gamma2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The alpha3-beta3 and the alpha3-gamma2 heterodimer were not detected, suggesting that the alpha3 chain can assemble with only beta3-gamma2 heterodimer to form a heterotrimer via disulfide bonds. These results are consistent with those of a previous report indicating that laminin chain assembly proceeds through the beta-gamma heterodimer to the alpha-beta-gamma heterotrimer in vivo. We suggest that the cell-free translation system is a valid system with which to study the mechanisms underlying laminin chain assembly. |
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