Expression and chain assembly of human laminin-332 in an insect cell-free translation system.
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.
Phan
Hoang-Phuong
HP
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, Japan.
Ezure
Toru
T
Ito
Masaaki
M
Kadowaki
Tatsuhiko
T
Kitagawa
Yasuo
Y
Niimi
Tomoaki
T
D000818:Animals; D015815:Cell Adhesion Molecules / Q000096:biosynthesis*; D002474:Cell-Free System; D004220:Disulfides; D006801:Humans; D007313:Insecta; D014176:Protein Biosynthesis; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D004220:Disulfides; D021122:Protein Subunits; D000097794:Kalinin