Expression and Chain Assembly of Human Laminin-332 in an Insect Cell-Free Translation System

Abstract

Laminins are a family of large heterotrimeric glycoproteins comprising α, β, and γ 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 β3-γ2 heterodimer and the α3-β3-γ2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The α3-β3 and the α3-γ2 heterodimer were not detected, suggesting that the α3 chain can assemble with only β3-γ2 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 β-γ heterodimer to the α-β-γ 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.