Ribulose-1,5-bisphosphate carboxylase–oxygenase (Rubisco) catalyses the first step in carbon fixation and is a strategic target for improving photosynthetic efficiency. In plants, Rubisco is composed of eight large and eight small subunits, and its biogenesis requires multiple chaperones. Here, we optimized a system to produce tobacco Rubisco in Escherichia coli by coexpressing chaperones in autoinduction medium. We successfully assembled tobacco Rubisco in E. coli with each small subunit that is normally encoded by the nuclear genome. Even though each enzyme carries only a single type of small subunit in E. coli, the enzymes exhibit carboxylation kinetics that are very similar to the carboxylation kinetics of the native Rubisco. Tobacco Rubisco assembled with a recently discovered trichome small subunit has a higher catalytic rate and a lower CO₂ affinity compared with Rubisco complexes that are assembled with other small subunits. Our E. coli expression system will enable the analysis of features of both subunits of Rubisco that affect its kinetic properties.