Picornaviruses are positive sense ribonucleic acid (RNA) genome containing viruses that produce a single polyprotein, which is co-translationally and post-translationally cleaved by viral proteases into individual proteins. The picornaviral 3ABC is a toxic protein and is difficult to express in heterologous systems. One of the methods to circumvent toxicity is to control the expression of the protein until sufficient biomass is accumulated. Lac promoter controlled T7 polymerase system is the most popular system but instability of expression is one of the major problems. We compared three different conditions for the expression of picornaviral 3ABC. The results show that the conventional media lead to leaky as well as lactose induced expression, and glucose-mediated repression can be used to overcome this obstacle, thus allowing for the expression of picornaviral 3ABC. This system could potentially be used for the prokaryotic expression of other toxic proteins.