The Gram-positive bacterium Lactococcus lactis is an ideal expression host for the overproduction of heterologous proteins in a functional form. L. lactis has recently been identified as an efficient Gram-positive cell factory for the production of recombinant proteins and the safety of this production system has been confirmed in multiple clinical trials. Key desirable features of L. lactis include its generally recognized as safe (GRAS) status, long history of safe use in food production, probiotic properties, absence of endotoxins, capacity to secrete stable recombinant protein to the growth medium, the presence of few proteases, and a diverse selection of cloning and inducible expression vectors. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. We have previously described the production of several Plasmodium falciparum antigens with varying degrees of predicted structural complexities, those which are considered difficult-to-produce proteins by using L. lactis pH-dependent inducible promoter (P170). The purpose of this chapter is to provide a detailed protocol for the expression of difficult-to-produce proteins, mainly high cysteine-rich proteins, in the soluble form in L. lactis from cloning of the target gene to the determination of expression levels and purification.