ABSTRACT
Vasoinhibin belongs to a family of proteins with antiangiogenic properties derived by proteolytic cleavage from the hormone prolactin (PRL). Vasoinhibin isoforms range from the first 79 to the first 159 residues of PRL. In an attempt to increase the yield of recombinant vasoinhibin and avoid incorrect intra- and inter-disulfide bond formation, the cDNA sequence comprising the first 123 amino acids of human PRL, in which cysteine 58 was or not mutated to serine, was codon-optimized. The optimized constructs achieved a 6-fold increase in mRNA expression but showed no change in protein production and reduced protein secretion when expressed in human embryo kidney (HEK293T/17) cells. Limited vasoinhibin levels associated with the activation of the unfolded protein response (UPR) and endoplasmic reticulum-associated degradation (ERAD) as revealed by the upregulation of UPR (Bip, Xbp-1, and Chop) and ERAD (Hrd1, Os9, and Sel1l) target genes. Mutation to serine introduced a new N-glycosylation site and associated with increased glycosylation and release of glycosylated vasoinhibin isoforms having reduced antiangiogenic properties. We conclude that overexpression and excessive glycosylation lead to protein degradation and reduced bioactivity, respectively, negatively affecting the production of recombinant vasoinhibin in mammalian cells.
