Expression and secretion of SSX2, a Cancer-Testis (CT) antigen, was investigated in Pichia pastoris as a means to develop a potential immunotherapy. Humoral and cellular immune responses against SSX2 gene products suggest that SSX2 is an attractive vaccine target for cancer immunotherapy. SSX2 was detected intracellularly in Pichia despite the addition of the Saccharomyces cerevisiae alpha mating factor signal. Deletion of the C-terminal nuclear retention signal (NLS) of SSX2, or SSX2NORD, however, significantly improved SSX2 secretion. Indirect immunofluorescence indicated that SSX2 with the NLS did not locate to the nucleus, but formed protein aggregates. Our experimental results further suggest that SSX2 with the NLS was misfolded in the ER and subjected to ER-associated protein degradation, while deletion of NLS facilitates correct folding of SSX2 inside the ER, therefore, improving its secretion. The important SSX2 epitopes have been reported at the N terminus of the protein, SSX2NORD is considered as a possible vaccine formats. Therefore, production of SSX2NORD was later scaled up in a 2 L fermentor that incorporated with a fed-batch protocol by maintaining a methanol concentration around 1 g/L. Decreasing the cultivation temperature from 25 ℃ to 16 ℃ improved protein stability in the supernatant. Furthermore the feed of glycerol/methanol mix was found to be crucial for cell growth during the transitional phase from glycerol (batch) utilization to methanol (induction) utilization. In this process, after 116 hours cultivation, the wet cell weight of P. pastoris reached 230 mg/ml and the SSX2NORD was produced at a yield of 50 mg/L.