Improved secretion of the cancer-testis antigen SSX2 in Pichia pastoris by deletion of its nuclear localization signal Journal Article
| Authors: | Huang, C. J.; Anderson, K. A.; Damasceno, L. M.; Ritter, G.; Old, L. J.; Batt, C. A. |
| Article Title: | Improved secretion of the cancer-testis antigen SSX2 in Pichia pastoris by deletion of its nuclear localization signal |
| Abstract: | The cancer-testis (CT) antigen synovial sarcoma X break point 2 (SSX2) was expressed in Pichia pastoris as a means to produce a delayed-type hypersensitivity skin test reagent for monitoring SSX2-specific anti-cancer immune responses. SSX2 was detected intracellularly in P. pastoris despite the addition of the Saccharomyces cerevisiae alpha-mating factor secretion signal. Increasing the SSX2 gene copy number did not improve its secretion but did enhance intracellular SSX2 levels. SSX2 with its C-terminal nuclear localization signal (NLS) deleted (SSX2NORD), however, was secreted. Indirect immunofluorescence indicated that SSX2 containing the NLS did not translocate to the nucleus but accumulated in the endoplasmic reticulum (ER). Experimental results further suggested that SSX2 containing the NLS was misfolded in the ER, while deletion of the NLS facilitated correct folding of SSX2 inside the ER and improved its secretion. Production of SSX2NORD was scaled-up to a 2-L fermentor using a fed-batch protocol to maintain methanol at a concentration of 1 g L -1. Decreasing the cultivation temperature from 25 °C to 16 °C improved protein stability in the culture supernatant. In this process, after 120 h cultivation, the wet cell weight of P. pastoris reached 280 mg mL-1, and the yield of SSX2NORD was 21.6 mg L-1. © 2009 Springer-Verlag. |
| Keywords: | sequence deletion; nonhuman; proteins; neoplasm proteins; culture medium; protein; protein stability; immunofluorescence; biotechnology; endoplasmic reticulum; immune response; antigen; antigens; antigens, neoplasm; cell culture; saccharomyces cerevisiae; cancer testis antigen; nucleotide sequence; recombinant proteins; testicular neoplasms; temperature; yeast; synovial sarcoma; repressor proteins; nuclear localization signal; deletion mutant; secretion; fluorescent antibody technique, indirect; nuclear localization signals; concentration of; methanol; cancer-testis antigen; delayed-type hypersensitivity; pichia pastoris; recombinant protein secretion; ssx2; break-points; culture supernatant; delayed type hypersensitivity; fed batches; fermentors; gene copy number; indirect immunofluorescence; p. pastoris; fermentation; scale up; supernatant; synovial sarcoma x break point 2; hypersensitivity, delayed; pichia |
| Journal Title: | Applied Microbiology and Biotechnology |
| Volume: | 86 |
| Issue: | 1 |
| ISSN: | 0175-7598 |
| Publisher: | Springer |
| Date Published: | 2010-03-01 |
| Start Page: | 243 |
| End Page: | 253 |
| Language: | English |
| DOI: | 10.1007/s00253-009-2275-2 |
| PUBMED: | 19826807 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Export Date: 20 April 2011" - "CODEN: AMBID" - "Source: Scopus" |
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