Borth Nicole, Mattanovich Diethard, Kunert Renate, Katinger Hermann
Institute for Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
Biotechnol Prog. 2005 Jan-Feb;21(1):106-11. doi: 10.1021/bp0498241.
Previous work has shown that a human-antibody-producing recombinant CHO cell line did not increase its intracellular content of protein disulfide isomerase (PDI) and heavy chain binding protein (BIP) according to the increasing expression of antibody. It was also found that the intracellular assembly of light and heavy chain is a major limiting factor for overall cell specific productivity, as secretion rates improve with higher light chain expression levels and heavy chain accumulates intracellularly when too little light chain is present. As these CHO cells had a significantly lower intracellular PDI content compared to that of hybridoma cells, these results have led us to try to overcome the limitation in the posttranslational assembly in the endoplasmatic reticulum. Recombinant CHO cells were transfected with PDI or BIP alone or in combination, and the effect on intracellular light and heavy chain content and specific production rate was determined. Overexpression of BIP, both alone and in combination with PDI, reduced the specific secretion rate, whereas PDI, when overexpressed alone, caused an increase of product secretion rate.
先前的研究表明，一株产生人抗体的重组CHO细胞系并不会随着抗体表达量的增加而提高其细胞内蛋白质二硫键异构酶（PDI）和重链结合蛋白（BIP）的含量。研究还发现，轻链和重链的细胞内组装是总体细胞比生产率的主要限制因素，因为随着轻链表达水平的提高，分泌率会提高，而当轻链含量过少时，重链会在细胞内积累。由于这些CHO细胞的细胞内PDI含量明显低于杂交瘤细胞，这些结果促使我们尝试克服内质网中翻译后组装的限制。将重组CHO细胞单独或联合转染PDI或BIP，并测定其对细胞内轻链和重链含量以及比生产率的影响。单独或与PDI联合过表达BIP均会降低比分泌率，而单独过表达PDI则会导致产物分泌率增加。
