A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells

被引:19
作者
Ailor, E
Pathmanathan, J
Jongbloed, JDH
Betenbaugh, MJ [1 ]
机构
[1] Johns Hopkins Univ, Dept Chem Engn, Baltimore, MD 21218 USA
[2] Univ Connecticut, Sch Med, Farmington, CT 06030 USA
[3] Univ Groningen, Dept Mol Genet, NL-9751 HH Haren, Netherlands
基金
美国国家科学基金会;
关键词
D O I
10.1006/bbrc.1999.0233
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the signal peptide so a baculovirus containing a signal peptidase from Bacillus subtilis (SipS) was constructed for expression studies. When the wild type SipS was coexpressed with scFv, preprocessed scFv fragments were no longer detected in insect cell lysates. Conversely, co-expression of scFv alone or with an inactive mutant SipS resulted in at least 30% of the intracellular polypeptide in an unprocessed form at 3 days post infection. Production of scFv in the medium was also enhanced in the presence of SipS; however, low secretion levels indicate the presence of a post-processing bottleneck. (C) 1999 Academic Press.
引用
收藏
页码:444 / 450
页数:7
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