Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli.
The correct folding of a protein is a pre-requirement for its proper posttranslational modification. The Escherichia coli Sec pathway, in which preproteins, in an unfolded, translocation-competent state, are rapidly secreted across the cytoplasmic membrane, is commonly assumed to be unfavorable for their modification in the cytosol. Whether posttranslationally modified recombinant preproteins can be efficiently transported via the Sec pathway, however, remains unclear. ACP and BCCP domain (BCCP87) are carrier proteins that can be converted into active phosphopantetheinylated ACP (holo-ACP) and biotinylated-BCCP (holo-BCCP) by AcpS and BirA, respectively. In the present study, we show that, when ACP or BCCP87 is fused to the C-terminus of secretory protein YebF or MBP, the resulting fusion protein preYebF-ACP, preYebF-BCCP87, preMBP-ACP or preMBP-BCCP87 can be modified and then secreted. Our data demonstrate that posttranslational modification of preYebF-ACP, preYebF-BCCP87 preMBP-ACP and preMBP-BCCP87 can take place in the cytosol prior to translocation, and the Sec machinery accommodates these previously modified fusion proteins. High levels of active holo-ACP and holo-BCCP87 are achieved when AcpS or BirA is co-expressed, especially when sodium azide is used to retard their translocation across the inner membrane. Our results also provide an alternative to achieve a high level of modified recombinant proteins expressed extracellularly.
College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, PR China.
Nan
N
Chen
Fu-Lin
FL
Hong
Hai-Hong
HH
Wang
Qi-Hang
QH
Yuan
Wan-Yan
WY
Ma
Xu-Na
XN
Gao
Rui
R
Shi
Rui-Juan
RJ
Zhang
Chang-Sheng
CS
Sun
Sheng-Bin
SB
Wang
D000103:Acetyl-CoA Carboxylase / Q000235:genetics; D000213:Acyl Carrier Protein / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism; D054889:Fatty Acid Synthase, Type II / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D000213:Acyl Carrier Protein; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C508772:YebF protein, E coli; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein