A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus.
Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6×His tag, as shown with pSeSD-N-lacS. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon Sulfolobus can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site.
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Nan
N
Peng
Ling
L
Deng
Yuxia
Y
Mei
Dongqing
D
Jiang
Yongmei
Y
Hu
Mariana
M
Awayez
Yunxiang
Y
Liang
Qunxin
Q
She
D001089:Arabinose / Q000378:metabolism*; D019848:Gene Expression Regulation, Archaeal / Q000187:drug effects*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D016944:Sulfolobus / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D001089:Arabinose