Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts.
This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours.
Ahn
Jin-Ho
JH
Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, Seoul 151-742, Korea.
Hwang
Mi-Yeon
MY
Lee
Kyung-Ho
KH
Choi
Cha-Yong
CY
Kim
Dong-Myung
DM
D002474:Cell-Free System; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D003062:Codon; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins