Changes of primary sequence and secondary structure proximal to the 5' end of the stop codon substantially increases the expression of the variable region of an antibody in E. coli.
The sequence context at the 5' end of the stop codon may influence the efficiency of termination and translation. To increase the expression of a designed variable region of an antibody (named as VH5) against tumor necrosis factor alpha (TNFalpha), two nucleotides (TC) at 25 and 26 nucleotides (nt) upstream of termination codon were substituted with AG, respectively. The free energy of 70 nt (arbitrarily defined from the 32 nt upstream of termination codon to 38 nt downstream) was changed from -13.5 kcal mol-1 to -17.3 kcal mol-1. The expression level was increased from 1+/-0.3% to 10+/-1.2% of total cellular protein. Although the precise mechanism of this phenomenon remains to be elucidated, this report provides an alternative means to increase the expression of a foreign gene in E. coli.
Qin
Weisong
W
Institute of Basic Medical Sciences, P.O. Box 130(3), Beijing, 100850, P.R. China.
Feng
Jiannan
J
Li
Yan
Y
Lin
Zhou
Z
Shen
Beifen
B
D024506:5' Flanking Region; D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D018388:Codon, Terminator / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D009690:Nucleic Acid Conformation; D015202:Protein Engineering / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D000906:Antibodies; D018388:Codon, Terminator; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha