Figure 1
From: Control of total GFP expression by alterations to the 3′ region nucleotide sequence
Effect of the native hydrophilic C-terminus of GFP on total and soluble protein expression. To obtain GFP expression vectors carrying the native hydrophilic C-terminal sequence of GFP (MDELYK; 6 aa; hy, +0.35) with a native stop codon (TAA), we constructed the C-terminal peptide LeuGlu(LE)-6×His (6H; 6 aa; hy, –0.28)-deleted clones of GFP-Stop(TAA)-# (where # = XhoI-6×His, the deleted non-coding nucleotide sequence of LeuGlu [Xho I restriction site]-6×His and is indicated by italics beyond the stop codon), ME6-GFP-Stop(TAA)-#, and MK6-GFP-Stop(TAA)-# from the control clones of GFP-LE-6H-Stop(TGA), ME6-GFP-LE-6H-Stop(TGA), and MK6-GFP-LE-6H-Stop(TGA), respectively, as described in the Methods. The coding region of the Xho I restriction site (CTCGAG; LeuGlu)-6×His(CAC) tag, derived from pET22b(+), is referred to as LeuGlu-6×His (LE-6H), and the non-coding region of the same sequence beyond the stop codon is #. Approximately 50 μg of total protein and a counter volume of the soluble fraction were used for fluorescence measurements as described in the Methods. All cultures were performed in triplicate, and results represent the mean value of at least duplicate experiments. Error bars indicate standard deviations. Lanes: 1, GFP-LE-6H-Stop(TGA), control; 2, GFP-Stop(TAA)- #; 3, ME6-GFP-LE-6H-Stop(TGA), control; 4, ME6-GFP-Stop(TAA)- #; 5, MK6-GFP-LE-6H-Stop(TGA), control; and 6, MK6-GFP-Stop(TAA)- #.