Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis

Abstract

To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-Change Site-Directed Mutagenesis Kit protocol (Stratagene, La Jolla, CA). Our new protocol consists of (i) a PCR reaction using an in vitro technique, LDA (ligation-during-amplification), (ii) a DPN:I treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik Change Kit protocol introduces mutations at a single site, requiring two complementary mutagenic oligonucleotides, our new protocol requires only one mutagenic oligonucleotide for a mutation site, and can introduce mutations in a plasmid at multiple sites simultaneously. A targeting efficiency >70% was consistently achieved for multiple-site mutagenesis. Furthermore, the new protocol allows random mutagenesis with degenerative primers, because it does not use two complementary primers. Our mutagenesis strategy was successfully used to alter the fluorescence properties of green fluorescent protein (GFP), creating a new-color GFP mutant, cyan-green fluorescent protein (CGFP). An eminent feature of CGFP is its remarkable stability in a wide pH range (pH 4-12). The use of CGFP would allow us to monitor protein localization quantitatively in acidic organelles in secretory pathways.

Figure 1

Schematic diagram of the multiple-site mutagenesis protocol. The two mutagenic primers are designed on a minus strand. The methylated template DNA strands are shown by thin lines; the in vitro-synthesized DNA strands by thick lines. The plus and minus strands are drawn by broken and solid lines, respectively. Xs indicate introduced mutations. ‘>>>’ means that the species on the left is dominant in the reaction mixture.

Figure 2

Fluorescence excitation (A) and emission (B) spectra of EGFP and its mutants: EGFP-Y66W, EGFP-T203Y and EGFP-Y66W/T203Y in cuvettes. Fluorescence was measured in 50 mM HEPES/KOH pH 7.5 at a protein concentration of 1 µM. They are depicted by green, blue, yellow and cyan lines, respectively. All spectra were normalized to a maximal value of 1.0. Differential excitation of fluorescent colonies was performed using two wavelengths, 450 and 490 nm, which are indicated by vertical red lines in (A). A band-pass filter (535DF25) is shown by a box drawn by a red dashed line.

Figure 3

An excitation ratio image (490/450) of fluorescent streaked bacterial cells producing EGFP (top left), EGFP-Y66W (bottom right), EGFP-T203Y (bottom left) and EGFP-Y66W/T203Y (top right).

Figure 4

An excitation ratio image (490/450) of fluorescent colonies expressing EGFP with the mutagenized substitutions Y66W and T203Y.

Figure 5

Excitation (dotted lines) and emission (solid lines) spectra of ECFP (cyan), ECGFP (black) and EGFP (green). All amplitudes have been normalized to a maximum value of 1.0.

Figure 6

pH-dependency of absorbance (A, C and E) and fluorescence emission (B, D and F) spectra of EGFP (A and B), ECFP (C and D) and ECGFP (E and F). Excitation was at 450 nm for EGFP, 430 nm for ECFP and 450 nm for ECGFP.

Figure 7

(A) pH-dependency of normalized peak amplitudes of absorbance of EGFP (488 nm), ECFP (435 nm) and ECGFP (463 nm). (B) pH-dependency of fluorescence emission of EGFP, ECFP and ECGFP. Because the emission spectrum of ECFP changes its shape with pH, the peak areas were normalized and plotted.