Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure.

Ulrike Schoetz,Steffen Heuer,David Ng,Wolfgang Beisker,Enrico Kühn,Horst Zitzelsberger,Jutta Pauli,Nikolaos C Deliolanis,Reinhard W Köster,Randolph B Caldwell,Ute Resch-Genger,Sebastian D Fugmann

doi:10.1371/journal.pone.0107069

Abstract

With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model.

Highlights

Fluorescent protein (FP) technology has become a ubiquitous research tool and with ever expanding applications, the need for new and improved FPs displaying unique features and specific spectral properties is growing
DT40 has with 1.361025 mutations/ bp/generation [14] a high and stable mutation rate at the immunoglobulin light chain locus, guaranteeing constant diversification of any gene cloned into this region
The precursor FP used, eqFP615, was transfected into the DT40 cell in a targeting construct that site integrates by homologous recombination as previously described [12]

Summary

Introduction

Fluorescent protein (FP) technology has become a ubiquitous research tool and with ever expanding applications, the need for new and improved FPs displaying unique features and specific spectral properties is growing. For intravital and whole-body imaging applications, the truly sought after FPs are those absorbing/emitting nearer and into the infrared (IR) spectrum [1,2]. Light attenuation in the 590–630 nm range drops 400 times for 8 mm of tissue [4], making FPs with strong absorbance at $630 nm of particular interest. Further attenuating the window of opportunity is the variation in tissue densities effectively giving an upper limit in the 930–1000 nm range after which absorption by lipid and water become interfering factors [5]. For advanced imaging applications such as optical fluorescence tomography and photoacoustic (or optoacoustic) tomography the effective diagnostic and therapeutic window is in the 650– 900 nm range [6]

Methods

Results

Conclusion