Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment

Abstract

Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology.

Molecular imaging with engineered physiology

“…Plasmids pEF-ENTR A (Addgene #17427) and pLenti X1 Zeo (Addgene #17299) were gifts from Eric Campeau51. Bacterial expression plasmids Z503, Z507 and Z508 were cloned using the Golden Gate method52 by assembling synthetic DNA sequences (IDT, Coralville, IA, USA) and PCR amplicons into the backbone of pEF-ENTR A. cfSGFP2 is a cysteine-free variant of the GFP53. CGRP is the alpha-isoform of human CGRP.…”

Characterization of Russell Bodies Accumulating Mutant Antithrombin Derived from the Endoplasmic Reticulum

“…The details on the construction of a plasmid for ss-cfSGFP2 (cysteine-free SGFP2 with a signal sequence of α1-antitrypsin) were reported previously. 26) Cell Culture, Establishment of Stably Expressing Cell Lines and Transfection Chinese hamster ovary (CHO) cells were cultured with Ham's F-12 medium containing 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 . 24) To prepare CHO cells in which the expression of wild type AT-GFP or mutant AT(C95R)-GFP is controlled with doxycycline, CHO cells expressing Tet repressor protein were transfected with 3.0 µg of pTRE2hyg/AT-GFP and pTRE2hyg/AT(C95R)-GFP, respectively, using the Effectene transfection reagent.…”

“…The details were basically the same as reported 26) except that the fluorescence light was split into two parts by a 50% beam splitter and detected by two single-photon avalanche diodes. The resulting two signals, F 1 and F 2 , were cross-correlated to yield the normalized second order autocorrelation function, G (2) (τ), with the correlation time τ,…”

Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway

“…Currently, two green FPs (superfolder GFP [9,10], and cfSGFP2 [11]) and the mFruit family [12] of FPs are available for use within the secretory pathway, however no unmodified blue variants exist. Creation of a secretory pathway blue FP will increase investigators’ ability to assay a greater number of reporters simultaneously and could provide a potential FRET pair [13].…”