The mKate fluorescent protein expressed by Leishmania mexicana modifies the parasite immunopathogenicity in BALB/c mice.

Parasites have been engineered to express fluorescent reporter proteins, yet the impact of red fluorescent proteins on Leishmania infections remains largely unknown. We analysed the infection outcome of Leishmania mexicana parasites engineered for the constitutive expression of mKate protein and evaluated their immunogenicity in BALB/c mice. Infection of BALB/c mice with mKate transfected L. mexicana (LmexmKate ) parasites caused enlarged lesion sizes, leading to ulceration, and containing more parasites, as compared to LmexWT . The mKate protein showed immunogenic properties inducing antibody production against the mKate protein, as well as enhancing antibody production against the parasite. The augmented lesion sizes and ulcers, together with the more elevated antibody production, were related to an enhanced number of TNF-α and IL-1ß producing cells in the infected tissues. We conclude that mKate red fluorescent protein is an immunogenic protein, capable of modifying disease evolution of L. mexicana.

Enhanced Tolerance against a Fungal Pathogen and Insect Resistance in Transgenic Tobacco Plants Overexpressing an Endochitinase Gene from Serratia marcescens.

In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.

Photoactivatable Blue Fluorescent Protein.

Photoactivatable and photoswitchable fluorescent proteins (FPs) are sophisticated molecular tools that in combination with super-resolution microscopy are helping to elucidate many biological processes. Through the Y66H mutation in the chromophore of the violet fluorescent protein SumireF, we created the first photoactivatable blue fluorescent protein (PA-BFP). This protein is rapidly activated over ordinary UV transilluminators at 302 or 365 nm in irreversible mode and by direct exposition to sunlight. The maximum excitation and emission wavelengths of this protein, centered at 358 and 445 nm, respectively, resemble the values of DAPI-the blue stain widely used in fluorescence microscopy to visualize nucleic acids in cells. Therefore, the immediate use of PA-BFP in cellular biology is clear because the technology required to follow this new genetically encoded reporter at the microscopic level has already been established. PA-BFP can potentially be used together with other photoactivatable fluorescent proteins of different colors to label multiple proteins, which can be simultaneously tracked by advanced microscopic techniques.

A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins.

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M-1  cm-1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.

Structural Factors Enabling Successful GFP-Like Proteins with Alanine as the Third Chromophore-Forming Residue.

GFP-like proteins from lancelets (lanFPs) is a new and least studied group that already generated several outstanding biomarkers (mNeonGreen is the brightest FP to date) and has some unique features. Here, we report the study of four homologous lanFPs with GYG and GYA chromophores. Until recently, it was accepted that the third chromophore-forming residue in GFP-like proteins should be glycine, and efforts to replace it were in vain. Now, we have the first structure of a fluorescent protein with a successfully matured chromophore that has alanine as the third chromophore-forming residue. Consideration of the protein structures revealed two alternative routes of posttranslational transformation, resulting in either chromophore maturation or hydrolysis of GYG/GYA tripeptide. Both transformations are catalyzed by the same set of catalytic residues, Arg88 and Glu35-Wat-Glu211 cluster, whereas the residues in positions 62 and 102 shift the equilibrium between chromophore maturation and hydrolysis.

CiPerGenesis, A Mutagenesis Approach that Produces Small Libraries of Circularly Permuted Proteins Randomly Opened at a Focused Region: Testing on the Green Fluorescent Protein.

Circularly permuted proteins (cpPs) represent a novel type of mutant proteins with original termini that are covalently linked through a peptide connector and opened at any other place of the polypeptide backbone to create new ends. cpPs are finding wide applications in biotechnology because their properties may be quite different from those of the parental protein. However, the actual challenge for the creation of successful cpPs is to identify those peptide bonds that can be broken to create new termini and ensure functional and well-folded cpPs. Herein, we describe CiPerGenesis, a combinatorial mutagenesis approach that uses two oligonucleotide libraries to amplify a circularized gene by PCR, starting and ending from a focused target region. This approach creates small libraries of circularly permuted genes that are easily cloned in the correct direction and frame using two different restriction sites encoded in the oligonucleotides. Once expressed, the protein libraries exhibit a unique sequence diversity, comprising cpPs that exhibit ordinary breakpoints between adjacent amino acids localized at the target region as well as cpPs with new termini containing user-defined truncations and repeats of some amino acids. CiPerGenesis was tested at the lid region G134-H148 of green fluorescent protein (GFP), revealing that the most fluorescent variants were those starting at Leu141 and ending at amino acids Tyr145, Tyr143, Glu142, Leu141, Lys140, and H139. Purification and biochemical characterization of some variants suggested a differential expression, solubility and maturation extent of the mutant proteins as the likely cause for the variability in fluorescence intensity observed in colonies.

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression.

Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.

Spiked Genes: A Method to Introduce Random Point Nucleotide Mutations Evenly throughout an Entire Gene Using a Complete Set of Spiked Oligonucleotides for the Assembly.

In vitro mutagenesis methods have revolutionized biological research and the biotechnology industry. In this study, we describe a mutagenesis method based on synthesizing a gene using a complete set of forward and reverse spiked oligonucleotides that have been modified to introduce a low ratio of mutant nucleotides at each position. This novel mutagenesis scheme named "Spiked Genes" yields a library of clones with an enhanced mutation distribution due to its unbiased nucleotide incorporation. Using the far-red fluorescent protein emKate as a model, we demonstrated that Spiked Genes yields richer libraries than those obtained via enzymatic methods. We obtained a library without bias toward any nucleotide or base pair and with even mutations, transitions, and transversion frequencies. Compared with enzymatic methods, the proposed synthetic approach for the creation of gene libraries represents an improved strategy for screening protein variants and does not require a starting template.

LanFP10-A, first functional fluorescent protein whose chromophore contains the elusive mutation G67A.

Since Green Fluorescent Protein (GFP) was first successfully expressed in heterologous systems in 1994, many genes encoding other natural autofluorescent proteins (AFPs) have been cloned and subsequently modified by protein engineering to improve their physicochemical properties. Throughout this twenty-two-year period, glycine 67 (Gly67) has been regarded as the only amino acid in the entire protein family that is essential for the formation of the different reported chromophores. In this work, we demonstrate that a synthetic gene encoding LanFP10-A, a natural protein encoded in the genome of the lancelet Branchiostoma floridae containing the G67A mutation, produces a heterologous, functional yellow fluorescent protein when expressed in E. coli. In contrast to LanFP10-A, LanFP6-A, a second GFP-like protein found in the lancelet genome that also contains the natural G67A mutation, was non-fluorescent.

Elimination of redundant and stop codons during the chemical synthesis of degenerate oligonucleotides. Combinatorial testing on the chromophore region of the red fluorescent protein mKate.

Although some strategies have been reported for the elimination of stop and redundant codons during the chemical synthesis of degenerate oligonucleotides, incorporating an expensive cocktail of 20 trimer-phosphoramidites is currently a commonly employed and straightforward approach. As an alternative option, we describe here a cheaper strategy based on standard monomer-phosphoramidites and a simplified resin-splitting procedure. The accurate division of the resin, containing the growing oligonucleotide, into four columns represents the key step in this approach. The synthesis of the degenerate codon NDT in column 1, loaded with 60% of the resin, produces 12 codons, while a degenerate codon VMA in column 2, loaded with 30% of the resin, produces 6 codons. Codons ATG and TGG, independently synthesized in columns 3 and 4, respectively, and loaded with 5% each, completes the 20 different codons. The experimental frequency of each mutant codon in the library was assessed by randomizing 12 contiguous codons that encode for amino acids located in the chromophore region of the enhanced red fluorescent protein mKate-S158A. Furthermore, randomization of three contiguous codons that encode for the amino acids Phe62, Met63, and Tyr64, which are equivalent to Phe64, Ser65, and Tyr66 in GFP, gave rise to some red and golden yellow fluorescent mutants displaying interesting phenotypes and spectroscopic properties. The absorption and emission spectra of two of these mutants also suggested that the complete maturation of the red and golden yellow chromophores in mKate proceeds via the formation of a green-type chromophore and a cyan-type chromophore, respectively.