Expression of codon optimized genes in microbial systems: current industrial applications and perspectives.
Front Microbiol. 2014;5:21
Authors: Elena C, Ravasi P, Castelli ME, Peirú S, Menzella HG
The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent “silent” nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.
PMID: 24550894 [PubMed - as supplied by publisher]
Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.
MAbs. 2013 Dec 11;6(2)
Authors: Valdés-Alemán J, Téllez-Sosa J, Ovilla-Muñoz M, Godoy-Lozano E, Velázquez-Ramírez D, Valdovinos-Torres H, Gómez-Barreto RE, Martinez-Barnetche J
High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.
PMID: 24492293 [PubMed - as supplied by publisher]
A 6′-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF—HC Pseudohydrogen Bonds.
J Org Chem. 2014 Jan 14;
Authors: Dugovic B, Leumann CJ
The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6′ (6′F-bc-T) has been achieved. Key steps of the synthesis were an electrophilic fluorination / stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an N-iodo-succinimide induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. Tm measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in Tm were in the range of +1-2 degC compared to thymidine and +1-3degC compared to a standard bc-T residue. Structural investigations of the 6′F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a non-classical CF-HC hydrogen bond. In contrast, structural investigations in solution by 1H- and 13C-NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. Based on these results we put forward the hypothesis that these weak CF-HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle chi of the 6′F-bcT nucleoside.
PMID: 24422513 [PubMed - as supplied by publisher]
[Progress in gene synthesis technology].
Sheng Wu Gong Cheng Xue Bao. 2013 Aug;29(8):1075-85
Authors: Feng M, Wang L, Tian J
Gene synthesis is the most fundamental and widely used tec…
[Preface for special issue on synthetic biology (2013)].
Sheng Wu Gong Cheng Xue Bao. 2013 Aug;29(8):1041-3
Authors: Chen G
Synthetic biology has developed quickly worldwide. In this …
Synthesis of nucleotide-amino acid conjugates designed for photo-CIDNP experiments by a phosphotriester approach.
Beilstein J Org Chem. 2013;9:2898-909
Authors: Abramova TV, Morozova OB, Silnikov VN, Yurkovsk…
A novel inducible expression system for the functional study of toxic gene in bacteria.
World J Microbiol Biotechnol. 2013 Dec 11;
Authors: Guo J, Jia R
The cloning and expression of toxic proteins in bacteria have posed a great challenge because of the leaky expression in inducible expression systems. Using artificial gene synthesis and clone screening methods, we identified a mutant T5 promoter, which significantly reduced leaky expression of lac operator. The mutant T5 promoter contains two T deletions at -35 region and may reduce promoter activity. A bacterial lethal gene, Φ174 lytic gene E, was successfully cloned in this system and expressed in the presence of isopropyl β-D-1-thiogalactopyranoside. The system is compatible with existing T5 inducible expression systems and can be used for the controlled expression of toxic proteins.
PMID: 24326910 [PubMed - as supplied by publisher]
COStar: A D-star Lite-based Dynamic Search Algorithm for Codon Optimization.
J Theor Biol. 2013 Dec 4;
Authors: Liu X, Deng R, Wang J, Wang X
Codon optimized genes have two major adva…
Immunological characterization of a Modified Vaccinia Virus Ankara vector expressing the Human Papilloma Virus 16 E1 protein.
Clin Vaccine Immunol. 2013 Dec 4;
Authors: Remy-Ziller C, Germain C, Spindler A, Hoffmann C, Silvestre N, Rooke R, Bonnefoy JY, Préville X
Women showing a normal cytology but diagnosed with a persistent high-risk Human Papilloma Virus (HR-HPV) infection have a higher risk than non-infected women to develop high grade cervical intraepithelial neoplasia and cervical cancer. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in HPV life cycle, represent very interesting candidates. Both proteins are necessary to maintain coordinated viral replication and gene synthesis during the differentiation process of the epithelium, and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we performed evaluation of a new active targeted immunotherapeutic, a Modified Vaccinia Virus Ankara (MVA) vector containing the E1 sequence of HPV16, aiming at inducing cellular immune responses with potential to help and clear persistent HPV-16 related infection. We carried out an extensive comparative time course analysis of the cellular immune response induced by different schedule of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16E1-specific CD8(+) T cells, but led to the differentiation of multi-functional effector T cells with high cytotoxic capacity. This study provides proof of concept that a MVA expressing HPV16E1 can induce robust and long lasting E1-specific responses and warrants further development of this candidate.
PMID: 24307238 [PubMed - as supplied by publisher]