On predicting highy the y expressed genes for Escherichia coli based on mRNA microarray data

Highly expressed genes [HEG] are genees available in the organism, which carry the preferred codons for the expression system. Identifying HEG helps to find preferred codons and use them in the gene optimization to express target protein. Currently, HEG-DB are the only database to store HEG data of many strains of microorganisms, but the data are still not updated and maintained. Therefore, our research is carried out to predict HEG in the E. coli K-12 MG1655 strain based on reference sets that are the mostly used ribosomal protein coding genes and genes with high transcription levels from microarray data proposed by the research. Next, the results of HEG from the two above reference sets, HEG-RP and HEG-mRNA, were compared. Finally, we analyzed and compared the HEG that the project predicted with HEG from HEG-DB database. The results from RP and 100-mRNA reference sets were completely identical and were better than data from HEG-DB in the number of HEGs, CAI values and the number of genes contributing to important metabolic pathways. The results showed that it was possible to use reference sets from mRNA microarray data instead of ribosomal protein reference sets in HEG prediction

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

Cloning, high-level gene expression and bioinformatics analysis of SP15 and LeIF from Leishmania major and Iranian Phlebotomus papatasi saliva as single and novel fusion proteins: a potential vaccine candidate against leishmaniasis

Background Early exacerbation of cutaneous leishmaniasis is mainly affected by both the salivary and Leishmania parasite components. Little is known of the vaccine combination made by immunogenic proteins of sandfly saliva (SP15) with Leishmania parasites (LeIF) as a single prophylactic vaccine, namely SaLeish. Also, there are no data available to determine the species-specific sequence of SP15 isolated from the Iranian Phlebotomus papatasi. Methods Integrated bioinformatics and genetic engineering methods were employed to design, optimize and obtain a vector–parasite-based vaccine formulation in a whole-length fusion form of LeIF-SP15 against leishmaniasis. Holistic gene optimization was initially performed to obtain a high yield of pure ‘whole-SaLeish’ expression using bioinformatics analyses. Genomic and salivary gland RNAs of wild-caught P. papatasi were extracted and their complementary DNA was amplified and cloned into pJET vector. Results The new chimeric protein of whole-SaLeish and randomly selected transcripts of native PpIRSP15 (GenBank accession nos. MT025054 and MN938854, MN938855 and MN938856) were successfully expressed, purified and validated by immunoblotting assay. Furthermore, despite the single amino acid polymorphisms of PpIRSP15 found at positions Y23 and E73 within the population of wild Iranian sandflies, antigenicity and conservancy of PpIRSP15 epitopes remained constant to activate T cells. Conclusions The SaLeish vaccine strategy takes advantage of a plethora of vector–parasite immunogenic proteins with potential protective efficacy to stimulate both the innate and specific cellular immune responses against Leishmania parasites.

Brain cell somatic gene recombination and its phylogenetic foundations

A new form of somatic gene recombination (SGR) has been identified in the human brain that affects the Alzheimer's disease gene, amyloid precursor protein (APP). SGR occurs when a gene sequence is cut and recombined within a single cell's genomic DNA, generally independent of DNA replication and the cell cycle. The newly identified brain SGR produces genomic complementary DNAs (gencDNAs) lacking introns, which integrate into locations distinct from germline loci. This brief review will present an overview of likely related recombination mechanisms and genomic cDNA-like sequences that implicate evolutionary origins for brain SGR. Similarities and differences exist between brain SGR and VDJ recombination in the immune system, the first identified SGR form that now has a well-defined enzymatic machinery. Both require gene transcription, but brain SGR uses an RNA intermediate and reverse transcriptase (RT) activity, which are characteristics shared with endogenous retrotransposons. The identified gencDNAs have similarities to other cDNA-like sequences existing throughout phylogeny, including intron-less genes and inactive germline processed pseudogenes, with likely overlapping biosynthetic processes. gencDNAs arise somatically in an individual to produce multiple copies; can be functional; appear most frequently within postmitotic cells; have diverse sequences; change with age; and can change with disease state. Normally occurring brain SGR may represent a mechanism for gene optimization and long-term cellular memory, whereas its dysregulation could underlie multiple brain disorders and, potentially, other diseases like cancer. The involvement of RT activity implicates already Food and Drug Administration–approved RT inhibitors as possible near-term interventions for managing SGR-associated diseases and suggest next-generation therapeutics targeting SGR elements.

Expression Enhancement of Trastuzumab in CHO Cells Using Codon Optimization and Promoter Selection for Mammalian Expression Vector

Trastuzumab is recombinant humanized monoclonal IgG antibody used in the treatment of metastatic breast cancer. It blocks the overexpression of tyrosine kinase Her 2 family receptors and downregulation of pathways. Genetic optimizations like gene optimization and expression vector optimization are the key elements to enhance monoclonal antibody expression. Expression vector optimization includes major parameters like selection of right promoters, poly A tail selection of genome integrating elements. Codon optimization is gene designing approach without affecting the amino acid sequence of the protein. Current research work involves development of codon optimization algorithm for trastuzumab heavy and light chain gene and comparative analysis of expression in different vectors.

A selectable system to evaluate synthetic gene optimization features

Heterologous gene expression – transferring genes from a natural cell or origin to another host cell, often across species – is a fundamental technique in biological research as well as biotechnological and pharmaceutical manufacturing. Optimization of gene sequences, therefore, is a critical factor when enhanced protein yield is needed. For any given protein, an enormous diversity of nucleotide sequences comprising the vast combinatorics of possible codon combinations could theoretically be used to encode the same amino acid sequence. The process of “codon optimization” typically replaces certain codons thought to be suboptimal with a more optimal codon encoding the same amino acid. However, such methods cannot address the enormity of possible nucleotide sequences possible to encode a given protein, or to decipher potential factors in addition to codon usage that may affect gene expression. Here we utilize the Sh ble gene encoding zeocin resistance in a system comprising bioinformatic synthetic gene production and an antibiotic selection platform. We find that supposedly codon optimized genes do not produce enhanced antibiotic resistance, suggesting that other factors are more important in synthetic gene optimization and heterologous gene expression.

Reference Gene Optimization for Circadian Gene Expression Analysis in Human Adipose Tissue

A hallmark of biology is the cyclical nature of organismal physiology driven by networks of biological, including circadian, rhythms. Unsurprisingly, disruptions of the circadian rhythms through sleep curtailment or shift work have been connected through numerous studies to positive associations with obesity, insulin resistance, and diabetes. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) measures oscillation in messenger RNA expression, an essential foundation for the study of the physiological circadian regulatory network. Primarily, measured oscillations have involved the use of reference gene normalization. However, the validation and identification of suitable reference genes is a significant challenge across different biological systems. This study focuses on adipose tissue of premenopausal, otherwise healthy, morbidly obese women voluntarily enrolled after being scheduled for laparoscopic sleeve gastrectomy surgery. Acquisition of tissue was accomplished by aspiratory needle biopsies of subcutaneous adipose tissue 1 to 2 weeks prior to surgery and 12 to 13 weeks following surgery and an in-surgery scalpel-assisted excision of mesenteric adipose tissue. Each biopsy was sterile cultured ex vivo and serially collected every 4 h over approximately 36 h. The candidate reference genes that were tested were 18S rRNA, GAPDH, HPRT1, RPII, RPL13α, and YWHAZ. Three analytic tools were used to test suitability, and the candidate reference genes were used to measure oscillation in expression of a known circadian clock element (Dbp). No gene was deemed suitable as an individual reference gene control, which indicated that the optimal reference gene set was the geometrically averaged 3-gene panel composed of YWHAZ, RPL13α, and GAPDH. These methods can be employed to identify optimal reference genes in other systems.

Constructing a gene optimization program based on the ant colony optimization algorithm for Escherichia coli

In recombinant protein production, transferring a wild type gene of one organism into another expression host sometime resulted in a low gene expression due to incompatibility between the gene and the expression system. In that case, the target gene needed to be optimized to be more compatible with the expression system through gene optimization process in which nucleotide composition of original gene would be replaced by synonym codons while retaining the protein sequence. In existing gene optimization programs, many optimization algorithms have been applied, such as Genetic Algorithm or Sliding Window, to search for the optimized gene sequence. In this research, we applied the Ant Colony Optimization (ACO) algorithm to construct a gene optimization program. The results showed that the gene after optimization has been improved in codon usage, GC content and reduced the occurrence of factors reducing transcription and translation efficiencies such as polycodon, polynucleotide, repeated sequence, and Shine - Dalgarno sequence. Comparing with some current programs using a gene encoding for human insulin also proved the efficiency in the gene optimization this program. These results have demonstrated the capabilities of applying ACO algorithm in the gene optimization problem.