High level expression of human basic fibroblast growth factor in Escherichia coli: Evaluating the effect of the GC content and rare codons within the first 13 codons

High-level expression of recombinant human basic fibroblast growth factor in Escherichia coli presents research opportunities such as analysis of hbFGF expression after translation initiation region (TIR) mutagenesis. In our study, hbfgf-cDNA was expressed in three stains of E. coli comprising OrigamiB (DE3), BL21 (DE3) and modified strain carrying copies for rare codon tRNAs (BL21 (DE3)-codonplus- RP). During the course of these experiments, we investigated the role of rare codon replacement and ofGC content reduction in N-terminal, just downstream of the ATG start codon. As standardized procedure, two forward primers were designed for modification of N-terminal of hbfgf-cDNA. Nterminally modified genes were PCR amplified and cloned into the expression vector, pET-22b.Meanwhile, wild-type gene remarkably expressed in all the strains especially in codon plus strain, rare codon substituted hbFGF gene construct surprisingly displayed undetectable levels of protein production; modified gene construct with reduction in GC content of the first 13 codons contributes to 2.5 folds increased expression level. In addition, recombinant hbFGF were purified and the biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of NIH/3T3 cells. Purified rhbFGF exhibited proliferative activity comparable to commercial rhbFGF

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/ Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis

Application of PCR-RFLP to Rapid Identification of the Main Pathogenic Dermatophytes from Clinical Specimens

"nBackground: In the present study, a PCR-RFLP based molecular technique was designed to rapid identification of der­matophytes in clinical specimens. Skin scrapings obtained from human cases suspected to dermatophytosis were studied in or­der to identify involved etiological fungi."nMethods: In this experimental study, the specimens (skin scrapings) of patients referred to Mycology Department of Pas­teur Institute of Iran were inoculated on Petri dishes contained selective agar for pathogenic fungi (SAPF) and incubated at 25º C until visible growth of fungal colonies. The colonies were examined for standard morphological characteristics after visi­ble growth on the agar medium. A small portion of each fungal colony was further studied by restriction fragment length poly­morphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). PCR amplicons were electrophoresed on 2% agarose gel after digesting by different restriction enzymes including MvaI, HinfI and HaeIII."nResults: Among 160 clinical samples examined, 6 dermatophyte species including  Trichophyton mentagrophytes, T. ru­brum, T. verrucosum, T. tonsurans, Microsporum canis and Epidermophyton floccosum were finally identified based on the col­ony morphology and microscopic criteria. Specific PCR products and RFLP patterns for MvaI, HinfI and HaeIII en­zymes allowed the rapid identification and reliable differentiation of isolated dermatophytes at the genus or species level for 5-10 day-old colonies."nConclusions: The results showed that PCR-RFLP analysis of the ITS region of rDNA is a rapid and reliable tool which al­lows identification of major pathogenic dermatophytes isolated in this study at species level in young 5-10 day-old colonies. &nbsp

Chemoselective PEGylation of cysteine analogs of human basic fibroblast growth factor (hbFGF) - design and expression

Purpose: To improve the stability and bioactivity of human basic fibroblast growth factor (hbFGF) by site-specific pegylationMethods: Four new mutants of hbFGF were designed with substituted Asp68, Lys77, Glu78 and Arg81 with cysteine with the aid of bioinformatics technique, and then cloned into pET21a plasmid, transferred into E. coli BL21 (DE3). The expressed proteins were purified using cation exchange and heparin affinity chromatography. Cysteine analogs of hbFGF were PEGylated with 10 KDa PEG and purified using size exclusion chromatography. Mitogenic activity and resistance against denaturation agents were evaluated by MTT assay and fluorescence spectrophotometry, respectively, and the results obtained were compared with the non-PEGylated form.Results: Despite greater resistance against denaturation agent (1.2 M guanidine hydrochloride for denaturation of PEGylated mutants compared with 0.8 M for non-PEGylated forms), the mitogenic activities of the four mutants Asp68, Lys77, Glu78 and Arg81were retained at 79, 78.6, 83.3 and 75.6 %, respectively.Conclusion: PEGylated hbFGF shows decreased mitogenic activity and increased resistance against denaturation agent. Keywords: Bioinformatics, Fibroblast growth factor, Cysteine analog, PEGylation, Denaturation agent, Guanidine hydrochloride, Mitogenic activit