Expression Of Heterologous Gene Products Research Articles

Isolation ofGPDPromoter fromTremella fuciformisand Driving Expression ofEGFPGene

Glyceraldehyde-3-phosphate dehydrogenase (GPD) cDNA was cloned by RT-PCR using total RNA from Tremella fuciformis as template with a pair of degenerate primers. Then, a 500-bp 5'-upstream promoter region of the gene encoding GPD from T. fuciformis genomic DNA was isolated by thermal asymmetric interlaced PCR. The cloned promoter was fused to 5'-upstream of enhanced green fluorescent protein gene to construct T. fuciformis expression vector pCB-TEGFP with hygromycin gene as a selectable marker. Electroporation was performed to transfer plasmid DNA of pCB-TEGFP into yeast-like conidia from T. fuciformis. Molecular evidence, including PCR analysis, fluorescence detection, fluorescence spectra assay, and SDS-PAGE, indicated that the EGFP gene had been integrated into the genome of transgenic T. fuciformis strains and was expressed successfully. The results also showed that this promoter could be used to carry out regulated expression of heterologous gene products in T. fuciformis.

Heterologous expression of cyan and yellow fluorescent proteins from the Kluyveromyces lactis KlMAL21–KlMAL22 bi-directional promoter

We have identified the Kluyveromyces lactis maltase (KlMAL22) and maltose permease (KlMAL21) intergenic region as a candidate bi-directional promoter for heterologous gene expression. The expressions of cyan and yellow fluorescent proteins from, respectively, the KlMAL22 and KlMAL21 orientations of the promoter, were compared between two promoter variants during growth in media containing glucose, galactose or glycerol. Expression from both orientations of the native promoter was repressed during growth in glucose and galactose and was induced during growth in glycerol. Disruption of a putative Mig1p binding site caused some de-repression of the maltase orientation of the promoter by 48 h of growth in glucose. The KlMAL21-KlMAL22 bi-directional promoter can be used to carry out regulated expression of heterologous gene products.

Optimization of transfection conditions for expression of green fluorescent protein in Drosophila melanogaster S2 cells

Drosophila melanogaster S2 cells provide attractive features for efficient expression of heterologous gene products. We report the optimization of transfection conditions for transient expression of green fluorescent protein (GFP) in Drosophila S2 cells by lipofectin and electroporation. GFP expression in lipofectin transfection was optimum at a transfection time of 24 h, a 5:1 ratio of lipofectin to DNA, and a seeding density of 4 × 10 5 cells/cm 2. On the other hand, GFP expression from electroporation transfection was optimum at 1125 V/cm, 250 μF, DNA content of 20 μg, and a seeding density of 1.0 × 10 7 cells/cuvette. Use of the optimum lipofectin method for transient expression resulted in an approximately 3-fold more efficient transfection than the electroporation method. We also investigated the stable transformation of Drosophila S2 cells by using the lipofectin method. Stably transformed polyclonal cell populations expressing GFP were isolated after 4 weeks of selection by using hygromycin B. The optimum ratio of plasmid pAc5CPPA-GFP to pCoHygro for the cotransfection of cells was 49 : 1 for GFP expression. Recombinant GFP expressed in stably transformed S2 cells was found primarily in the intracellular fraction with a molecular weight of 28 000. The maximum GFP concentration in the S2 cells was approximately 9 mg/l. We demonstrated the expression of GFP in Drosophila S2 cells and its use as a reporter for the analysis of gene expression.

31] Baculovirus-mediated expression of human multidrug resistance cDNA in insect cells and functional analysis of recombinant P-glycoprotein

This chapter discusses the baculovirus-mediated expression of human multidrug resistance ( MDR) cDNA in insect cells. High-level expression of recombinant P-glycoprotein is described with varying degrees of success in several heterologous expression systems, including bacteria, yeast, and insect cells. Heterologous expression of MDR cDNAs in insect cells by recombinant baculovirus expression vectors is quite successful and has contributed significantly to the recent progress in the study of the structure–function relationship of the human MDR1 P-glycoprotein, its substrate interactions, and the role of its posttranslational modifications. The baculovirus expression system is safe because of the high species specificity of baculoviruses that infect insect cells exclusively. Baculoviruses have the capacity to package and express relatively large foreign genes. The wide use of the baculovirus expression system has lead to the development and commercial availability of novel technologies and experimental kits that allow for reliable and rapid expression of heterologous gene products as full-length proteins and as fusion proteins.

Analysis of heterologous gene expression in Xenopus blastomeres.

Like Xenopus oocytes, Xenopus cleavage blastomeres are ideal synthetic factories for the expression of heterologous gene products. For 6 h after fertilization, the embryos are transcriptionally quiescent (), but mRNA is recruited for translation at a rate greater than in the oocyte, and the protein products of exogenous mRNA and DNA, introduced by microinjection, are synthesized as efficiently as those of endogenous RNA (). This is an excellent system in which to examine the developmental function of heterologous genes, because one can express genes during developmentally inappropriate stages and in developmentally inappropriate regions. For example, to test whether a gene is involved in dorsal axis formation, researchers express the gene in a blastomere that produces ventral tissues and assay whether the blastomere’s fate changes to a dorsal one (–(). Spatial misexpression is possible in Xenopus because.

Yeast systems for the expression of heterologous gene products

Significant advances have been made over the past year in our understanding of some of the critical parameters affecting high-level production of heterologous proteins in yeast. Recent studies of plasmid stability, promoter strength and secretion efficiency are yielding potential improvements in expression.

Gene expression using Gram-negative bacteria

The technology for expressing heterologous gene products has developed to the point where it may be successfully applied to almost any Gram-negative bacterial system. Yet, when one thinks of gene expression in Gramnegative bacteria, Escherichia coli is the organism that typically first comes to mind. E. coli has been, and continues to be, the workhorse of the gene expression field. No other system has been developed that can not only express a large number of known gene products at levels sufficient for detailed biochemical analysis or product development, but also express undefined coding sequences (open reading frames) in amounts sufficient to determine the identity of the gene product and to characterize its function. Indeed, literally hundreds of papers are published each year in which it has been reported that genes of prokaryotic or eukaryotic origin have been cloned and expressed in E. coil The ability to express this myriad of genes has been aided by the development of numerous expression vector systems which use highly efficient transcriptional and translational regulatory signals to optimize production to levels that may reach 30% of total cellular protein (for a review of expression vector technology see [ 1] ). However, over the last year or two, the focus of improving expression in E, coli has shifted from the optimization of these regulatory signals to the manipulation of the coding sequence itself and the quality of the protein being produced. This review highlights advances made over the last two years in our ability to optimize the expression of heterologous gene products in E. coli with respect to both the quantity and quality (solubility, folding, activity, and homogeneity) of the proteins being produced. In addition, it will spotlight our emerging abilities to express heterologous gene products in other Gram-negative organisms where the aim is not so much to overproduce these proteins but rather to impart a useful new phenotype or function to these organisms.

New Perspectives on the Structure and Function of Ubiquitin

Ubiquitin has been suggested to play a key role in a wide variety of essential cellular functions ranging from differential regulation of gene expression to protein degradation. Recent studies on natural and synthetic ubiquitin gene fusions have led to important discoveries concerning novel functions for the ubiquitin system in cells, mechanisms of proteolytic processing, and the development of a ubiquitin fusion technology for augmenting the expression of heterologous gene products in bacteria and yeast. Furthermore, studies involving site-directed mutagenesis and two-dimensional NMR have proven ubiquitin to be an excellent model for protein engineering and have led to important discoveries concerning its mechanism of action in ATP-dependent proteolysis. Finally, the recent identification and characterization of ubiquitin carboxyl extension proteins as ribosomal proteins has opened up an even newer area of ubiquitin-related research and has helped to explain the mechanisms involved in increasing the expression of heterologous gene products made as ubiquitin fusions.

Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis

A 1.6 kb cDNA fragment encoding the mature part of the human tissue-type plasminogen activator (t-PA) was subcloned into a Bacillus subtilis dual plasmid expression system [Le Grice et al., Gene 55 (1987) 95–103]. Expression of the tPA gene in this vector was regulated by the inducible Escherichia coli lac elements, as well as a strong phage-T5-derived promoter and ribosome-binding site preceding the polylinker. The 5' end of the tPA gene corresponding to the N terminus of mature t-PA was fused in phase to the third codon present in the polylinker region of the expression vector, p602/22, to form p602-t-PA. B. subtilis containing p602-t-PA, when induced with isopropyl-β-D-thiogalactopyranoside, produced large amounts of immunoreactive t-PA (approx. 20 μg/ml). As expected, t-PA was not secreted into the culture media, but was localized in intracellular inclusion bodies and was found to be enzymatically inactive. However, enzymatic activity could be regained following complete reduction followed by slow oxidation of the solubilized inclusion bodies. The recombinant t-PA (rt-PA) showed, after purification, a smaller molecular size than melanoma t-PA, probably due to lack of glycosylation in the Bacillus system. Like melanoma t-PA, rt-PA exhibited tremendous stimulation of plasminogen activation in the presence of fibrin. Our results illustrate that B. subtilis, when supplied with the proper transcriptional/translational regulatory elements, can be an effective system for expression of heterologous gene products.