Two-dimensional Gel System Research Articles

Form follows function in geminiviral minichromosome architecture

A comprehensive survey on the viral minichromosomes of the begomoviruses Abutilon mosaic virus, tomato yellow leaf curl Sardinia virus, African cassava mosaic virus, Indian cassava mosaic virus (family Geminiviridae) during the course of infections in Nicotiana benthamiana is summarized. Using optimized one-dimensional and two-dimensional gel systems combined with blot hybridization and a standardized evaluation, discrete and heterogeneous virus-specific signals with different DNA forms were compared to trace functions of viral multiplication with inactive/active replication and/or transcription. A quantitative approach to compare the distantly related viruses during the course of infection with the aim to generalize the conclusions for geminiviruses has been developed. Focussing on the distribution of topoisomers of viral supercoiled DNA, which reflect minichromosomal stages, predominant minichromosomes with 12 nucleosomes, less with 13 nucleosomes and no with 11 nucleosomes were found. These results indicate that chromatin with only one open gap to bind transcription factors is the favourite form. The dynamics during infections in dependence on the experimental conditions is discussed with reference to the design of experiments for resistance breeding and molecular analyses.

Proteomic heterogeneity of nonfunctional pituitary adenomas

Nonfunctional pituitary adenoma (NFPA) is highly heterogeneous with different subtypes, which is the pathophysiological basis of its proteome heterogeneity. Two-dimensional gel electrophoresis-based comparative proteomics and systems biology-based pathway network analyses revealed the variations in proteomes and pathway networks among four NFPA subtypes. A set of differentially expressed proteins and pathway network data were common and specific to each NFPA subtype. Four common pathway systems were determined including MAPK-signaling abnormality, oxidative stress, mitochondrial dysfunction, and cell-cycle dysregulation. However, those molecular network systems are actually different among four NFPA subtypes including different protein-expression levels of most of nodes, different protein profiles, and different pathway network profiles. Those DEP profiles, DEP functional characteristics, and variations in signaling pathway systems provide the proteomic heterogeneity profile of human NFPAs. It is the useful data for discovery of effective biomarkers and targets to achieve personalized and precision medicine practice for an NFPA.

Identification of proteins expressed differently among surgically resected stage I lung adenocarcinomas

RationaleAmong patients with surgically resected stage I lung adenocarcinoma, some succumb to early recurrence, while others survive for more than 5years. Few markers to predict prognoses in these patients have been accepted. Recent advances in proteomic methodologies offer a unique chance to identify new candidate biomarkers. The aim of this study is to find differences in protein expression in resected lung cancer tissue of stage I adenocarcinoma from patients with no recurrence for more than 5years and from those with early recurrence. MethodsLung cancer tissues were obtained from 15 patients with pathologically confirmed stage I adenocarcinoma. The patients were divided into two groups, those with recurrence within 36months (early recurrence group, n=9) and those that were disease-free for over 5years (disease free group, n=6). Tissue proteins were separated by a two-dimensional electrophoresis long gel system (30×40cm) with set ranges (3-10NL) and examined by nano-LC-ESI-MS/MS. Western blot assays were performed to validate these proteins. ResultsTwelve protein spots were up-regulated and 8 were down-regulated in the disease-free group as compared with the recurrence group. Of the 12 up-regulated proteins, haptoglubin, tau-tubulin kinase-2 (TTBK2), thymidine phosphorylase, annexin-1, PIN1, CAPG, and SEC23 were validated by Western blot. Among the 8 down-regulated proteins, serpinB6 and trangelin-2 were validated. ConclusionsA total of 9 differentially expressed proteins were successfully extracted, identified, and confirmed from stage I lung adenocarcinoma tissues. The increased or decreased expression of these proteins according to prognosis may be the basis for further studies of proteomics in developing prognostic biomarkers.

Identification of nuclear structural protein alterations associated with seminomas

Currently, there are no specific markers available for the early detection and for monitoring testicular cancer. Based upon an approach that targets nuclear structure, we have identified a set of proteins that are specific for seminomas, which may then have clinical utility for the disease. Utilizing samples obtained from men with no evidence of testicular cancer (n = 5) as well as those with seminomas (n = 6), nuclear matrix proteins were extracted and separated using a high-resolution two-dimensional electrophoresis gel system. The proteins were identified by mass spectrometry analysis. These analyses revealed seven nuclear matrix proteins associated with the normal testes, which did not appear in the seminomas. In the seminomas, four nuclear matrix proteins were identified to be associated with the disease that were absent in the normal testes. Mass spectrometric and immunoblot analyses of these proteins revealed that one of the proteins identified in the normal testes appears to be StAR-related lipid transfer protein 7 (StARD7). In the non-seminoma tissues, one of the identified proteins appears to be cell division protein kinase 10 (CDK10). Both StarD7 and CDK10 could potentially be involved in cell differentiation and growth, and thus may serve as potential targets for therapy of prognostication of seminomas. This is the first study to examine the role of nuclear structural proteins as potential biomarkers in testicular cancer. We are currently examining the roles of some of the identified proteins as potential biomarkers for the disease.

Identification of Proteinaceous Inhibitors of a Cysteine Proteinase (an Arg-Specific Gingipain) from Porphyromonas gingivalis in Rice Grain, Using Targeted-Proteomics Approaches

Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors that this bacterium produces, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major determinants of the pathogenicity of P. gingivalis. Here, we report on our finding that there are inhibitors of these cysteine proteinases in a rice protein fraction. Comprehensive affinity chromatography and MS analyses resulted in the identification of 17 Arg-gingipain (Rgp)-interacting proteins in the rice endosperm. Of these, four proteins (i.e., a 26 kDa globulin, a plant lipid transfer/trypsin-alpha amylase inhibitor, the RA17 seed allergen, and an alpha amylase/trypsin inhibitor) were estimated to account for 90% of the Rgp inhibitory activity in the rice protein fraction, using a two-dimensional gel system of double-layer reverse zymography. In addition, a synthetic peptide derived from an Rgp-interacting protein, cyanate hydratase, could inhibit the growth of P. gingivalis and showed inhibitory activity against both the Arg- and Lys-gingipains. These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases.

Purification and characterization of two novel cysteine protease inhibitors, Eel-CPI-2 and Eel-CPI-3, in the skin mucus of the Japanese eel Anguilla japonica

We have discovered multiple acidic cysteine protease inhibitors, in addition to the known Eel-CPI-1, in the skin mucus extract of the Japanese eel Anguilla japonica by using the two-dimensional gel system of gelatin reverse zymography. Two of the acidic inhibitors, which we have named Eel-CPI-2 and Eel-CPI-3, were purified to homogeneity by anion exchange chromatography on a column of DEAE-Sepharose CL-6B, followed by fast protein liquid chromatography on Superdex 75 10/300 GL and HiTrap Q HP columns. The amino acid compositions of Eel-CPI-2 and Eel-CPI-3 were found to be almost identical and closely similar to that of the eel galectin AJL1. The molecular masses of Eel-CPI-2 and Eel-CPI-3 were elucidated to be 16,089.080 and 16,089.137 Da, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The apparent dissociation constant of Eel-CPI-2 and Eel-CPI-3 for cysteine protease papain was determined to be 1.79 × 10−7 and 1.05 × 10−7 M, respectively, by a quartz crystal microbalance technique.

Formation of calcium oxalate concentric precipitate rings in two-dimensional agar gel systems containing Ca 2+–RE 3+ (RE = Er, Gd and La)–C 2O 42−

The concentric precipitate rings are found in urinary stone section and may relate to Liesegang pattern and formation mechanism of urinary stone. Urinary stone is mainly composed of calcium oxalate, so it is significant to study the concentric precipitate rings. The influence of RE 3+ on the formation of calcium oxalate concentric precipitate rings is studied for the first time in two-dimensional agar gel systems. Precipitate rings are characterized by means of Fourier transform infrared spectrometry (FT-IR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and biomicroscope with digital camera. The results show concentric precipitate rings are made of calcium oxalate and RE 3+ promote the present of rings. The number of concentric rings are influenced by different RE 3+ and the reaction time. The agar gel systems are helpful for the ions to diffuse uniformly, and as a medium where calcium oxalate rings are formed. In comparison with Gd 3+ and La 3+ ions, the content and size of calcium oxalate monohydrate (COM) crystals are highest and biggest after the addition of Er 3+ ions, indicating that Er 3+ ions promote the formation of concentric rings effectively. However, in the similar systems containing Cu 2+, Fe 2+, Fe 3+and Co 2+, we found the ring only appeared in the system containing Fe 3+ ions. And the formation mechanism of the calcium oxalate concentric precipitate rings in two-dimensional agar gel systems containing rare earth ions is also discussed in the paper.

Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37°C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin β chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.

Clonal proteomics: one gene - family of proteins.

The work presented here attempts to consolidate our knowledge on cellular transcriptome and proteome. It takes into account that a typical activated cell (lymphocyte) contains 40 000 mRNA molecules at any time, and it represents about 5000 different molecular species of transcripts. Such a cell has about 1 000 000 000 protein molecules, some of them being present at 10 000 000 copies while others at a very low copy number (say 1 to 10 copies per cell). By studying cell free expression of individual cDNA clones (or pools of known complexity) we address to those rare molecular components that will remain undetected by the current analytical means. For our analysis we use cell free translation systems (wheat germ or rabbit reticulocyte origin) and we study polypeptide products originating from intact, or restriction endonuclease-treated cDNA clones. We conclude that in most instances expressed genes yield transcript(s) that translate into several, and often very numerous families of polypeptide species. In our ISODALT two-dimensional gel system we characterize the proteomic profile of the clonal polypeptide families in terms of their molecular mass, charge, multiple products, and appearance.

Back to the future: the human protein index (HPI) and the agenda for post-proteomic biology.

The effort to produce an index of all human proteins (the human protein index, or HPI) began twenty years ago, before the initiation of the human genome program. Because DNA sequencing technology is inherently simpler and more scalable than protein analytical technology, and because the finiteness of genomes invited a spirit of rapid conquest, the notion of genome sequencing has displaced that of protein databases in the minds of most molecular biologists for the last decade. However, now that the human genome sequence is nearing completion, a major realignment is under way that brings proteins back to the center of biological thinking. Using an influx of new and improved protein technologies--from mass spectrometry to re-engineered two-dimensional (2-D) gel systems, the original objectives of the HPI have been expanded and the time frame for its execution radically shortened. Several additional large scale technology efforts flowing from the HPI are also described.

Back to the future: The human protein index (HPI) and the agenda for post-proteomic biology

The effort to produce an index of all human proteins (the human protein index, or HPI) began twenty years ago, before the initiation of the human genome program. Because DNA sequencing technology is inherently simpler and more scalable than protein analytical technology, and because the finiteness of genomes invited a spirit of rapid conquest, the notion of genome sequencing has displaced that of protein databases in the minds of most molecular biologists for the last decade. However, now that the human genome sequence is nearing completion, a major realignment is under way that brings proteins back to the center of biological thinking. Using an influx of new and improved protein technologies – from mass spectrometry to re-engineered two-dimensional (2-D) gel systems, the original objectives of the HPI have been expanded and the time frame for its execution radically shortened. Several additional large scale technology efforts flowing from the HPI are also described.

Phosphorylation of human p53 on Thr-55.

The pleiotropic function of p53 is believed to be greatly influenced by phosphorylation, and several sites on p53 are known to be targets for distinct protein kinases. In this study, we observed that affinity-purified p53 from overexpressing cells was phosphorylated by a co-purified protein kinase in vitro. To identify phosphorylation site(s), the resulting phosphorylated p53 protein was subjected to primary and secondary proteolytic cleavage, and phosphopeptides were fractionated by a two-dimensional peptide gel system. Phosphoamino acid analysis and manual Edman degradation of the isolated phosphopeptides enabled us to unequivocally identify Thr-55 as the major phosphorylation site on p53. Furthermore, comparative phosphopeptide mapping data suggest that DNA-PK is not the kinase responsible for this phosphorylation. Significantly, using a phospho-specific antibody for Thr-55, we have shown that Thr-55 is indeed phosphorylated in vivo. These data define Thr-55 as a novel phosphorylation site and for the first time show threonine phosphorylation of human p53.

Complementation of the Saccharomyces cerevisiaePlasma Membrane H+-ATPase by a Plant H+-ATPase Generates a Highly Abundant Fusicoccin Binding Site

Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs. The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs. Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding.

Characterization of excised DNA intermediates associated with V(D)J recombination at the T-cell receptor delta locus.

Lymphocyte development requires the assembly of antigen receptor genes through the specialized process of V(D)J recombination. This process is initiated by cleavage at the junction between coding segments (V, D, and J) and the recombination signal sequences that border these segments, resulting in generation of double-strand break intermediates. We have used a two-dimensional gel system to characterize broken molecules arising from V(D)J recombination at the T-cell receptor (TCR) delta locus and have identified linear species excised by Ddelta1-Ddelta2 and V-Ddelta2 rearrangement in thymus DNA. Relatively few (approximately 10) V-Ddelta2-excised linear species were detected in DNA from fetal thymocytes. The sizes of these species corresponded to the estimated distances between Ddelta2 and the V gene segments utilized by gammadelta T cells and indicated that both Ddelta2-proximal and -distal V gene segments are targeted for V-Ddelta2 rearrangement. Similar-sized species were observed in DNA from thymocytes of scid mice in which T-cell development is arrested prior to TCR expression. Since previous studies suggest that the TCR alpha/delta locus encodes more than 100 V gene segments, our results indicate that a few select V gene segments are predominantly targeted for rearrangement to Ddelta2, and this primarily accounts for the restricted Vdelta gene repertoire of gammadelta T cells.

Two-dimensional gel electrophoretic analysis of vectorially labeled surface proteins of human spermatozoa.

The objective of this study was to identify the repertoire of proteins exposed on the surface of ejaculated human spermatozoa. High-resolution two-dimensional gel systems for separation of human sperm and seminal plasma proteins were developed using both isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) followed by polyacrylamide gel electrophoresis (IEF/PAGE, NEPHGE/PAGE). Proteins were visualized by silver staining of gels and by electroblotting followed by gold staining. The protein patterns were analyzed by computer after laser or camera scanning. One thousand three hundred ninety-seven sperm proteins with a molecular mass between 5 and 160 kDa and isoelectric points (pI) from 4 to 11 were catalogued from silver-stained gels loaded with approximately 0.25 mg of NP-40/urea extracts of sperm harvested by Percoll density gradient centrifugation, and 1191 proteins were resolved following extraction with SDS/3-[3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate/urea. Analysis of seminal plasma proteins obtained from vasectomized patients revealed over 300 silver-stained proteins, which aided the identification of sperm-coating proteins acquired from secretions of the accessory sex organs. Sperm surface proteins accessible to vectorial labeling with 125I or N-hydroxysuccinimide biotin were identified; 181 protein spots were radiolabeled with 125I, while 228 protein spots were biotinylated, including several groups of protein isoforms. Cytoskeletal and intra-acrosomal control proteins were not iodinated or biotinylated, thus verifying the surface specificity of both labeling methods. Ninety-eight sperm surface proteins were labeled by both iodine and biotin, and 22 sperm surface proteins, representing five groups of protein isoforms, were shown to contain phosphotyrosine. A composite computer image showing the position of the dually vectorially labeled sperm surface proteins was constructed, together with a table of the proteins' molecular weight, pI, and relative concentration. In addition, novel isoforms of actin, beta-tubulin, PH-20, and several phosphotyrosine-containing proteins were identified in human sperm.

Inactivation of topoisomerase I or II may lead to recombination or to aberrant replication termination on both SV40 and yeast 2 micron DNA.

Topoisomerase I is believed to be sufficient for early replication of circular viral genomes such as those of SV40 and of yeast plasmids. Topoisomerase II is required for the decatenation of the daughter genomes and probably also for fork elongation during the later stages of SV40 replication. Using the neutral-neutral two-dimensional gel system, we have followed the progression of replication of both SV40 and the yeast 2 micron plasmid under various conditions of topoisomerase inhibition. During SV40 replication, inhibition of topoisomerase II by VP16, VM26 or hypertonic shock (but not by merbarone), and inhibition of topoisomerase I by camptothecin all led to the accumulation of aberrant DNA structures containing two almost completely replicated genomes. These aberrant structures resembled either recombination intermediates or late Cairns structures in which the site of replication termination had shifted and now mapped to a continuum of sites throughout the genome. Replication of the 2 micron plasmid in a topoisomerase II- but not a topoisomerase I-deficient yeast gave rise to very similar structures. The data suggest that inactivation of topoisomerase I or II either stimulates recombination or, by differentially affecting replication fork progression, leads to aberrant replication termination.

Sequence and characterization of the bacteriophage T4 comC alpha gene product, a possible transcription antitermination factor.

We have sequenced a 1,340-bp region of the bacteriophage T4 DNA spanning the comC alpha gene, a gene which has been implicated in transcription antitermination. We show that comC alpha, identified unambiguously by sequencing several missense and nonsense mutations within the gene, codes for an acidic polypeptide of 141 residues, with a predicted molecular weight of 16,680. We have identified its product on one- and two-dimensional gel systems and found that it migrates abnormally as a protein with a molecular weight of 22,000. One of the missense mutations (comC alpha 803) is a glycine-to-arginine change, and the resulting protein exhibits a substantially faster electrophoretic mobility. The ComC alpha protein appears immediately after infection. Its rate of synthesis is maximum around 2 to 3 min postinfection (at 37 degrees C) and then starts to decrease slowly. Some residual biosynthesis is still detectable during the late period of phage development.

Nuclear matrins: identification of the major nuclear matrix proteins.

A preparative two-dimensional polyacrylamide gel system was used to separate and purify the major Coomassie blue-stained proteins from the isolated rat liver nuclear matrix. Approximately 12 major proteins were consistently found. Of these, 5 proteins represented identified proteins, including nuclear lamins A, B, and C, the nucleolar protein B-23, and residual components of core heterogeneous nuclear ribonucleoproteins. The remaining eight major proteins termed the nuclear matrins consisted of matrin 3 (125 kDa, slightly acidic), matrin 4 (105 kDa, basic), matrins D-G (60-75 kDa, basic), and matrins 12 and 13 (42-48 kDa, acidic). Peptide mapping and two-dimensional immunoblot studies indicate that matrins D-G compose two pairs of related proteins (matrins D/E and F/G) and that none of the matrins resemble the nuclear lamins or any of the other major proteins detected on our two-dimensional gels. Subfractionation immunoblot experiments demonstrated the nearly exclusive localization of matrins F/G and other matrins to the nuclear matrix fraction of the cell. These results were further supported by indirect immunofluorescence microscopy that showed a strictly interior nuclear localization of the matrins in intact cells in contrast to the peripherally located nuclear lamins. We conclude that the nuclear matrins are a major class of proteins of the nuclear matrix interior and are distinct from the nuclear lamins.

Frog cardiac calsequestrin. Identification, characterization, and subcellular distribution in two structurally distinct regions of peripheral sarcoplasmic reticulum in frog ventricular myocardium.

Calsequestrin is a calcium-binding protein known to sequester calcium accumulated in the sarcoplasmic reticulum (SR) of muscle cells during relaxation. In the present study, we used affinity-purified antibodies to chicken cardiac calsequestrin to identify a 60,000-Da calsequestrin in frog myocardium. Like previously identified cardiac calsequestrins, it is enriched in cardiac microsomes, it is enriched by biochemical procedures previously used to purify cardiac and skeletal calsequestrins, and it exhibits a pH-dependent shift in its apparent Mr on a two-dimensional gel system. Finally, the NH2-terminal amino acid sequence of this 60,000-Da immunoreactive protein purified by fast protein liquid chromatography was identical to that of rabbit skeletal and canine cardiac calsequestrin. Thus, we conclude that this protein corresponds to the calsequestrin isoform in frog ventricular muscle. Frog calsequestrin was localized in discrete foci present at the periphery but absent from the central regions of frog ventricular myocytes as determined by immunofluorescence labeling. Immunoelectron microscopic labeling demonstrated that calsequestrin was confined to the lumen of two structurally distinct regions of the SR, where it was localized in the subsarcolemmal region of the myofibers. One of these appeared to correspond to the terminal SR previously reported to be closely apposed to the sarcolemma of frog myofibers. The other region, although close to the sarcolemma, was not physically joined to it and appeared to correspond to corbular SR. It generally is believed that frog cardiac SR does not provide activator Ca2+ required for excitation-contraction coupling. However, the identification of a calsequestrin isoform very similar to mammalian cardiac calsequestrin that is confined to specialized regions of frog cardiac SR lends support to the idea that frog cardiac SR has the ability to store Ca2+ and thus function in some capacity in frog cardiac muscle contraction.

Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli

Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5′ end of the genes, and two ochre stop codons have been created at their 3′ ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector eaturing a tac promoter, and the pT7-5 vector featuring a T7-promoter. The overexpressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used.