Roseovarius halocynthiae sp. nov., isolated from the sea squirt Halocynthia roretzi.

A Gram-negative, motile, ovoid- to rod-shaped bacterial strain, designated MA1-10T, was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea. Strain MA1-10T grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2 % (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain MA1-10T clustered with Roseovarius crassostreae CV919-312T, with which it exhibited 97.1 % sequence similarity, at a bootstrap resampling value of 96.2 %. It exhibited 93.3-95.8 % 16S rRNA gene sequence similarity to the type strains of other recognized Roseovarius species. Strain MA1-10T contained Q-10 as the predominant ubiquinone and C18:1ω7c as the major fatty acid, which is consistent with data for the genus Roseovarius. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid and an unidentified lipid. The DNA G+C content of strain MA1-10T was 55.4 mol%. Mean DNA-DNA relatedness between strain MA1-10T and R. crassostreae DSM 16950T was 13 %. Differential phenotypic properties, together with phylogenetic and genetic distinctiveness, demonstrated that strain MA1-10T could be distinguished from all recognized Roseovarius species. On the basis of the data presented, strain MA1-10T is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius halocynthiae sp. nov. is proposed; the type strain is MA1-10T (=KCTC 23462T=CCUG 60745T).

Molecular characterization and transcriptional analysis of the olive flounder (Paralichthys olivaceus) YGHL1 gene in response to hypoxia and infection.

The hypoxia-inducible gene 1, YGHL1 from olive flounder (Paralichthys olivaceus) (fYGHL1) was cloned, and its structural organization and expression profiles were determined. A 1,400 kb full-length cDNA encoding a predicted polypeptide of 91 amino acids was sequenced. The fYGHL1 gene comprises three introns, four exons, and several transcriptional elements upstream of the transcriptional start site. The mRNA transcript is expressed in almost all tissues, with high expression in the intestine and brain of normal-conditioned fish, and is expressed constitutively in early developmental stages after hatching. The mRNA expression of fYGHL1 is highly regulated by hypoxia and E. tarda infection. The expression of fYGHL1 mRNA was down regulated in the gill, spleen, intestine, and stomach of flounder under hypoxic conditions, whereas the expression level was increased in flounder embryonic cells treated with the hypoxia-mimic CoCl(2) (a HIF-1 inducer). Pathogen challenge induced fYGHL1 expression in the spleen of juvenile fish. Taken together, these results suggest that fYGHL1 is a hypoxia-related gene with potential roles in the hypoxia response mechanism as well as in defense, immune responses, growth, and regulation of reproduction.

Molecular characterization of the autophagy-related gene Beclin-1 from the olive flounder (Paralichthys olivaceus).

Autophagy is an important cellular response to starvation and stress, and plays critical roles in embryogenesis, development, cell death, cancer, and immunity. Beclin-1 is one of the central regulators of autophagy in mammals. In the present study, we isolated a PoBeclin-1 cDNA from the olive flounder (Paralichthys olivaceus) by screening a flounder gill cDNA library and rapid amplification of cDNA ends (RACE). The PoBeclin-1 cDNA we isolated encodes a 447-amino acid polypeptide containing a conserved Bcl-2-binding domain. The deduced amino acid sequence of PoBeclin-1 showed high degrees of sequence identity (80.5-95.3%) with Beclin-1 from human, frog, mouse, zebrafish, and pufferfish. PoBeclin-1 transcripts were detected from 1 day post-hatching and were found to be ubiquitously expressed in the healthy flounder. Expression of PoBeclin-1 mRNA was increased in the kidney and spleen of flounders challenged with viral hemorrhagic septicemia virus (VHSV). When infected with VHSV, PoBeclin-1-overexpressing HINAE cells had low level (about 26%) of VHSV G transcripts compared to control cells. Taken together, these results suggest that PoBeclin-1 may play a role in the innate immune response to viral infection in the flounder.

Molecular cloning and mRNA expression of the liver-specific cathepsin L1 gene of the olive flounder, Paralichthys olivaceus.

We isolated a homolog of cathepsin L from a cDNA library of the olive flounder liver. The flounder cathepsin L1 transcript consisted of 1,221 bp that encoded a polypeptide of 334 amino acids. The overall identity between flounder cathepsin L1 and other cathepsin Ls was 50-64%, and flounder cathepsin L1 contained the highly conserved ERFNIN-motif. A phylogenetic tree indicated that flounder cathepsin L1 is in the same monophyletic group as zebrafish cathepsin Lc. RT-PCR analysis revealed that cathepsin L1 transcripts were expressed only in the liver. They were detected from 28 d post-hatching. Under starvation conditions, cathepsin L1 expression was decreased at 30 d.

ß-propeller phytase hydrolyzes insoluble Ca(2+)-phytate salts and completely abrogates the ability of phytate to chelate metal ions.

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the ß-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.

Paracoccus fistulariae sp. nov., a lipolytic bacterium isolated from bluespotted cornetfish, Fistularia commersonii.

A Gram-stain-negative, non-motile, non-spore-forming and short rod- or rod-shaped bacterial strain, designated 22-5(T), was isolated from a bluespotted cornetfish, Fistularia commersonii, and subjected to taxonomic study. Strain 22-5(T) grew optimally at 30 °C and in the presence of 2-5 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 22-5(T) belonged to the genus Paracoccus and joined the cluster comprising Paracoccus homiensis DD-R11(T) and Paracoccus zeaxanthinifaciens ATCC 21588(T), with which strain 22-5(T) exhibited 97.4 and 96.9 % 16S rRNA gene sequence similarity, respectively. Strain 22-5(T) exhibited 94.0-96.6 % 16S rRNA gene sequence similarity with the other type strains of species of the genus Paracoccus. Strain 22-5(T) contained Q-10 as the predominant menaquinone and C(18 : 1)ω7c as the predominant fatty acid. In this study, P. zeaxanthinifaciens KCTC 22688(T) also contained Q-10 as the predominant isoprenoid quinone. The DNA G+C content of strain 22-5(T) was 63.6 mol%. Strain 22-5(T) exhibited 44 and 32 % DNA-DNA relatedness to P. homiensis KACC 11518(T) and P. zeaxanthinifaciens KCTC 22688(T), respectively. On the basis of phenotypic, phylogenetic and genetic data, strain 22-5(T) is considered to represent a novel species of the genus Paracoccus, for which the name Paracoccus fistulariae sp. nov. is proposed. The type strain is 22-5(T) (=KCTC 22803(T) =CCUG 58401(T)).

Photobacterium gaetbulicola sp. nov., a lipolytic bacterium isolated from a tidal flat sediment.

A Gram-negative, motile, non-spore-forming and lipolytic bacterial strain, designated Gung47(T), was isolated from a tidal flat on the west coast of Korea. Strain Gung47(T) grew optimally at 30 °C and with 2-5 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain Gung47(T) belonged to the genus Photobacterium. Strain Gung47(T) exhibited 98.1 % 16S rRNA gene sequence similarity with Photobacterium rosenbergii LMG 22223(T) and 94.3-96.3 % similarity with other type strains of species of the genus Photobacterium. Strain Gung47(T) exhibited 47 % DNA-DNA relatedness to P. rosenbergii LMG 22223(T). Strain Gung47(T) contained Q-8 as the predominant ubiquinone and C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH, C(16 : 0) and C(18 : 1)ω7c as the major fatty acids. In this study, two closely related type strains, P. rosenbergii LMG 22223(T) and Photobacterium halotolerans LMG 22194(T), were also found to have Q-8 as the predominant ubiquinone. The DNA G+C content of strain Gung47(T) was 50.6 mol%. The differential phenotypic properties together with the phylogenetic and genetic distinctiveness of strain Gung47(T) demonstrated that this strain is distinguishable from recognized Photobacterium species. Therefore, strain Gung47(T) is considered to represent a novel species of the genus Photobacterium, for which the name Photobacterium gaetbulicola sp. nov. is proposed. The type strain is Gung47(T) (=KCTC 22804(T) =CCUG 58399(T)).

Characterization of the flounder IL-6 promoter and its regulation by the p65 NF-kappaB subunit.

Interleukin (IL)-6 plays important roles in the regulation of the immune response and inflammation in many cell types and its induction by bacterial endotoxins or cytokines is regulated at the transcriptional level. The present study demonstrates the isolation and characterization of the flounder IL-6 promoter sequence and its transcriptional regulation in olive flounder (hirame) natural embryo (HINAE) cells. The promoter region (-400 to +79 bp from the transcription initiation site) of the flounder IL-6 gene contains putative cis-regulatory elements for CCAAT-enhancer-binding proteins (C/EBP; -346 to -355 bp and -166 to -160 bp), cAMP response element binding (CREB; -81 to -85 bp), the activator protein 1 (AP-1; -56 to -62 bp), and NF-kappaB (-39 to -48 bp). Transfection of p65 stimulated the PoIL-6-luc-WT reporter, but not the PoIL-6-luc-kappaB mt reporter, and treatment with LPS augmented p65-stimulated reporter activity in HINAE cells. In contrast, transfection of C/EBPbeta or c-Jun failed to induce synergistic effects in the LPS-driven PoIL-6-luc-WT reporter activity. These results give us new insight into the regulation of flounder IL-6 transcription by the p65 NF-kappaB subunit.

An immune responsive complement factor D/adipsin and kallikrein-like serine protease (PoDAK) from the olive flounder Paralichthys olivaceus.

The cDNA encoding of a complement factor D/adipsin and kallikrein-like serine protease, designated PoDAK, was isolated from the olive flounder Paralichthys olivaceus. PoDAK cDNA encodes a polypeptide with 277 amino acids containing conserved catalytic triad residues of serine proteases. The amino acid sequence of PoDAK showed high similarity to the kallikrein-like protein of medaka, mammalian adipsin/complement factor D and tissue kallikrein homolog, KT-14 of trout, complement factor D of zebrafish, and shared 31.6-36.8% homology with complement factor D/adipsin known from other species, including mammals. Phylogenetic analysis revealed that PoDAK clustered with the kallikrein-like protein of medaka and mammalian adipsin/complement factor D and tissue kallikrein homolog KT-14 of trout. The expression of PoDAK mRNA was high in the gills and heart, moderate in muscle, liver, intestine, stomach, kidney, and spleen of healthy flounder, and increased in the kidney, liver, and spleen of flounder challenged by the viral hemorrhagic septicemia virus (VHSV) or Streptococcus iniae. In situ hybridization confirmed that PoDAK mRNA is localized in the kidney and heart of individuals infected with VHSV. Further investigations are needed to clarify the function of PoDAK in vivo and in vitro.

Cloning and expression analysis of cathepsin D in the olive flounder Paralichthys olivaceus.

We isolated a homolog of cathepsin D from the cDNA library of the olive flounder kidney. The olive flounder cathepsin D transcript consisted of 1,733 bp that encoded a polypeptide of 396 amino acids. The overall similarity between olive flounder cathepsin D and other cathepsin Ds was very high, with the highest amino acid sequence identity to barramundi perch (89%). RT-PCR revealed that cathepsin D was expressed in almost all tissues, with high expression in the liver, intestine, kidney, skin, and spleen. The accumulation of cathepsin D mRNA after bacterial infection, as determined by RT-PCR, was constitutive and increased greatly after bacterial infection.

Purification, characterization, and gene cloning of glucose-1-phosphatase from Citrobacter braakii.

Citrobacter braakii produced an intracellular acid glucose phosphatase (AgpC) which was purified 986 fold to homogeneity with the specific activity of 286 units/mg. AgpC hydrolyzed a wide variety of phosphorylated compounds with high activity for glucose-1-phosphate and glucose-6-phosphate. The optimum pH and temperature for the enzyme activity was pH 5.0 and 45 degrees C, respectively. The Km value for glucose-1-phosphate was 5.12 mM with a Vmax 27.8 U mg(-1). Its molecular weight was 46 kDa by SDS-PAGE gel and the sequence of N-terminal amino acid residues identified was Gln-Thr-Ala-Pro-Glu-Gly-Tyr-Gln-Leu-Gln. The glucose-1-phosphatase gene (agpC) was cloned from the C. braakii genomic library. This gene comprised 1,242 nucleotides and encoded a polypeptide of 413 amino acids. The result of its BLAST search showed a significant similarity with glucose-1-phosphatase from enterobacteria such as E. coli, Enterobacter, Shigella, and Salmonella.

Segregation distortion at a microsatellite marker in the olive flounder, Paralichtys olivaceus.

In the course of quantitative trait loci (QTL) analysis of the back cross (BC1) families of olive flounder (Paralichtys olivaceus), we observed significant segregation distortion at a microsatellite marker Poli9-58TUF in two crosses of informative progenies. The family 1 of the random BC1 progenies derived from a cross between a F1 male genotype (A/B) and a F1's female parent genotype (A/C) and the family 2 (A/C x A/C) displayed a strong bias in the locus from the Mendelian inheritance by the elimination of homozygous A/A genotype. The deleterious roles of the AA genotypes are suggested during the metamorphosis and it implies that the parents of these families carried a recessive gene or genes hampering development at an early stage because the offspring of the double heterozygote parents show the reduction in frequency or elimination of one homozygous class, which is an evidence for linkage between the genetic marker and gene(s) with recessive deleterious alleles. This data support a hypothesis that the region contains a recessive lethal gene or genes.

Expression Analysis of Lily Type Lectin Isotypes in the Rock Bream, Oplegnathus fasciatus: in the Tissue, Developmental Stage and Viral Infection.

Lectins belong to the pattern-recognition receptors (PRRs) class and play important roles in the recognition and elimination of pathogens via the innate immune system. Recently, it was reported that lily-type lectin-1 is involved when a pathogen attacks in the early immune response of fish. However, this study is limited to information that the lectin is involved in the innate immune response against viral infection. In the present study, the lily-type lectin-2 and -3 of Oplegnathus fasciatus (OfLTL-2 and 3) have been presented to be included B-lectin domain and two D-mannose binding sites in the amino acid sequence that an important feature for the fundamental structure. To investigate the functional properties of OfLTLs, the tissue distribution in the healthy rock bream and temporal expression during early developmental stage analysis are performed using quantitative real-time PCR. OfLTL-2 and 3 are predominantly expressed in the liver and skin, but rarely expressed in other organ. Also, the transcripts of OfLTLs are not expressed during the early developmental stage but its transcripts are increased after immune-related organs which are fully formed. In the challenge experiment with RBIV (rock bream iridovirus), the expression of OfLTLs was increased much more strongly in the late response than the early, unlike previously known. These results suggest that OfLTLs are specifically expressed in the immune-related tissues when those organs are fully formed and it can be inferred that the more intensively involved in the second half to the virus infection.

Expression Pattern of Early Growth Response Gene 1 during Olive Flounder (Paralichthys olivaceus) Embryonic Development.

The early growth response protein 1 (Egr-1) is a widely reported zinc finger protein and a well known transcription factor encoded by the Egr-1 gene, which plays key roles in many aspects of vertebrate embryogenesis and in adult vertebrates. The Egr-1 expression is important in the formation of the gill vascular system in flounders, which develops during the post-hatching phase and is essential for survival during the juvenile period. However, the complete details of Egr-1 expression during embryo development in olive flounder are not available. We assessed the expression patterns of Egr-1 during the early development of olive flounders by using reverse transcription polymerase chain reaction (RT-PCR) analysis. Microscopic observations showed that gill filament formation corresponded with the Egr-1 expression. Thus, we showed that Egr-1 plays a vital role in angiogenesis in the gill filaments during embryogenesis. Further, Egr-1 expression was found to be strong at 5 days after hatching (DAH), in the development of the gill vascular system, and this strong expression level was maintained throughout all the development stages. Our findings have important implications with respect to the biological role of Egr-1 and evolution of the first respiratory blood vessels in the gills of olive flounder. Further studies are required to elucidate the Egr-1-mediated stress response and to decipher the functional role of Egr-1 in developmental stages.

Transcriptional Onset of Lysozyme Genes during Early Development in Olive Flounder (Paralichthys olivaceus).

The immune system in teleost fish is not completely developed during embryonic and larval stages, therefore effective innate mechanisms is very important for survival in such an environment. However, the knowledge of the development of immune system assumed to be restricted. In many species, lysozymes have been considered as important genes of the first line immune defense. The early detection of lysozyme mRNA in previous reports, led to the investigation of its presence in oocytes. As a result, c-type lysozyme mRNA transcripts were detected in unfertilized oocytes indicating maternal transfer. Therefore, we investigated the expression patterns of lysozymes in flounder, including the matured oocyte. In our results, c-type lysozyme mRNA was first detected in unfertilized oocyte stage, observed the significantly decreased until hatching stage, and was significantly increased after hatching stage. On the other hand, g-type lysozyme mRNA transcripts were first detected at late neurula stage, and the mRNA level was significantly increased after 20 dph. It may be suggest that maternally supplied mRNAs are selectively degraded prior to the activation of embryonic transcription. This study will be help in understanding the maturation and onset of humoral immunity during development of olive flounder immune system.

Development and Validation of Single Nucleotide Polymorphism (SNP) Markers from an Expressed Sequence Tag (EST) Database in Olive Flounder (Paralichthys olivaceus).

To successful molecular breeding, identification and functional characterization of breeding related genes and development of molecular breeding techniques using DNA markers are essential. Although the development of a useful marker is difficult in the aspect of time, cost and effort, many markers are being developed to be used in molecular breeding and developed markers have been used in many fields. Single nucleotide polymorphisms (SNPs) markers were widely used for genomic research and breeding, but has hardly been validated for screening functional genes in olive flounder. We identified single nucleotide polymorphisms (SNPs) from expressed sequence tag (EST) database in olive flounder; out of a total 4,327 ESTs, 693 contigs and 514 SNPs were detected in total EST, and these substitutions include 297 transitions and 217 transversions. As a result, 144 SNP markers were developed on the basis of 514 SNP to selection of useful gene region, and then applied to each of eight wild and culture olive flounder (total 16 samples). In our experimental result, only 32 markers had detected polymorphism in sample, also identified 21 transitions and 11 transversions, whereas indel was not detected in polymorphic SNPs. Heterozygosity of wild and cultured olive flounder using the 32 SNP markers is 0.34 and 0.29, respectively. In conclusion, we identified SNP and polymorphism in olive flounder using newly designed marker, it supports that developed markers are suitable for SNP detection and diversity analysis in olive flounder. The outcome of this study can be basic data for researches for immunity gene and characteristic with SNP.

Expression Pattern of Early Growth Response Gene 1 during Olive Flounder (Paralichthys olivaceus) Embryonic Development.

The early growth response protein 1 (Egr-1) is a widely reported zinc finger protein and a well known transcription factor encoded by the Egr-1 gene, which plays key roles in many aspects of vertebrate embryogenesis and in adult vertebrates. The Egr-1 expression is important in the formation of the gill vascular system in flounders, which develops during the post-hatching phase and is essential for survival during the juvenile period. However, the complete details of Egr-1 expression during embryo development in olive flounder are not available. We assessed the expression patterns of Egr-1 during the early development of olive flounders by using reverse transcription polymerase chain reaction (RT-PCR) analysis. Microscopic observations showed that gill filament formation corresponded with the Egr-1 expression. Thus, we showed that Egr-1 plays a vital role in angiogenesis in the gill filaments during embryogenesis. Further, Egr-1 expression was found to be strong at 5 days after hatching (DAH), in the development of the gill vascular system, and this strong expression level was maintained throughout all the development stages. Our findings have important implications with respect to the biological role of Egr-1 and evolution of the first respiratory blood vessels in the gills of olive flounder. Further studies are required to elucidate the Egr-1-mediated stress response and to decipher the functional role of Egr-1 in developmental stages.

Shewanella upenei sp. nov., a lipolytic bacterium isolated from bensasi goatfish Upeneus bensasi.

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R(T), was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R(T) belonged to the genus Shewanella. Strain 20-23R(T) exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192(T), Shewanella haliotis DW01(T), and Shewanella chilikensis JC5(T), respectively. Strain 20-23R(T) exhibited 93.1-96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3-98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R(T) contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20-23R(T), S. algae KCTC 22552(T) and S. haliotis KCTC 12896(T) were similar; major components were iso-C(15:0), C(16:0), C(16:1) ω7c and/or iso-C(15:0) 2-OH and C(17:1) ω8c. The DNA G+C content of strain 20-23R(T) was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20-23R(T), together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R(T) represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20-23R(T) (=KCTC 22806(T) =CCUG 58400(T)).

A novel cold-adapted esterase from Salinisphaera sp. P7-4: gene cloning, overproduction, and characterization.

Salinisphaera sp. P7-4 was isolated from the intestine of silver whiting, Sillago japonicas caught in the Pacific Ocean, and the esterase gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (951 bp) corresponded to a protein of 316 amino acid residues with a molecular weight of 34,443. The esterase had 46 and 44% identities with the esterase enzymes of Ralstonia eutropha JMP134 and Rhodopseudomonas palustris HaA2, respectively. The primary structure of P7-4 esterase showed the conserved catalytic triad (Ser, Asp, His), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein P7-4 was successfully expressed in Escherichia coli in a biologically active form. The enzyme showed high catalytic activity at low temperatures (5-25° C) with an activation energy of 2.18 kcal/mol. This result indicated that the esterase from Salinisphaera sp. P7-4 is a new cold-adapted enzyme. The enzyme preferentially hydrolyzed acyl-group chains with short chain lengths of ≤10 carbon. Metal ions such as Cd2(+), Co2(+), Cu2(+), Hg2(+), Ni2(+) and Zn2(+) inhibited enzymatic activity. Additionally, EDTA has no effect on its activity, whereas inhibition was observed with PMSF, a serine hydrolase inhibitor.

N-terminal pI determines the solubility of a recombinant protein lacking an internal transmembrane-like domain in E. coli.

We examined whether the isoelectric point (pI) of the Nterminal region of the recombinant protein 7xMefp1 acts as a universal index for expression of the protein in soluble form in E. coli. Expression analysis of 7xMefp1 fused to various N-terminal sequences with pI values ranging from 2.73 to 13.35 yielded three pI range-specific curves (acidic, neutral, and alkaline curves at pI 2.73-3.25, 4.61-9.58, and 9.90-13.35, respectively) for soluble expression (by facilitated diffusion) as a proportion of total protein. For neutral N-termini (pI 4.61-9.58), the total amount of rMefp1 expressed was minimally affected by DeltaG(RNA) for unfolding the mRNA secondary structure. The highly hydrophilic nature of longer N-terminal sequences with strongly acidic and alkaline pI values reduced the translation of rMefp1-encoding transcripts, thereby reducing the amount of soluble rMefp1 produced. After characterizing both feedback and non-feedback regulation in the acidic, alkaline, and neutral pI ranges, we suggest that three different pI range-specific soluble expression curves exist for the recombinant protein, each defined by specific ranges of the leader sequence pI values.