Production of recombinant buffalo (Bubalus bubalis) and goat (Capra hircus) growth hormones from genetically modified E. coli strains

The growth hormone cDNAs of Indian reverine buffalo (Bubalus bubalis) and beetal goat (Capra hircus) were cloned in Escherichia coli through RT-PCR technique. Nucleotide sequencing revealed several silent mutations in both cDNAs and only one amino acid change in the case of goat when compared to reported bovine (Bos taurus) sequence. The high level expression of both the polypeptide hormones was achieved in E. coli (> or =30% of soluble intracellular proteins) through the construction of two-cistronic gene expression system. The solubilisation of recombinant growth hormones from inclusion bodies and subsequent oxidation to correctly folded monomeric form was also carried out. A combination of reverse-phase HPLC and non-reducing SDS-PAGE was successfully applied to distinguish between reduced and oxidised forms of growth hormones. A moderate yield ( approximately 40% of starting material, with potential for upscaling), two-step purification process comprising of hydrophobic interaction and ion-exchange chromatographies was developed. The process eliminates the need for costly, laborious and time-consuming steps of ultrafiltration and dialysis, as reported earlier for the purification of many recombinant animal growth hormones. The biophysical, biochemical and functional analyses of purified refolded polypeptides showed that the hormones produced in this study were identical to natural pituitary bovine growth hormone.     

The Cpn10(1) Co-Chaperonin of A. thaliana Functions Only as a Hetero-Oligomer with Cpn20

The A. thaliana genome encodes five co-chaperonin homologs, three of which are destined to the chloroplast. Two of the proteins, Cpn10(2) and Cpn20, form functional homo-oligomers in vitro. In the current work, we present data on the structure and function of the third A. thaliana co-chaperonin, which exhibits unique properties. We found that purified recombinant Cpn10(1) forms inactive dimers in solution, in contrast to the active heptamers that are formed by canonical Cpn10s. Additionally, our data demonstrate that Cpn10(1) is capable of assembling into active hetero-oligomers together with Cpn20. This finding was reinforced by the formation of active co-chaperonin species upon mixing an inactive Cpn20 mutant with the inactive Cpn10(1). The present study constitutes the first report of a higher plant Cpn10 subunit that is able to function only upon formation of hetero-oligomers with other co-chaperonins.     

Enhanced TGFbeta1 maturation in high five cells coinfected with recombinant baculovirus encoding the convertase furin/pace: improved technology for the production of recombinant proproteins in insect cells

One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFbeta1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFbeta1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFbeta1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFbeta1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFbeta1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFbeta1 by coexpressing prohTGFbeta1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFbeta1 increased in a dose-dependent fashion reaching up to 7. 8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFbeta1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide.     

Recombinant goldfish growth hormones (gfGH-I and -II) expressed in Escherichia coli have similar biological activities

Complementary DNA regions coding for two different mature goldfish growth hormones (gfGH-I and gfGH-II) with four and five cysteine residues were cloned into the bacterial expression vector, pRSETA. The recombinant gfGH-I (five cysteines) and -II (four cysteines) were produced in Escherichia coli as the fusion proteins carrying N-terminal 6XHis tag, which facilitates purification by using metal chelating affinity chromatography under denaturing condition with urea. The recombinant hormones were further refolded by gradually removing the urea. Native gfGH was also purified from goldfish pituitary glands and served as a positive control in the present study. The native and recombinant hormones were tested in goldfish hepatic radioreceptor assay and in vitro Spi 2.1 promoter activation assay. Our results showed that the two recombinant gfGHs are biologically active, and they have similar biological activities despite their having different cysteine contents.     

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in insect cells using recombinant baculovirus.

Molt-inhibiting hormone (MIH) negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in insect cells using recombinant baculovirus. Insect Sf9 cells were transfected with recombinant baculovirus containing a DNA insert encoding the C. sapidus MIH prohormone (signal sequence plus mature hormone). The construct was designed to yield a mature, fully processed recombinant MIH (recMIH). Several baculovirus recombinants showing no contamination with wild-type viral DNA were subsequently analyzed for their ability to direct expression of recMIH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from infected cells revealed time-dependent expression of two proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature hormone. Western blot results (using antiserum against MIH of Carcinus maenas) indicated that the proteins were MIH-immunoreactive. N-Terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and molecular mass. Cell lysates containing the recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. We anticipate the recombinant peptide will prove useful for studies of the structure and function of MIH.     

Syntheses of recombinant yellowtail and flounder growth hormones in Escherichia coli.

For syntheses of recombinant yellowtail and flounder growth hormones (r-yGH and r-fGH) in E. coli, expression plasmids were constructed. The expression level of r-yGH and r-fGH in the host cells were very high, reaching 15 and 8% of the total protein, respectively. These product proteins were accumulated in inclusion bodies in the cells. The recombinant hormones were isolated from the pellets ina glutathione reduction/oxidation buffer. The refolded hormones were further purified by DEAE-Toyopearl 650M chromatography to homogeneity. The purified r-yGH and r-fGH were composed of 188 and 174 amino acid residues, respectively, having amino-terminal sequences starting with methionine. The recombinant hormones had potent growth-promoting activities on juvenile rainbow trout Salmo gairdneri in a dose-dependent manner.     

Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419).

We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and gamma-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes (Ki = 3 x 10(-10) M). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.     

Syntheses of Recombinant Yellowtail and Flounder Growth Hormones in Escherichia coli

For syntheses of recombinant yellowtail and flounder growth hormones (r-yGH and r-fGH) in E. coli, expression plasmids were constructed. The expression level of r-yGH and r-fGH in the host cells were very high, reaching 15 and 8% of the total protein, respectively. These product proteins were accumulated in inclusion bodies in the cells. The recombinant hormones were isolated from the pellets in a glutathione reduction/oxidation buffer. The refolded hormones were further purified by DEAE-Toyopearl 650M chromatography to homogeneity. The purified r-yGH and r-fGH were composed of 188 and 174 amino acid residues, respectively, having amino-terminal sequences starting with methionine. The recombinant hormones had potent growth-promoting activities on juvenile rainbow trout Salmo gairdneri in a dose-dependent manner.     

Endothelial cell signalling induced by trans-sialidase from Trypanosoma cruzi

The protozoan responsible for Chagas' disease, Trypanosoma cruzi , expresses on its surface an unusual trans -sialidase enzyme thought to play an important role in host–parasite interactions. Trans -sialidase is the product of a multigene family encoding both active and inactive proteins. We have demonstrated that despite lacking enzymatic activity due to a single mutation, Tyr342-His, inactive trans -sialidase displays sialic acid binding activity, with identical specificity to that of its active analogue. In this work we demonstrate that binding of a recombinant inactive trans -sialidase to molecules containing α2,3-linked sialic acid on endothelial cell surface triggers NF-κB activation, expression of adhesion molecules and upregulation of parasite entry into host cells. Furthermore, inactive recombinant trans -sialidase blocks endothelial cell apoptosis induced by growth factor deprivation. These results suggest that inactive members of the trans -sialidase family play a role in endothelial cell responses to T. cruzi infection.     

液胶色谱柱复性在低分子量尿激酶原突变体（DscuPA—32K）中的应用

将构建一种具溶栓和抗栓以重功能尿激酶原突变体（DscuPA-32K)基因,在大肠杆菌中进行表达。由于DscuPA-32K分子较大并且表达量较高,目的的性质基本以包涵体的形式存在。包涵体中的蛋白质是无活性的蛋白质,为了获得有活性的蛋白质,就需要对包涵体进行变性及复性。尝试了一种新的凝胶色谱柱复性方法,并通过柱复性方法与常规的稀释复性方法进行了比较,发现柱复性方法明显优于稀释复性方法,具有成本低,效率高,并对目的的蛋白质（DscuPA-32K)进行了初步纯化等优点,尤其对酶这一类容易失活降解的蛋白质进行复性时,很值得进行推广应用。     

A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins

We have developed and evaluated a highly parallel protein expression and purification system using ORFs derived from the pathogenic bacterium Streptococcus pneumoniae as a representative test case in conjunction with the Gateway cloning technology. Establishing high throughput protein production capability is essential for genome-wide characterization of protein function. In this study, we focused on protein expression and purification outcomes generated from an expression vector which encodes an NH(2)-terminal hexa-histidine tag and a COOH-terminal S-tag. Purified recombinant proteins were validated by SDS-PAGE, followed by in-gel digestion and identification by MALDI-TOF/TOF analysis. Starting with 1360 sequence-validated destination clones we examined correlation analyses of expression and solubility of a wide variety of recombinant proteins. In total, 428 purified proteins (31%) were recovered in soluble form. We describe a semi-quantitative scoring method using an S-tag assay to improve the throughput and efficiency of expression and solubility studies for recombinant proteins. Given a relatively large dataset derived from proteins representing all functional groups in a microbial genome we correlated various protein characteristics as they relate to protein expression outcomes.     

Role of growth-hormone and insulin-like growth-factor-binding proteins.

Some peptide hormones are associated with specific, high-affinity plasma proteins. The major binding protein (BP) for growth hormone (GH) in humans is a circulating fragment of the GH membrane receptor, consisting of the hydrophilic, extracellular portion of that transmembrane glycoprotein. The circulating levels of GH-BP mirror the levels of GH receptors. There are 4 well-characterized insulin-like growth factor (IGF)-BPs. One IGF-binding component in plasma is a fragment of the extracellular portion of the IGF-II/mannose-6-phosphate receptor, analogous to the GH-BP. The 3 other cloned IGF-BPs form a homologous family of proteins with differences in structure, glycosylation and hormonal control that suggest differences in function. The GH- and IGF-BPs play a major role in the metabolism and biological action of these peptide hormones.     

Identification and characterization of novel splice variants of the human EPM2A gene mutated in Lafora progressive myoclonus epilepsy

The EPM2A gene, defective in the fatal neurodegenerative disorder Lafora disease (LD), is known to encode two distinct proteins by differential splicing; a phosphatase active cytoplasmic isoform and a phosphatase inactive nuclear isoform. We report here the identification of three novel EPM2A splice variants with potential to code for five distinct proteins in alternate reading frames. These novel isoforms, when ectopically expressed in cell lines, show distinct subcellular localization, interact with and serve as substrates of malin ubiquitin ligase-the second protein defective in LD. Two phosphatase active isoforms interact to form a heterodimeric complex that is inactive as a phosphatase in vitro, suggesting an antagonistic function for laforin isoforms if expressed endogenously in significant amounts in human tissues. Thus alternative splicing could possibly be one of the mechanisms by which EPM2A may regulate the cellular functions of the proteins it codes for.     

Anti-aggregatory effect of cyclodextrins in the refolding process of recombinant growth hormones from Escherichia coli inclusion bodies

Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin show a positive effect on the aggregation suppression of both proteins. The influence of different methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin suppress not only folding-related, but also temperature-related aggregates formation of both proteins. Circular dichroism experiments (monitoring of protein solution turbidity by registering high tension voltage) showed that the onset temperature of aggregation of both growth hormones increased with increasing 2-hydroxypropyl-β-cyclodextrin concentration. In conclusion, cyclodextrins have perspectives in biotechnology of veterinary growth hormones not only for protein production, but also for its storage.     

Restoring the Procofactor State of Factor Va-like Variants by Complementation with B-domain Peptides

Coagulation factor V (FV) circulates as an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domain. Conserved basic and acidic sequences within the B-domain appear to play an important role in keeping FV as an inactive procofactor. Here, we utilized recombinant B-domain fragments to elucidate the mechanism of this FV autoinhibition. We show that a fragment encoding the basic region (BR) of the B-domain binds with high affinity to cofactor-like FV(a) variants that harbor an intact acidic region. Furthermore, the BR inhibits procoagulant function of the variants, thereby restoring the procofactor state. The BR competes with FXa for binding to FV(a), and limited proteolysis of the B-domain, specifically at Arg¹⁵⁴⁵, ablates BR binding to promote high affinity association between FVa and FXa. These results provide new insight into the mechanism by which the B-domain stabilizes FV as an inactive procofactor and reveal how limited proteolysis of FV progressively destabilizes key regulatory regions of the B-domain to produce an active form of the molecule.     

包涵体蛋白体外复性的研究进展

外源基因在大肠杆菌中高水平表达时 ,通常会形成无活性的蛋白聚集体即包涵体。包涵体富含表达的重组蛋白 ,经分离、变性溶解后须再经过一个合适的复性过程实现变性蛋白的重折叠 ,才能够得到生物活性蛋白。近年来 ,发展了许多特异的策略和方法来从包涵体中复性重组蛋白。最近的进展包括固定化复性以及用一些低分子量的添加剂等来减少复性过程中蛋白质的聚集 ,提高活性蛋白的产率。     

拟南芥 MeIAA 抗性突变体的筛选和初步图位克隆分析

生长素是最重要的植物激素之一, 参与了植物生长发育的各个方面。植物体内游离的IAA是生长素的主要活性形式, 在IAA甲基转移酶1(IAMT1)的作用下, IAA可以转变为IAA甲酯 (MeIAA)。MeIAA本身没有活性, 在植物体内的MeIAA酯解酶作用下可以重新转变为IAA。 MeIAA是非极性分子, 能够在植物体内自由扩散。利用MeIAA的这种特殊性质筛选突变体, 可以分离到MeIAA代谢途径或者IAA途径中新的成分。我们对拟南芥种子进行EMS诱变, 通过观察黑暗下下胚轴的生长情况, 筛选MeIAA的抗性突变体。我们成功分离到了8株可能的抗性突变体, 并对其中的一个Methyl -IAA resistant 1 (mir1) 突变体进行了深入分析。MeIAA抗性突变体的筛选将为进一步了解MeIAA的代谢、IAA稳态调控和响应机理提供新的材料。     

Increased disulphide dimer formation of latent associated peptide fusions of TGF-β by addition of l-cystine

The development of novel protein therapeutics relies on the ability to express appreciable amounts of correctly folded recombinant proteins. Latent IFN-β is engineered using the latency-associated peptide (LAP) of transforming growth factor β1 (TGF-β1) to maintain IFN-β in a biologically inactive form until such time as it is released at sites of inflammation by matrix metalloproteinase activity (see Adams et al., 2003). CHO cells cultured in suspension were used for expression of latent IFN-β to allow medium scale transient transfection. However, the recombinant protein expressed in this system consisted of a mixture of properly linked disulphide dimers and monomers. The ratio of dimer:monomer produced could be significantly altered towards increased dimer production by the addition of l-cystine to the CHO culture medium. The total yield of latent IFN-β was increased by co-transfection of plasmid coding for the simian virus (SV) 40 large T antigen to the plasmid with the SV40 origin of replication expressing latent IFN-β DNA. These results provide valuable new insights for developing protocols to produce substantial quantities of latent cytokine dimers in CHO cells in suspension.