Bacterial Expression Plasmid Research Articles

Recombinant NY‐ESO‐1 Cancer Antigen: Production and Purification under cGMP Conditions

The cancer‐testis antigen, NY‐ESO‐1, has been engineered into a bacterial expression plasmid which incorporates a His6‐tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15‐litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY‐ESO‐1 tagged protein isolated as solubilised inclusion bodies. A three‐step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY‐ESO‐1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.

Assembly of Human Immunodeficiency Virus Precursor Gag Proteins

To investigate the mechanism by which human immunodeficiency virus (HIV) precursor Gag (PrGag) proteins assemble to form immature virus particles, we examined the in vitro assembly of MACANC proteins, composed of the PrGag matrix, capsid, and nucleocapsid domains. In the absence of other components, MACANC proteins assembled efficiently at physiological temperature but inefficiently at lower temperatures. However, the addition of RNA reduced the temperature sensitivity of assembly reactions. Assembly of MACANC proteins also was affected by pH because the proteins preferentially formed tubes at pH 6.0, whereas spheres were obtained at pH 8.0. Because neither tubes nor spheres were amenable to analysis of protein-protein contacts, we also examined the membrane-bound assemblies of MACANC proteins. Interestingly, MACANC proteins organized on membranes in tightly packed hexameric rings. The observed hexamer spacing of 79.7 A is consistent with the notion that more PrGag proteins assemble into virions than are needed to provide capsid proteins for mature virus cores. Our data are also consistent with a model for PrGag contacts in immature virions where capsid hexamers are tightly packed, where nucleocapsid domains align beneath capsid C-terminal domains, and where matrix domains form trimers at the nexus of three neighbor hexamers.

Ric-3 Promotes Functional Expression of the Nicotinic Acetylcholine Receptor α7 Subunit in Mammalian Cells

Expression of functional, recombinant alpha7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor alpha7 subunit. In addition to confirming the previously reported effect on alpha7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional alpha7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface alpha-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the alpha7 subunit. In contrast, co-expression of alpha7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous alpha7 receptors. Immunoprecipitation studies demonstrate that alpha7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of alpha7 protein on the plasma membrane of cells lacking ric-3, but surface alpha-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of alpha7 receptors in HEK293 cells.

E2A-PBX1 Interacts Directly with the KIX Domain of CBP/p300 in the Induction of Proliferation in Primary Hematopoietic Cells

The E2A gene encodes DNA-binding transcription factors, called E12 and E47, involved in cell specification and maturation. E2A is also involved in a chromosomal translocation that leads to the expression of an oncogenic transcription factor called E2A-PBX1 in cases of acute leukemia. In the work described here, we elucidate the interaction between E2A-PBX1 and transcriptional co-activators. We confirm that the E2A portion can interact with CBP and PCAF and map required elements on E2A and CBP. On CBP, the interaction involves the KIX domain, a well characterized domain that mediates interactions with several other oncogenic transcription factors. On E2A, the interaction with CBP requires conserved alpha-helical domains that reside within activation domains 1 and 2 (AD1 and AD2, respectively). Using purified, recombinant proteins, we show that the E2A-CBP interaction is direct. Notwithstanding the previously demonstrated ability of AD1 and AD2 to function independently, some of our findings suggest functional cooperativity between these two domains. Finally, we show that the CBP/p300-interactive helical domains of E2A are important in the induction of proliferation in cultured primary bone marrow cells retrovirally transduced with E2A-PBX1. Our findings suggest that some aspects of E2A-PBX1 oncogenesis involve a direct interaction with the KIX domain of CBP/p300.

Gene amplification by PCR and subcloning into a GFP‐fusion plasmid expression vector as a molecular biology laboratory course*

A novel experimental sequence for the advanced undergraduate laboratory course has been developed at Earlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment, undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in a bacterial expression plasmid over the course of a single semester in a weekly 3-h laboratory period. Students designed PCR primers for amplification of the selected gene using computational DNA sequence analysis tools. During the experimental portion of the course, students amplified and ligated the target DNA into a commercially available GFP expression vector. Following transformation of the ligation product, plasmids were harvested from the resulting bacterial colonies and were analyzed by restriction digestion to confirm the creation of the chimeric GFP-DNA. This course gave students valuable experience with commonly used molecular techniques in an authentic research project. In addition, students gained experience with experimental design and execution. The techniques presented here are flexible and can be generalized for use with almost any DNA sequence and expression vector. This series also serves as an example of how faculty can adapt their ongoing research projects to the undergraduate laboratory.

Expression and purification of the recombinant diphtheria fusion toxin DT 388IL3 for phase I clinical trials

A genetically engineered fusion toxin targeted to acute myeloid leukemia (AML) blasts was designed with the first 388 amino acid residues of diphtheria toxin with an H-M linker fused to human interleukin-3. The cDNA was subcloned in the pRK bacterial expression plasmid and used to transform BLR (DE3) Escherichia coli. A single transformed colony was grown in Superbroth with ampicillin; bacteria were centrifuged at an OD 650 of 1.3; master cell bank aliquots of bacteria in 30% glycerol/Superbroth were frozen and stored at −80 °C. Master cell bank bacteria were diluted 1500-fold into Superbroth and recombinant protein was induced with 1 mM IPTG at an OD 650 of 0.6. After two additional hours of fermentation, inclusion bodies were isolated, washed, and denatured in guanidine hydrochloride and dithioerythritol. Recombinant protein was refolded by diluted 100-fold in cold buffer with arginine and oxidized glutathione. After dialysis, purified protein was obtained after anion-exchange, size exclusion on FPLC, and polymyxin B affinity chromatography. The final material was filter sterilized, aseptically vialed, and stored at −80 °C. Seventy-five 3-L bacterial culture preparations were made and pooled for the AT-1 batch (568 mL) and twenty-four 3-L bacterial culture preparations were made and pooled for the AT-2 batch (169 mL). The final product was characterized by Coomassie Plus protein assay, Coomassie-stained SDS–PAGE, limulus amebocyte lysate endotoxin assay, human AML TF/H-ras cell cytotoxicity assay, sterility, tandem mass spectroscopy, IL3 receptor binding affinity, ADP ribosylation activity, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, peptide mapping, stability, HPLC TSK3000, N-terminal sequencing, E. coli DNA contamination, C57BL/6 mouse toxicity, cynomolgus monkey toxicity, and immunohistochemistry. Yields were 25.7 ± 5.6 mg/L bacterial culture of denatured fusion toxin. After refolding and chromatography, final yields were 20 ± 11% or 5 mg/L. Vialed product was sterile. Batches were in 0.25 M sodium chloride/5 mM Tris, pH 8, and had protein concentrations of 1.8–1.9 mg/mL. Purity by SDS–PAGE was 99 ± 1%. Aggregates by HPLC were <1%. Potency revealed a 48 h IC 50 of 6–8 pM on TF/H-ras cells. Endotoxin levels were 1 eu/mg. The remaining chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The LD 10 in mice was 250 μg/kg/day every other day for six doses. The MTD in monkeys was 60 μg/kg/day every other day for six doses. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at −80 and −20 °C. Further, the drug was stable at 4 and 25 °C in the plastic syringe and administration tubing for 24 h and at 37 °C in human serum for 24 h. The synthesis of this protein drug should be useful for production for clinical phase I/II clinical trials and may be suitable for other diphtheria fusion toxins indicated for clinical development.

Immunotherapy of human cervical high-grade cervical intraepithelial neoplasia with microparticle-delivered human papillomavirus 16 E7 plasmid DNA

Objective: The purpose of this study was to evaluate the safety of the administration of a bacterial expression plasmid encoding a 13 amino acid sequence that is highly homologous with human papillomavirus E7 within poly (lactide-co-glycolide) microparticles (ZYC101) in women with HLA A2+ antigen and persistent cervical intraepithelial neoplasia grade 2/3 and human papillomavirus 16. Study Design: Fifteen women entered an institutional review board–approved dose-escalating phase I study with the use of three levels of blood monitoring and urine studies, Papanicolaou tests, and colposcopy. Escalation required no serious adverse events. Immunologic responses were evaluated in peripheral blood with the use of human papillomavirus peptide–stimulated interferon gamma enzyme-linked immunosorbent assay for T-cell reactivity. In cervical secretions, immunoglobulin A anti-human papillomavirus 16 E2 concentrations were measured. Three doses every 3 weeks were followed 4 weeks later by surgical excision. Results: No serious adverse events occurred. Five women had complete histologic responses; 11 women had human papillomavirus–specific T-cell responses. Four of five complete histologic responses developed immunoglobulin A anti-E2–specific antibody. Conclusion: ZYC101 warrants further investigation because of a 33% complete histologic responses, a 73% immunologic response, and no serious adverse events.

Mechanisms of dCMP Transferase Reactions Catalyzed by Mouse Rev1 Protein

The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction. Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus. The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides. Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the V(max) value. Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at a V(max) similar to that for dCMP.

Set of vectors for the expression of histidine-tagged proteins in vaccinia virus recombinants.

Vaccinia virus expression vectors are widely used to direct the expression of proteins in eukaryotic cells. Here, we describe a new set of plasmid vectors designed for the expression of histidine-tagged proteins in the vaccinia system. To facilitate the rapid isolation of virus recombinants, the plasmids contain a viral gene (F13L) that serves as an efficient selection marker based on virus plaque phenotype. Histidine codons and restriction sites derived from pET-16b bacterial expression plasmid were included, thus facilitating the transfer of genes between E. coli and vaccinia expression plasmids. Plasmids in which the gene is placed downstream of either a strong vaccinia virus or a T7 promoter were constructed, allowing for constitutive or conditional expression, respectively, of the foreign protein.

T-Cell Reactivity to the P2C Nonstructural Protein of a Diabetogenic Strain of Coxsackievirus B4

Enteroviruses are proposed as initiating factors in the etiology of Type 1 diabetes mellitus (Type 1 DM). Molecular mimicry between the autoantigen glutamic acid decarboxylase 65 (GAD65) and the coxsackievirus B4 (CVB4) nonstructural protein P2C is frequently cited as a mechanism by which this virus triggers the disease, but little is known about the immunogenicity of this viral protein in humans, mainly due to the problem of obtaining highly pure preparations of P2C. We generated large amounts of highly pure, soluble P2C protein, coupled to the fusion partner maltose binding protein (MBP–P2C) using the PMAL-c2 bacterial expression plasmid and a two-step purification system comprising amylose resin and ion exchange. Using purified viral protein we show that specific T-cell responses against P2C are detected in the blood of healthy donors and Type 1 DM patients. Proliferation responses to P2C were detected only in subjects also demonstrating T-cell proliferation to CVB4 Vero cell lysates. However, in additional cases T-cell responses to P2C were detectable through the release of interferon-γ or interleukin-4 in individuals who did not make proliferative responses. Taken together, our data show that the P2C nonstructural protein of CVB4 is targeted by T cells during the antiviral immune response and may trigger the production of T helper 1 and T helper 2 cytokines. The availability of pure, immunogenic P2C should allow the putative role of antiviral responses in the development of autoimmune diabetes to be investigated.

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC50 of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID50 of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.

Further Biochemical and Kinetic Characterization of Human Eukaryotic Initiation Factor 4H

A cDNA encoding human eukaryotic initiation factor (eIF) 4H was subcloned into a bacterial expression plasmid for purification of recombinant protein. Recombinant human eIF4H (heIF4H) was purified to greater than 95% homogeneity and shown to have similar physical characteristics to eIF4H purified from rabbit reticulocyte lysate as described previously. Functional studies have revealed that recombinant heIF4H functions identically to rabbit eIF4H in stimulating protein synthesis, and the ATP hydrolysis and helicase activities of eIF4A. More detailed enzymatic studies revealed that eIF4H increases the affinity of eIF4A for RNA by 2-fold, but has no effect on the binding of ATP by eIF4A. eIF4H stimulates the helicase activity of eIF4A at least 4-fold, and it is postulated that this stimulation occurs through increasing the processivity of eIF4A. Northern blot analysis shows that eIF4H is expressed ubiquitously in human tissues, and displays different levels of expression in given tissues relative to eIF4B. Secondary structure analysis of heIF4H by circular dichroism suggest that eIF4H has a mostly beta-sheet structure, which appears similar to other RNA recognition motif-containing proteins. Finally, it is suggested that eIF4H functions in translation initiation through protein-protein interactions that possibly stabilize conformational changes that occur in eIF4A during RNA binding, ATP hydrolysis, and RNA duplex unwinding.

Biosynthesis of a Head-to-Tail Cyclized Protein with Improved Biological Activity

Head-to-tail (backbone) peptide cyclization involves condensing the Nand C-termini of a peptide together and is typically used to rigidify structure and improve in ViVo stability.1 Although backbone cyclization is routinely used to modify the properties of small bioactive peptides as part of drug discovery programs, its application to proteins remains largely unexplored.2 This is primarily because the synthetic methodologies developed for the cyclization of small peptides cannot be easily extended to much larger protein systems. Nonetheless, protein cyclization remains of considerable interest to the protein engineering and protein folding communities3 since it is expected to provide useful insights into the importance of the Cand N-termini of a protein in folding, structural stability, and, consequently, biological activity. In the present work, we describe a novel biosynthetic process, based on an intramolecular chemical ligation reaction, which allows the cyclization of recombinant polypeptide precursors. This approach has been successfully applied to the generation of a circular version of a Src homology 3 (SH3) domain with improved biological activity over the wild-type protein. Several strategies for the preparation of circular polypeptides from unprotected linear precursors have been described. In pioneering studies in this area, Creighton and co-workers used a chemical cross-linking approach to prepare a backbone cyclized version of bovine pancreatic trypsin inhibitor.2a More recently, chemical2b,c,4 and enzymatic2d intramolecular ligation methods have been developed which allow linear synthetic peptides to be efficiently cyclized under aqueous conditions. However, the requirement for synthetic peptide precursors has limited these chemical/enzymatic cyclization approaches to relatively small polypeptide systems. One potential solution to this size problem would be to use recombinant polypeptides as precursors, thereby allowing large polypeptides and even proteins to be backbonecyclized. In principle, it should be possible to generate a circular recombinant protein by using an intramolecular version of the native chemical ligation approach.5 Native chemical ligation involves the chemoselective reaction that occurs between an N-terminal cysteine residue in one peptide and an R-thioester group within a second peptide, resulting in the formation of a normal peptide bond. Importantly, incorporation of both of these reactive moieties within the same synthetic polypeptide leads to efficient backbone cyclization.2b,c,4 Considerable progress has been made in generating recombinant polypeptides for use in native chemical ligation reactions: Verdine and co-workers have used a mutagenesis/factor Xa proteolysis strategy to generate reactive recombinant N-terminal cysteine proteins,6 while we and others have shown that recombinant polypeptide R-thioesters can be prepared by chemically intercepting a naturally occurring proteinsplicing reaction.7 To date, recombinant polypeptide segments have been used only in intermolecular native chemical ligation reactions.7 Here we describe a straighforward route to backbonecyclized recombinant polypeptides based on an intramolecular version of native chemical ligation (Figure 1). As a demonstration of our biosynthetic cyclization strategy, we attempted to cyclize the N-terminal SH3 domain from the c-Crk adaptor protein.8 This 57-residue protein domain possesses a globular structure9 composed of five !-strands which position the Nand C-termini in close proximity.10 A bacterial expression plasmid was prepared in which the gene corresponding to the SH3 domain (residues Y136 to Y190 of murine c-Crk) was cloned into a commercially available intein expression system.11 The SH3 domain was modified at the DNA level to append the sequence MIEGRC at the N-terminus and to add an extra Gly residue at the C-terminus. The MIEGRC motif contains a factor Xa proteolysis site and allows the generation of an N-terminal Cys upon in Vitro proteolysis.6,7e Importantly, this motif acts as a cysteine-protecting group, preventing premature ligation reactions.7e The addition of the C-terminal Gly residue was carried out to improve the kinetics of cyclization as well as to stabilize the formation of the new loop between the Nand C-termini.12 The recombinant fusion protein (1) was expressed in Escherichia coli to give, after affinity purification, a major component on SDS-PAGE with the correct apparent molecular weight (Figure 2A). A secondary band representing less than 10% of total protein was also detected. This was assigned as the intein-CBD protein fragment and is presumably due to some premature cleavage of the fusion protein (1). As illustrated in Figure 1, cyclization of the recombinant SH3 domain was achieved by simply treating the fusion protein (1) adsorbed on chitin beads with factor Xa protease at pH 7.2 for 10 h. This proteolysis step afforded N-terminal Cys fusion protein (2) which spontaneously reacted in an intramolecular fashion to yield the corresponding circular SH3 domain (3) with concomitant cleavage from the beads. As shown in Figure 2A and B, the cyclization process was extremely clean, allowing the circular SH3 domain (3) to be readily purified

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos.

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.

Synergistic effect of formulated plasmid and needle-free injection for genetic vaccines.

A plasmid-based gene expression system was complexed with protective, interactive, and non-condensing (PINC) polymer system and administered with Medi-Jector, a needle-free injection device (NFID), to achieve high and sustained levels of antigen-specific antibodies in blood circulation. Human growth hormone (hGH) or bacterial beta-galactosidase gene expression plasmids driven by a cytomegalovirus (CMV) promoter were formulated in saline or complexed with a PINC polymer, polyvinylpyrrolidone (PVP), and intramuscularly or subcutaneously administered into dogs and pigs using a 22-gauge needle or a NFID. The hGH-specific IgG titers in serum were measured by an ELISA. Beta-galactosidase expression was measured in injected muscles by an enzymatic assay or immunohistochemistry. The effect of NFID on DNA stability and topology was assessed by gel electrophoresis. Intramuscular (i.m.) or subcutaneous (s.c.) injection of a hGH expression plasmid pCMV-hGH (0.05-0.5 mg/kg) in dogs and pigs elicited antigen-specific IgG antibody titers to expressed hGH. With both routes of injection, pDNA delivery by a NFID was superior to pDNA injection by needle. The magnitude of hGH-specific IgG titers with NFID was 15-20-fold higher than needle injection when pDNA was complexed with PVP, and only 3-4-fold higher with pDNA in saline. The transfection efficiency in the injected muscle, as measured by beta-galactosidase expression, following i.m. injection of pCMV-betagalactosidase/PVP, was not significantly different between needle and NFID-injected groups. These data demonstrate that the combination of pDNA/ PVP complexes and a NFID act synergistically to achieve high and sustained levels of antigen-specific IgG response to expressed antigen. This gene delivery approach may offer advantage over needle injection of naked DNA for the development of genetic vaccines.

A Novel Assay Reveals a Role for SolubleN-Ethylmaleimide-sensitive Fusion Attachment Protein in Mannose 6-Phosphate Receptor Transport from Endosomes to the Trans Golgi Network

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.

Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis.

The complete protein-coding region of the human relaxin-like factor (RLF; formerly Ley-I-L) was cloned by reverse transcription-polymerase chain reaction from human testis and subcloned into a bacterial expression plasmid for the production of recombinant human RLF in Escherichia coli. Polyclonal antibodies were raised against the recombinant RLF, as well as against a peptide epitope from the B-domain of the RLF polypeptide. Antibodies were used for immunohistochemistry of Bouin-fixed, paraffin-embedded samples of human testis tissues. Specific immunoreactivity was located exclusively in the Leydig cells with a consistent high intensity of staining, showing similar spatial distribution to other Leydig cell markers, such as the luteinizing hormone (LH) receptor and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and to the pattern of RLF mRNA shown by in-situ transcript hybridization. In biopsy samples from patients with severe disturbances of spermatogenesis, RLF staining intensity was consistently high in all cases, unlike staining for 3 beta-HSD which varied considerably between patients. Immunostaining for RLF would thus appear to offer an interesting new marker for Leydig cells in human testis samples.

Expression and characterization of recombinant rat parathyroid hormone-related peptide (1–141) and an amino-terminally-truncated analogue (38–141)

We have synthesized and purified recombinant parathyroid hormone related peptide (PTHrP (1–141)) and PTHrP (38–141) using an E. coli system that requires minimal purification. The cDNAs encoding PTHrP (1–141) and PTHrP (35–141) respectively were inserted into the multiple cloning site of the pTrcHis-B bacterial expression plasmid. The PTHrP encoded sequences were thereby fused at their NH 2-termini to six histidine residues within the fusion protein. The recombinant plasmids were transfected into E. coli cells and PTHrP synthesis was induced by addition of 1 mM isopropyl- β- d-thiogalactopyranoside (IPTG) at 37°C. The recombinant fusion proteins were purified by binding of the histidine residues to a nickel column followed by gradient elusion and dialysis. PTHrP (1–141) was released from its fusion protein by cyanogen bromide cleavage, whereas PTHrP (38–141) was released by enzymatic digestion with enterokinase. This rapid isolation method resulted in pure PTHrP (1–141) and (38–141) as judged by SDS-polyacrylamide gel electrophoresis and NH 2-terminal sequence analysis. PTHrP (1–141) stimulated cAMP accumulation and mobilized intracellular calcium ([Ca 2+] i) in UMR106 osteoblast-like cells, and stimulated phosphate transport in OK/E renal cells, whereas PTHrP (38–141) was inert in these bioassays. Availability of PTHrP and its NH 2-terminally truncated analogue, which lacks the sequence necessary for its hypercalcemic actions, will enable their biological activities to be examined in greater detail.

Molecular characterization of xyn3, a member of the endoxylanase multigene family of the rumen anaerobic fungus Neocallimastix frontalis.

Different cDNAs designated xyn3 and xyn4 were isolated from an expression library of the anaerobic rumen fungus Neocallimastix frontalis. Xyn3 was further characterized and was shown to contain a single open reading frame of 1821 bp coding for a protein, XYN3, of 607 amino acids (Mr 66 000). The predicted primary structure of XYN3 consisted of two large reiterated regions of 223 amino acids with a high degree of identity (88.3%). Each domain of XYN3, XYN3A and XYN3B, showed significant homology with fungal and bacterial xylanases belonging to the endoxylanase family 11. XYN3 and XYN3A were cloned in a bacterial expression plasmid harbouring a 6 His-C terminal tag and the recombinant proteins XYN3 and XYN3A were purified from Escherichia coli. The recombinant proteins had Mr of 66 800 and 34 000 respectively and hydrolysed xylan to xylo-oligosaccharides. Analysis of truncated forms of XYN3 confirmed that the full-length protein contained two catalytic domains displaying similar substrate specificity. Western-blot analysis using antiserum raised against XYN3A showed that the N. frontalis xylanase was not submitted to post-translational maturation. XYN3A antiserum recognized similar polypeptides in the culture medium of two other rumen fungi, Piromyces rhizinflata and Caecomyces communis.

Mutagenesis study to ameliorate the bacterial expression of phosphoprotein P of vesicular stomatitis virus

The phosphoprotein gene of vesicular stomatitis virus, a Rhabdovirus, has been inserted into bacterial expression plasmids containing the Escherichia coli tac promoter and ribosome binding site (RBS). A low level of expression of the protein was detected. Sequence analysis showed the presence of 15 nucleotides in the spacer region i.e., between the Shine- Dalgarno sequence and ATG. Alteration of the distance and the sequence in the spacer region by oligonucleotide-directed mutagenesis revealed a correlation among the expression levels, acces- sibility of the RBS and requirement for a minimum spacing of at least 7 nucleotides between the Shine-Dalgarno sequence and ATG for optimal gene expression.