Megumi Kosaka

Human Betacellulin, a Member of the EGF Family Dominantly Expressed in Pancreas and Small Intestine, is Fully Active in a Monomeric Form

Betacellulin (BTC) was found to be expressed mainly in human pancreas and small intestine. This finding suggests that BTC possesses some specific function distinguished from the other members of epidermal growth factor (EGF) family. To clarify this function, the released form of human BTC has been expressed in E.coli, purified, and characterized. The recombinant human BTC was produced as an inclusion body. This material was dissolved in guanidine-HCl under reducing conditions, refolded, and purified through sequential liquid chromatography. Purified BTC was electrophoresed under reducing conditions and a molecular size of 18 kDa was determined, which is the supposed size of a dimer of the peptide. However, chemical analysis failed to show a covalently linked dimer. The molecular mass of BTC analyzed by mass spectrometry revealed it to be 9 kDa, which is consistent with theoretical value for a monomer. Recombinant BTC showed growth promoting activity for mouse fibroblasts and rat aortic smooth muscle cells which was equivalent to EGF On the other hand, BTC was found to exhibit a growth inhibitory effect on the cells overexpressing EGF receptor.

Purification and Characterization of a Recombinant Human Cripto-1 Protein

Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors. To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E. coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein. We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites. Expression of the modified rhCR-1 protein in E. coli was limited to the inclusion bodies. The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence. Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation. By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethylammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent. This modification facilitates solubilization of the protein when rhCR-1 is denatured. The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer. The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit beta-casein expression, to stimulate the tyrosine phosphorylation of Shc and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper‐like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild‐type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L‐RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X‐ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L‐RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L‐ and 2L‐RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L‐RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L‐RNase 1) was also evaluated. N4L‐RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper‐like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates.

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

Molecular cloning and expression of human ribonuclease 4 cDNA

A cDNA coding for human ribonuclease 4 was isolated from a pancreas cDNA library and sequenced. This cDNA (996 bp) includes an entire open reading frame encoding mature protein (119 aa) following signal peptide (28 aa). Expression of mature protein in Escherichia coli showed an apparent molecular mass of about 16 kDa, which was slightly lower than the mature form of human RNase 1, in SDS-PAGE.

Field methane tracking with a portable and real-time open-gas monitor based on a cw-driven Pb-salt diode laser

Abstract An atmospheric gas monitor with a PbSnTe diode laser has been developed. The open, 1 m optical path in a single-folded configuration enables local and non-contact measurement in a portable instrument. The accuracy is dependent on optical noise and a technique in diode-current modulation was effective in suppressing it. Analysis of the system noise based on the experimental data was made and a 10 ms interval was sufficient to attain an accuracy of 3 ppm for methane. Field measurements were made and a rapid fluctuation in the density of atmospheric methane density was recorded.

Analytical Description of Tunable Diode Laser Derivative Spectrometry

The instrument functions are investigated, and a quantitative description is given of how a second-derivative spectrometer employing a tunable diode laser attains the power to suppress spectral noise. As a result, it is found that the power can be improved by modifying the source-frequency modulation profile. The essence of this approach is to make the profile at its extreme as sharp as possible, and a hyperbolic sine profile with suitable parameters is proposed as the optimal profile that can be attained in practice.

Transient cell proliferation with polyethylenimine-cationized N-terminal domain of simian virus 40 large T-antigen.

Polyethylenimine (PEI) cationization is a powerful strategy for protein transduction into cells. In this study, we attempted the artificial regulation of cell proliferation by protein transduction of the N-terminal domain (1-132 amino acids) of the simian virus 40 large T-antigen (SVLT-N), which inactivates retinoblastoma family proteins but not p53. To deliver SVLT-N into cells, we employed an indirect cationization method by forming a complex of biotynylated SVLT-N through disulfide bonds (biotin-SS-SVLT-N) and PEI-cationized avidin (PEI600-avidin). Using this complex, SVLT-N was transduced into the nucleus of confluent and quiescent Balb/c 3T3 cells and was found to be complexed with a cellular target protein, pRb. Furthermore, SVLT-N transduction induced cell proliferation in spite of confluent conditions. Because SVLT-N thus transduced into cells gradually degraded and was not detectable after a 4-d incubation, transiently transformed cells were obtained by this method. These results suggest that oncogene protein transduction technology has great potential for in vitro regulation of cell proliferation.

High Sensitivity Short-Path Monitoring of Trace Gases Employing PbSnTe Tunable Diode Laser

A system for atmospheric trace gas monitoring is described. The absorption spectrum in the infrared region of a specific gas is involved. A lead-salt diode laser is employed for the tunable light source and the short optical path of less than one meter allows the equipment to be portable. Fast measurement can be performed thanks to be built-in microcomputer and specially developed electronic system. Reliability is achieved because a correlational algorithm is calculated by the computer avoiding the ambiguity of the absolute laser frequency. Gas-density data is obtained every few seconds. Experiments were executed for methane and a sensitivity of 0.3 ppmm with S/N=1 was attained being limited by parasitic etalon fringes. A higher sensitivity down to 0.01 ppmm is possible provided that a transversally mono-modal laser is employed.

Uniformly cationized protein efficiently reaches the cytosol of mammalian cells

Protein cationization techniques are powerful protein transduction methods for mammalian cells. As we demonstrated previously, cationized proteins with limited conjugation to polyethylenimine have excellent ability to enter into cells by adsorption-mediated endocytosis [Futami, J., et al. (2005) J. Biosci. Bioeng. 99, 95-103]. In this study, we show that proteins with extensive and uniform cationization covering the protein surface reach the cytoplasm and nucleus more effectively than proteins with limited cationic polymers or proteins that are fused to cationic peptides. Although extensive modification of carboxylates results in loss of protein function, chicken avidin retains biotin-binding ability even after extensive amidation of carboxylates. Using this cationized avidin carrier system, the protein transduction ability of variously cationized avidins was investigated using biotinylated protein as a probe. The results revealed that cationized avidins bind rapidly to the cell surface followed by endocytotic uptake. Small amounts of uniformly cationized avidin showed direct penetration into the cytoplasm within a 15 min incubation. This penetration route seemed to be energy dependent and functioned under cellular physiological conditions. A biotinylated exogenous transcription factor protein that penetrated cells was demonstrated to induce target gene expression in living cells.