Production of recombinant human bile salt-stimulated lipase in Pichia pastoris.

Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris. Human BSSL has 16 successively repeated sequences in the carboxy terminal region. The sequence consists of 11 amino acid residues. The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression. The clone used for fermentation was a transformant of GS115 (his4) integrated with four copies of the expression cassette containing the modified hBSSL cDNA. Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation. A sufficient glycerol feed at 30 degrees C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days. On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20 degrees C and pH 5.7. Under these conditions, 0.8-1 g of rhBSSL was secreted in 1 liter of the medium. By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction. The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography.

Comparison of three signals for secretory expression of recombinant human midkine in Pichia pastoris.

The secretion signals of Saccharomyces cerevisiae alpha mating factor, human midkine itself, and Pichia pastoris acid phosphatase, were tried for the expression of human midkine under the control of the AOX1 gene promoter in P. pastoris. Approximately 28 mg/l, 1.5 mg/l, and 0.2 mg/l of midkine were secreted by using the a mating factor pre-pro-sequence, the midkine signal sequence, and the phosphatase signal sequence in flask cultures, respectively.

An approach to the removal of yeast specific O-linked oligo-mannoses from human midkine expressed in Pichia pastoris using site-specific mutagenesis.

Human midkine is expressed and secreted in the medium under the control of an AOX1 gene promoter in Pichia pastoris using its own secretion signal. The midkine precursor is properly processed to yield the correct amino-terminus of mature midkine. However, more than half of the product receives yeast specific mannosylations. The sites for the mannosylations were determined to be the three threonine residues in the carboxy-terminal region of human midkine. In order to obtain non-mannosylated midkine, alanine residues were substituted for the three threonine residues by site specific mutagenesis. HPLC and mass spectrometry confirmed that the mutant midkine contained almost no mannose residues. Despite the amino acid substitutions in the carboxy-terminal region, mutant human midkine, promoted CHO cell proliferation as well as normal midkine.

Production of recombinant human midkine in yeast, Pichia pastoris.

Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of the AOX1 gene promoter. The expression of midkine was efficiently induced by methanol in a high cell density fermentation. Approximately 0.3 g/l culture of midkine accumulated in the cells by 72 h after induction. When the cells were disrupted, midkine was recovered in an insoluble form, and was insoluble even in the presence of 7 M urea. The precipitate was dissolved in the buffer solution (pH 8) containing 8 M guanidine hydrochloride, 10 mM dithiothreitol, 1 mM EDTA and 50 mM Tris-Cl, and then, midkine was renatured by dialysis at high concentration against the buffer solution (pH 8) containing 0.5 M sodium chloride and 20 mM Tris-Cl. The renatured midkine was recovered using a SP-Sepharose column, and purified further by Heparin-Sepharose column chromatography. Approximately 64 mg/l culture of the purified midkine was obtained. The amino acid sequence of amino-terminus and the amino acid composition of midkine were the same as those of Met-midkine that has a methionine residue at the amino-teminus. Mass spectrometry of purified Met-midkine showed a mass of 13370.7 Da (average), almost the theoretical mass for it. The Met-midkine enhanced the proliferation of Chinese hamster ovary (CHO) cells.

Molecular cloning of a cDNA and a gene encoding an immunomodulatory protein, Ling Zhi-8, from a fungus, Ganoderma lucidum.

A large amount of the novel immunomodulatory protein Ling Zhi-8 (LZ-8) is synthesized in the mycelia of Ganoderma lucidum (Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K., and Tsunoo, H. (1989) J. Biol. Chem. 264, 472-478). A cDNA and a gene for LZ-8 were isolated and characterized. The mixed oligonucleotide probes for LZ-8 cDNA were designed from the results of protein sequencing (Tanaka, S., Ko, K., Kino, K., Tsuchiya, K., Yamashita, A., Murasugi, A., Sakuma, S., and Tsunoo, H. (1989) J. Biol. Chem. 264, 16372-16377) and were used for screening the mycelial cDNA library. The nucleotide sequence of the cloned cDNA confirms the amino acid sequence of LZ-8 that was previously determined by protein sequencing. The clones containing the LZ-8 gene (lz-8) were obtained from the mycelial genomic DNA library using the cDNA probe. Two CCAAT-like sequences and one TATA box were found at the upstream region of the postulated transcription initiation site of lz-8. A small intron (61 nucleotides long) divided lz-8 into two exons at the 5'-untranslated region. The other characteristic sequences were also found around the postulated transcription initiation site and around the poly(A) additional site.

Complete amino acid sequence of an immunomodulatory protein, ling zhi-8 (LZ-8). An immunomodulator from a fungus, Ganoderma lucidium, having similarity to immunoglobulin variable regions.

The complete amino acid sequence of a novel immunomodulatory protein, ling zhi-8 (LZ-8), isolated from a fungus, Ganoderma lucidium (Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K., and Tsunoo, H. (1989) J. Biol. Chem. 264, 472-478), was determined by protein sequencing. The polypeptide consists of 110 amino acid residues with an acetylated amino end and has a molecular mass of 12,420 Da including an amino-end blocking group. There is no attachment site for an Asn-linked oligosaccharide chain, consistent with the very low carbohydrate content of LZ-8. These results indicate that the native form of LZ-8 with a molecular mass of 24 kDa is a homodimer of the LZ-8 polypeptide whose sequence is described here. Furthermore, the LZ-8 chain shows considerable similarity to the variable region of immunoglobulin heavy chain both in its sequence and in its predicted secondary structure. The interesting possibility that LZ-8 is related to an ancestral protein of the immunoglobulin superfamily is also discussed.

Discrimination and quantitative analysis of wild type and point mutant early region 1B genes of adenovirus using oligodeoxyribonucleotide hybridization probes.

One of the cytocidal (cyt) mutants of adenovirus 2 (Ad2), cyt15, has been shown to have two point mutations separated by 36 bases in its early region 1B (E1B) gene that encodes the 19K (Mr, 19,000) protein. A part of the cyt15 E1B gene, including one of the point mutations, was probed with a 23-base long oligodeoxyribonucleotide (ME1B23). Another oligonucleotide probe of the same length was used for the corresponding region of the wild type (wt) E1B gene (E1B23). Over the 32P-end labeled oligonucleotide probe, a 25-fold molar excess of a competitor (1) (the unlabeled oligonucleotide of each counterpart) was added to the hybridization mixture. The E1B gene with or without a point mutation could easily be distinguished from its counterpart and quantitated in the presence of its counterpart under the hybridization conditions employed. Under the stringent wash conditions, the E1B gene of wt Ad2 could be completely discriminated from even a 100-fold molar excess of cyt15 E1B gene with the 32P-labeled E1B23 probe. The wt E1B gene could also be quantitated in the presence of a 100-fold molar excess of the mutant E1B gene.

Unique properties of Cd-binding peptides induced in fission yeast, Schizosaccharomyces pombe.

Metallothioneins, a class of low molecular weight cysteine-rich proteins that bind heavy metal ions, have been found in various eucaryotic organisms. When fission yeasts are grown in the presence of high concentration of CdCl2, large amounts of Cd-binding peptides (Cd-BP1 and Cd-BP2) are synthesized. Cd-BP1 (MW 4000) contains 4 mole of small unit peptide (cadystin, MW 771), 6 mole of Cd2+, and 1 mole of the labile sulfide; on the other hand, Cd-BP2 (MW 1800) contains 2 mole of cadystin and 2 mole of Cd2+. While Cd-BP2 shows similarities to mammalian Cd-thioneins in UV and CD spectra, Cd-BP1 has a characteristic shoulder at 265 nm in the UV absorption spectrum and shows two marked Cotton bands at 257 nm (negative) and 275 nm (positive). These characteristics of Cd-BP1 are not found in the other Cd-thioneins. When Cd-BP1 is acidified (pH 2.0) and successively neutralized, a shoulder of 265 nm in the UV spectrum and a Cotton band at 275 nm disappear, and the molecular weight changes from 4000 to 1800, with simultaneous loss of the labile sulfide. While the reconstituted complex without labile sulfide showed the characteristics of Cd-BP2, the reconstituted complex in the presence of labile sulfide indicated partial reconstitution of Cd-BP1. The UV and CD spectra differences between reconstituted and native Cd-BP1 suggest the requirement for some additional molecular architecture including another peptide-Cd2+ interaction. Induction of cadystin synthesis is almost exclusive for Cd, but an exception is a small amount of cadystin also induced by the higher concentration of CuCl2 (2.5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Application of synthetic oligonucleotides to the diagnosis of human genetic diseases.

Under appropriate conditions synthetic oligonucleotide hybridization probes display essentially absolute hybridization specificity. That is, every nucleotide must form a Watson-Crick base pair in order that the probe forms a stable duplex. All of the non-Watson-Crick base pairs, including G-T, have a destabilizing effect. Thus, it is possible to choose stringent conditions of hybridization such that, while a perfectly matched duplex between an oligonucleotide and complementary DNA will form, duplexes mismatched at one or more position will not. Mutations in a single base in the DNA sequence of a gene can and do result in genetic diseases. The hybridization of oligonucleotides to the region of DNA containing these base changes would be affected by the mutations and thus, oligonucleotide hybridization provides a means of detecting single base changes. In an attempt to develop a non-radioactive method for the detection of human genetic diseases, we have prepared biotinylated-oligonucleotides by an enzymatic method. An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3'-terminus was prepared by a primer extention reaction using E. coli DNA polymerase I (Klenow fragment). The probe could be specifically and tightly bound with Avidin D in 1 M NaCl. It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing 5 non-consecutive non-complementary bases. The hybridized biotinylated probe could be visualized by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmol) was used. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3' end is described.

Formation of cadmium-binding peptide allomorphs in fission yeast.

It has been reported that two kinds of Cd-binding peptide (Cd-BP1 and Cd-BP2) are induced in fission yeast upon exposure to Cd, and that they consist of the same unit peptide (cadystin), but Cd-BP1 binds 1.5 times more Cd atoms per cadystin than Cd-BP2 (Murasugi, A., Wada, C., & Hayashi, Y. (1981) J. Biochem. 90, 1561-1564). The relative amount of each allomorphic Cd-BP in the cell varied with time after induction and with the concentration of Cd in the induction medium. Further, the production of acid-labile sulfide in the cell increased greatly upon exposure to Cd and varied with time after Cd addition and with Cd concentration in the medium, as in the case of Cd-BP1. Since Cd-BP1 contains labile sulfide, the increase of labile sulfide production together with the increase of cellular Cd concentration may be the driving force to form Cd-BP1, resulting in the increase of the relative amount of Cd-BP1.

Biotin-labeled oligonucleotides: enzymatic synthesis and use as hybridization probes.

An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3' terminus was prepared by a primer extention reaction using Escherichia coli DNA polymerase I (Klenow fragment). For efficient synthesis of the probe, it was necessary to add about 16-fold molar excess of the template oligonucleotide (pentadecanucleotide) to the primer oligonucleotide (nonadecanucleotide) in the reaction mixture and to continue the reaction for 2.5 hr at 4 degrees C. The probe was purified by polyacrylamide gel electrophoresis under denaturing conditions. The probe could be specifically and tightly bound with Avidin D (Vector Laboratories) in 1 M NaCl. It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing five consecutive noncomplementary bases. The hybridized biotinylated probe could be detected by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmoles) was used. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3' end is described.

Occurrence of acid-labile sulfide in cadmium-binding peptide 1 from fission yeast.

Two kinds of Cd-binding peptides (Cd-BP1 and Cd-BP2) are induced in fission yeast upon addition of CdCl2 to the culture medium (l). It was also reported that Cd-BP1 and Cd-BP2 consisted of the same components, unit peptides (Cys3, Glu3, Gly1) and Cd atoms, though the respective amounts of components in each molecule were different (1, 2). Now, we have found that Cd-BP1 contains about 1 mol of acid-labile sulfide per mol, and Cd-BP2 contains no labile sulfide. The existence of the labile sulfide explains the unique physicochemical characteristics of Cd-BP1. Since acid-labile sulfide has not been found in metallothioneins or other metallothionein-like metal-binding proteins, the occurrence of labile sulfide in Cd-BP1 is the first instance in this field.

Cadmium-binding peptide induced in fission yeast, Schizosaccharomyces pombe.

When S. pombe is cultured in a medium containing a high concentration of CdCl2 (1 mM), it grows for over 20 h accumulating Cd2+ in the cells. Simultaneously, Cd-binding peptides (Cd-BP1 and -BP2) are synthesized, and accumulated depending on the time after addition of Cd2+ to the culture medium. Apparent molecular weights of Cd-BP1 and -BP2 are 4,000 and 1,800, respectively. Both Cd-BPs are composed of common unit peptides, confirmed by chemical and physicochemical analyses.