Masato Kuriyama

A novel procedure for the preparation of biologically active recombinant peptides using a cyanylation reaction

A cysteine specific cleavage reaction was used for the preparation of biologically active peptides from recombinant fusion proteins. The fusion protein through cysteine was prepared by a recombinant DNA technology and then treated with cyanylating reagents such as 2-nitro-5-thiocyanatobenzoic acid (NTCB) and 1-cyano-4-(dimethylamino) pyridinium tetrafluoroborate (DMAP-CN) to release the desired product. As an example, we have selected a glucagon-like peptide 1 (7-37) (termed insulinotropin). We constructed an expression vector for a fusion protein in which insulinotropin and human basic fibroblast growth factor (hbFGF) mutein (abbreviated as CS 23) are connected by cysteine and then expressed it in Escherichia coli cells. The fusion protein, after refolding, was purified by heparin affinity chromatography, since CS23 has a strong affinity for heparin. The affinity-purified fusion protein was treated with NTCB or DMAP-CN to give crude insulinotropin, which was then purified by reversed phase (rp) high-performance liquid chromatography (HPLC). From various criteria such as amino acid analysis, amino acid sequence and the biological activity, the purified material obtained was found to be methionylated insulinotropin (Met-insulinotropin) with full activity. The specificity and simplicity of the present method make it versatile and convenient for the preparation of biologically active peptides.

Secretion of active human lysozyme by Acremonium chrysogenum using a Fusarium alkaline protease promoter system

We constructed expression vectors for Acremonium chrysogenum using a Fusarium alkaline protease promoter region and tested their potential as secretion systems for foreign proteins using the human (h)-lysozyme gene as an indicator. The gene encoding h-lysozyme was linked to the coding region of (1) the carboxy terminal of the alkaline protease pre peptide, (2) the carboxy terminal of the prepro peptide, (3) three amino acids of the mature protein preceded by the prepro peptide and (4) the carboxy terminal of chicken lysozyme signal peptide, inserted into the genomic DNAs of A. chrysogenum and expressed under the control of the alkaline protease promoter. The transformants of A. chrysogenum with each of these plasmids secreted enzymatically active h-lysozyme. A maximum yield in excess of 40 mg l-1 was obtained when h-lysozyme was linked to the carboxy terminal of alkaline protease prepro peptide. The majority of the amino terminal sequence of the purified h-lysozyme from the culture supernatant was identical with that of authentic h-lysozyme, but it showed some heterogeneity.

Controlled expression of human basic fibroblast growth factor mutein CS23 in Escherichia coli under a bacteriophage T7 promoter

A DNA sequence coding for human basic fibroblast growth factor (hbFGF) mutein CS23 was inserted downstream of a T7 promoter in pBR322. The plasmid was introduced into Escherichia coli MM294 lysogenized with a bacteriophage λ having the T7 RNA polymerase gene under the control of the lacUV5 promoter. When 0.4 mM IPTG was added to induce gene expression in this system, the accumulation of hbFGF mutein CS23 was very rapid at the beginning, but it quickly stopped. Therefore, the total amount was low, and most of the product was inactive due to the formation of inclusion bodies. By controlling the expression of the T7 RNA polymerase at an appropriate level, we succeeded in accumulating a large amount of the mutein in the cells, all in a soluble and active form. By adding 0.04 mM IPTG, more than 1.5 g per liter of hbFGF mutein CS23 was accumulated under optimum culture conditions.

Stabilization of a recombinant plasmid in yeast

Abstract When Saccharomyces cerevisiae AH22R − -2075 harboring pGLD p31-RcT which encodes a modified hepatitis B virus-surface antigen P31 (rHBsAg P31 mutien) coding gene and β-isopropylmalate dehydrogenase gene ( LEU2 ) was cultivated on a large scale in a leucine-lacking medium, rHBsAg non-producing colonies appeared at high frequency. Restriction enzyme analysis and Southern blot analysis with LEU2 probe revealed that the cells from these non-producing colonies had lost the expression plasmid, pGLD p31-RcT, and had unusual 2 μm DNA containing the LEU2 gene. This was caused by homologous recombination between 2 μm DNA and pGLD p31-RcT which contains a 2 μm DNA fragment and the LEU2 gene. Stabilization of the recombinant plasmid was achieved by the following three approaches: (i) reconstruction of the expression plasmid by separating the LEU2 gene from the 2 μm DNA fragment on the plasmid; (ii) selection of plasmid-stabilized clones by taking advantage of the differences in growth rate between producing and non-producing clones; and (iii) addition of a high concentration of l -histidine to the medium.

High-level expression of human fibroblast growth factor-9 N33 in Escherichia coli

Abstract We constructed plasmids carrying the human fibroblast growth factor-9 N33 (hFGF-9 N33) gene under the control of the T7 promoter in pBR322. These plasmids were introduced into Escherichia coli MM294 lysogenized with bacteriophage λ with the T7 RNA polymerase gene under the control of the lacUV5 promoter. We compared the expression level of hFGF-9 N33 in the transformed E. coli MM294(DE3) pETGAF25 and MM294(DE3) pTG931 which are ampicillin- and tetracycline-resistant strains, respectively. Twice as much hFGF-9 N33 was produced by MM294(DE3) pTG931 as by MM294(DE3) pETGAF25 , although the growth rates of these strains were similar. The expression plasmid pTG941, a derivative of pTG931, was constructed by reduring the size of the long non-coding region between the stop codon of the hFGF-9 N33 gene and the T7 terminator. The amount of hFGF-9 N33 expressed in MM294(DE3) pTG941 was twice that expressed in MM294(DE3) pTG931 , although the growth rates of these strains were similar. The expression level reached a maximum when 10 mg/l of isopropyl β- d -thiogalactopyranoside, an inducer, was added to the culture at 250 Klett units of growth. Feeding glucose to the culture increased the final growth level and caused a 40% increase in the amount of hFGF-9 N33 produced. Over 0.5 g/l of hFGF-9 N33 was produced in a large-scale culture by improving the expression plasmids and optimizing the culture conditions.

A human-human hybridoma secreting anti-Pseudomonas aeruginosa exotoxin-A monoclonal antibody with highly potent neutralizing activity

A hybridoma secreting human monoclonal antibody (MAB) against Pseudomonas aeruginosa exotoxin A (PEA) was constructed by fusing Epstein-Barr virus-transformed peripheral blood lymphocytes with human B lymphoblastoid cell line TAW-925. The human-human hybridoma stably produced human IgG2 MAB at the rate of 0.4–0.5 μg/ml per 106 cells per day for more than six months, and the MAB was capable of neutralizing the in vitro cytotoxic and in vivo lethal effects of PEA with approximately 100-and 70-fold, respectively, higher activity than serum polyclonal antibody preparations.

Initiation methionine of recombinant interleukin-2 is completely processed in vivo by replacement of the second proline residue

A portion of the recombinant interleukin-2 (rIL-2) produced in Escherichia coli had an amino (N-) terminal methionine. We found that this partial processing of the N-terminal methionine was caused by the proline residue, which is the second amino acid of IL-2. Alanine or histidine was substituted for proline by site-directed mutagenesis. There were no marked differences between each mutein and rIL-2 in the amount of product, accumulation states or bioactivity. However, these muteins had no additional methionine at the amino terminus.