Satoru Misawa

Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies.

Overexpression of cloned or synthetic genes in Escherichia coli often results in the formation of insoluble protein inclusion bodies. Within the last decade, specific methods and strategies have been developed for preparing active recombinant proteins from these inclusion bodies. Usually, the inclusion bodies can be separated easily from other cell components by centrifugation, solubilized by denaturants such as guanidine hydrochloride (Gdn-HCl) or urea, and then renatured through a refolding process such as dilution or dialysis. Recent improvements in renaturation procedures have included the inhibition of aggregation during refolding by application of low molecular weight additives and matrix-bound renaturation. These methods have made it possible to obtain high yields of biologically active proteins by taking into account process parameters such as protein concentration, redox conditions, temperature, pH, and ionic strength.

High-level expression of Mycoplasma arginine deiminase in Escherichia coli and its efficient renaturation as an anti-tumor enzyme.

The arginine deiminase (AD) gene was cloned from Mycoplasma arginini and expressed in the cytosol of Escherichia coli as inclusion bodies with an expression level of at least 20% of the total bacterial proteins. The inclusion bodies were solubilized with 6 M guanidine hydrochloride (Gdn-HCl) under reducing conditions, in order to avoid incorrect disulfide-bond formation of the recombinant (r-) AD molecules, and renaturation was performed under various refolding conditions. The optimum renaturation conditions were found to be incubation for 90 h at pH 7.5 and 15 degrees C. The resulting completely refolded r-AD was purified to homogeneity by anion-exchange and arginine-affinity chromatography and its activity yield was 72.5%. The specific activity of the purified r-AD was comparable to and its amino acid composition was identical to those of Mycoplasma AD, and NH2-terminal sequence analysis revealed that its methionine residue corresponding to the translation initiation codon had been removed completely. Anti-tumor activity analyses showed that r-AD inhibited the growth of two mouse cell lines, hepatoma MH134 and fibrosarcoma Meth A, strongly in vitro at concentrations in excess of 10 ng ml-1. Moreover, when MH134-implanted mice were given single intravenous injections of r-AD at doses of 50 mg kg-1 and higher, their survival times were prolonged significantly. These results, taken together, indicate that the enzymatic properties and biological actions of r-AD were highly consistent with those of Mycoplasma AD.

Chemical modification by polyethylene glycol of the anti-tumor enzyme arginine deiminase from Mycoplasma arginini.

Ammo acid‐degrading enzymes are known to inhibit the growth of tumor cells in culture by depleting amino acids in the medium. Here we demonstrate that arginine deiminase (EC 3.5.3.6) from Mycoplasma arginini had stronger growth‐inhibitory activity against all 4 kinds of tumor cell lines tested than L‐asparaginase and arginase, which are well‐known anti‐tumor enzymes. Next, chemical modification of the arginine deiminase molecule with polyethylene glycol was shown to enhance its potency as an anti‐tumor enzyme. The percentage of modified amino groups per molecule was estimated to be 51% of the total amino groups, and the average molecular weight was estimated to be about 400,000 by gel‐filtration HPLC. The enzymic activity of the modified enzyme was 25.5 units/mg protein, which was equivalent to 57% of that of the native enzyme. The modified enzyme strongly inhibited growth of a mouse hepatoma cell line, MH134, at a concentration of more than 10 ng/ml, showing almost the same dose‐response curve as the native enzyme. When a bolus of 5 units of the modified enzyme was intravenously injected into male BDF1 mice, L‐arginine in the blood completely disappeared within 5 rain, and remained undetectable for more than 8 days. On the other hand, in the case of bolus injection of the same number of units of native enzyme, the plasma L‐arginine level recovered up to 66% of the control level at 8 days. These results suggest that this modified enzyme has a longer plasma clearance time and may be more effective as a new anti‐tumor agent than the native enzyme.

On-column refolding and characterization of soluble human interleukin-15 receptor α-chain produced in Escherichia coli

Interleukin-15 receptor alpha-chain (IL-15Ralpha) is a member of the new cytokine receptor family, which possesses the sushi domain. To investigate the biochemical and biophysical characteristics of soluble human IL-15Ralpha (shIL-15Ralpha), shIL-15Ralpha was recombinantly expressed in Escherichia coli. The shIL-15Ralpha containing a six histidine-tag was expressed as inclusion bodies, which were solubilized with urea, immobilized on a Ni-nitrilotriacetic acid column, and refolded by a decreasing gradient of urea concentration. The refolded shIL-15Ralpha exhibited a highly flexible structure, neutralized human interleukin-15-induced cell proliferation effectively, and bound to its ligand with the same affinity as human IL-15Ralpha on the cell surface, as demonstrated by circular dichroism, a cell proliferation assay, and surface plasmon resonance, respectively. Thus, we succeeded in refolding shIL-15Ralpha to an active form on an affinity column.

Isolation and Characterization of Monoclonal Antibodies That Inhibit Hepatitis C Virus NS3 Protease

ABSTRACT A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (Ki = 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependentcis processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent transprocessing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.

Synthesis and structure–activity relationship of diarylamide urea derivatives as selective inhibitors of the proliferation of human coronary artery smooth muscle cells

A series of diarylamide urea derivatives were synthesized and evaluated for their inhibitory activities against human coronary artery smooth muscle cells (SMCs) and human coronary artery endothelial cells (ECs). Compound 2o was superior to the lead compound, Tranilast, in terms of its potency of the inhibitory activity and cell selectivity.

Synthesis and Structure-Activity Relationship of Diarylamide Derivatives as Selective Inhibitors of the Proliferation of Human Endothelial Cells

A series of diarylamide urea derivatives were synthesized and evaluated for their inhibitory activities against human coronary artery endothelial cells (ECs) and human coronary artery smooth muscle cells (SMCs). Compound was superior to Tranilast, in terms of both cell selectivity and the potency of its inhibitory activity toward the proliferation and angiogenesis of ECs.

Crystal structure of human dual specificity phosphatase, JNK stimulatory phosphatase‐1, at 1.5 Å resolution

Human JNK stimulatory phosphatase‐1 (JSP‐1) is a novel member of dual specificity phosphatases. A C‐terminus truncated JSP‐1 was expressed in Escherichia coli and was crystallized using the sitting‐drop vapor diffusion method. Thin‐plate crystals obtained at 278 K belong to a monoclinic space group, C2, with unit‐cell parameters a = 84.0 Å, b = 49.3 Å, c = 47.3 Å, and β = 119.5°, and diffract up to 1.5 Å resolution at 100 K. The structure of JSP‐1 has a single compact (α/β) domain, which consists of six α‐helices and five β‐strands, and shows a conserved structural scaffold in regard to both DSPs and PTPs. A cleft formed by a PTP‐loop at the active site is very shallow, and is occupied by one sulfonate compound, MES, at the bottom. In the binary complex structure of JSP‐1 with MES, the conformations of three important segments in regard to the catalytic mechanism are not similar to those in PTP1B. JSP‐1 has no loop corresponding to the Lys120‐loop of PTP1B, and tryptophan residue corresponding to the substrate‐stacking in PTP1B is substituted by alanine residue in JSP‐1. Proteins 2007.

Existence of β-methylnorleucine in recombinant hirudin produced by Escherichia coli

Abstract A gene encoding for hirudin, a potent thrombin inhibitor, was expressed in Escherichia coli , which is the most widely used host. When the recombinant hirudin analog, CX-397, was overproduced by E. coli (600 mg l −1 ) in the absence of nutrient amino acids in the culture medium, the presence of two derivatives in the final product was observed with extremely increased retention times on reverse-phase high-performance liquid chromatography. Each derivative was due to methylation of an isoleucine residue at Ile29 or Ile59 in the CX-397. The structure was deducible as β-methylnorleucine (βMeNle; (2 S ,3 S )-2-amino-3-methylhexanoic acid). The modification pathway of βMeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain. Additionally, βMeNle is synthesized by mutants of Serratia marcescens that belong to the same family, Enterobacteriaceae, as E. coli (J. Antibiot. 34 (1981a) 1278). These findings suggest that the lack of nutrient amino acids in the culture medium leads to the synthesis of βMeNle in E. coli , which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein.

Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4: tolerance of a CDR peptide to conformational changes of a target

We have synthesized and characterized peptides derived from complementarity‐determining regions (CDRs) of 8D4, a mouse monoclonal antibody against NS3 protease domain of hepatitis C virus. 8D4 inhibits enzymatic activity without its cofactor, NS4A peptide. One of the synthetic peptides derived from CDRs, CDR1 of the heavy‐chain (CDR‐H1) peptide strongly inhibited NS3 protease activity competitively in the absence of NS4A and non‐competitively in the presence of NS4A. Moreover, cyclic CDR‐H1 peptides bridged by disulfide inhibited NS3 protease more potently. The chain length of the CDR‐H1 peptide is critical for strong inhibition, even when the peptide is circularized. This finding suggests the importance of peptide conformation. In contrast to a cognate antibody molecule, CDR‐derived peptides may provide good ligands for target molecules by having a tolerance to conformational changes of the targets caused by cofactor binding or mutation.