Yuuki Morimoto

A novel protease obtained from FBS-containing culture supernatant, that processes single chain form hepatocyte growth factor to two chain form in serum-free culture

The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.

In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) to apolipoprotein AI in rabbits

In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) from E. coli to apolipoprotein (apo) AI was investigated. rh-Met-proapo AI was labeled with 125I, and then administered intravenously to rabbits. Blood was sampled periodically for 6 days. The plasma decay curves of radioiodinated rt-Met-proapo AI were similar to those of human mature apo AI (fractional catabolic rate (FCR); 1.018 +/- 0.090/day vs. 0.976 1 0.031/day, respectively). In vivo conversion of rh-Met-proapo AI to mature apo AI was examined by autoradiography of the isoelectric focusing (IEF) slab gel, i.e., the HDL fraction from each sampling point was semiquantitatively applied to IEF. It was found that the radioactivity of rh-Met-proapo AI migrated to more acidic isoproteins, the conversion was complete within 24 h, and the FCR of rh-Met-proapo AI was 9.20 +/- 1.34/day. Although the plasma decay curves of both human pro (rh-Met-proapo AI) and mature apo AI were significantly steeper than those of rabbit mature apo AI4 and apo AI5 (FCR; 0.703 +/- 0.027/day and 0.795 +/- 0.031/day, respectively), the conversion rate of human rt-Met-proapo AI to mature apo AI in rabbit was assumed to be 1:1. In vitro incubation of rh-Met-proapo AI with rabbit serum produced mature apo AI isoproteins, as determined by the apo AI immunoblotting method. Prediction of the amino acid sequence at the NH2 terminus of rabbit proapo AI showed that the prosegment consisted of an alpha helix with a high probability of a beta turn at Pro9, which is close to that in humans. Thus, (1) the proteolytic cleavage of proapo AI is an extracellular event, (2) the converting enzyme in rabbits can also process human proapo AI, (3) this converting enzyme does not specifically and directly attack the Gln6-Asp7 bond which links the carboxyl-terminal residue of the hexapeptide to the amino-terminal residue of human mature apo AI. The conformation of proapo AI at the NH2 terminus (alpha helix of the prosegment and a beta turn at Pro9) may have a key role in this cleavage, and (4) the examination of rh-Met-proapo AI in rabbits helps to explain the early events of HDL biogenesis.

High-level secretion of human apolipoprotein E produced in Escherichia coli : use of a secretion plasmid containing tandemly polymerized ompF-hybrid gene

A gene encoding the mature form of human apolipoprotein E (h-apoE) was fused to the secretion signal coding sequence of the Escherichia coli major outer membrane protein F (ompF) which was preceded by a consensus Shine-Dalgarno sequence. Two copies of this hybrid gene were inserted tandemly into an expression vector and expressed in E. coli under the transcriptional control of two tac promoters regulated by lac repressors. By the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to the growth media, cells synthesized h-apoE at the level of 27.2 micrograms per A600 and up to 22% of the total cellular protein. The h-apoE produced by E. coli was processed precisely, secreted into the periplasmic space and formed protein aggregates there. However, despite aggregation, they were easily dissolved in water and actively formed protein-lipid complexes with dimyristoyl phosphatidyl choline (DMPC). These results demonstrated that E. coli cells are able to synthesize and secrete a large amount of active h-apoE using a prokaryotic signal sequence.

High level production of human proapo A-I by fed-batch culture of recombinant Escherichia coli

Abstract Three Escherichia coli strains, two recA − strains (DH1 and YK537) and one recA + strain (KS476) harboring human proapo A-I expression plasmid pUS(pAI), were cultivated in fed-batch mode using a synthetic medium and the amounts of human proapo A-I accumulation were compared under various cultivation conditions. In the expression plasmid, nine proapo A-I genes were tandemly ligated downstream of the tac promoter. Experimental results indicated that selection of the host strain and cultivation temperature was important. Among the three E. coli strains checked, strain DH1 yielded the most effective production of human proapo A-I at 30°C.

High-level production and secretion of a mouse-human chimeric Fab fragment with specificity to human carcino embryonic antigen in Escherichia coli

A high-level secretion system for the production of mouse-human chimeric antibody 21B2 (MHC 21B2) Fab fragment specific for human carcino embryonic antigen (hCEA) in Escherichia coli has been constructed. The genes encoding a light chain and an Fd fragment (a variable region and the CH1 domain of a heavy chain) of a mouse-human chimeric antibody were directly fused to the signal peptide of the E. coli ompF gene sequence. E. coli cells containing expression vectors in which each of the two genes are located downstream of a separate tac promoter were able to secrete the light chain and Fd fragment as two of their major cellular proteins. The signal peptides were efficiently removed from the primary products by post-translational processing, although they formed insoluble aggregates, possibly in the periplasm. In high-cell-density culture experiments using a jar fermentor, the amount of light chain and Fd fragment produced was at levels of up to 2.88 g/l and 1.28 g/l culture, respectively. By optimizing the conditions that encourage correct folding, formation of disulphide bonds, and association of the light chain with the Fd fragment, we have established a procedure that can purify, refold, and combine aggregated products to electrophoretically homogeneous Fab fragment with a yield of approximately 47%. Fab fragment produced in this manner shows essentially the same antigen-binding activity and specificity to hCEA as the parental mouse antibody 21B2 (MoAb 21B2).

High-level expression of human proapolipoprotein A-I in Escherichia coli using expression plasmids containing tandemly polymerized proapolipoprotein A-I structural genes

SummaryA gene coding human proapolipoprotein A-I (proapo A-I) was inserted into a plasmid with a consensus ribosome binding sequence in Escherichia coli. One to three copies of this gene were tandemly inserted to construct proapo A-I expression plasmids, pMTpAI, pMT(pAI)2 and pMT(pAI)3, respectively. The cells harbouring pMT(pAI)3, could produce proapo A-I at a level of 49 μg/ml A600 and up to approx. 48% of the total cellular protein. The product was soluble in E. coli, and formed protein-lipid complexes with dimyristoyl phosphatidyl choline, which formed stacked disc structures. Analyses of the rec proapo A-I formed in the bacteria was identical to human proapo A-I except for methionine at the N-terminus.

Effects of substitutions of glycine and asparagine for serine132 on activity and binding of human lipoprotein lipase to very low density lipoproteins

For studying the role of Ser132 in the putative catalytic site of human lipoprotein lipase (LPL), mutant LPL cDNAs expressing LPLs with amino acid substitutions of Gly or Asn for Ser132 were obtained by site‐directed mutagenesis, and were expressed in COS‐1 cells. Considerable amounts of LPL enzyme protein mass were detected in the culture medium of COS‐1 cells transfected with wild‐type LPL, LPL‐Gly132, or LPL‐Asn132. LPL‐Gly132 hydrolyzed Triton X‐100‐triolein and tributyrin as effectively as wild‐type LPL, whereas LPL‐Asn132 showed no activity. LPL‐Asn132 bound to very low density lipoproteins as effectively as wild‐type LPL.

High level production of 3-cyano-6-hydroxypyridine from 3-cyanopyridine by Comamonas testosteroni MCI2848

Summary We optimized culture and reaction conditions for the production of 3-cyano-6-hydroxypyridine (6-CHP) from 3-cyanopyridine (3-CP) by resting cells of Comamonas testosteroni MCI2848. High reaction activity was induced by the addition of 6-halo-nicotinic acid, as inducer, to the culture medium. The productivity by the intact cells was increased by addition of nicotinic acid or 6-hydroxy nicotinic acid to the reaction mixture. Intact cells of this strain incubated at 30°C in an optimal reaction mixture containing 5 mM sodium nicotinate produced 57.2 g/l of 6-CHP with 100% reaction-selectivity for 40 h.

Identification and Analysis of The Hepatocyte Growth Factor Activator

The recombinant human hepatocyte growth factor(r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA(r-CHO) was the two chain form in fetal bovine serum(FBS) containing culture. However, in serum-free culture, single chain form HGF was detected. We purified the protease from the culture supernatant that proteolytically processed single chain r-hHGF to two chain active form r-hHGF. When this protease was added to serum-free culture of the r-CHO cells, the proteolytic processing was completely achieved. Then we purified from human serum a novel serine protease(HGF activator) responsible for this process, and cloned cDNA from human liver cDNA library. The nucleotide sequence showed the HGF activator to consist of multiple domains homologous to those in the blood coagulation factor XII. Then from human plasma we purified the precursor of HGF activator which did not activate single chain HGF. The precursor was cleaved in vitro by thrombin into active form. It led us to conclude that HGF activator is present in plasma as an inactive zymogen and that the zymogen is activated by thrombin.