Tatsurou Shibui

Secretion of a functional Fab fragment in Escherichia coli and the influence of culture conditions

SummaryGenes encoding a light chain and an Fd region (a variable region and a CH1 domain of a heavy chain) of a mouse-human chimeric antibody with specificity for human carcinoembryonic antigen (CEA) were fused to a DNA segment coding for the signal peptide of Escherichia coli ompF. E. coli cells harbouring an expression vector containing these genes downstream of a tac promoter were able to secrete a Fab fragment of the antibody efficiently. When the cells were cultured at 37° C and the inducer (isopropyl-\-d-thiogalactopyranoside, IPTG) concentration was 1 mm (standard conditions), production of functional Fab was very low (medium; 200 ng/l culture and periplasm; <90 ng/l culture). In order to optimize functional Fab production, we examined the influence of culture conditions (i.e. temperature and the inducer concentration) on secretion of the product. It was found that a 12.7-fold higher amount of Fab fragment could be produced at 30° C using 0.1 mm IPTG, as compared with standard conditions. Under these optimal conditions, functional Fab accumulated in the periplasm and culture medium for 10 h after induction and the total production level was found to reach approximately 4.5 mg/l culture.

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 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).

Periplasmic production of human pancreatic prokallikrein in Escherichia coli

SummaryThe human pancreatic prokallikrein gene has been fused to the DNA sequence coding for the signal peptide of the Escherichia coli major outer membrane protein F (OmpF) and expressed under the control of tac promoter in E. coli. By induction with isopropyl-β-d-thiogalactopyranoside, the cells produced prokallikrein very efficiently. The fused OmpF signal peptide was verified as being processed correctly at the cleavage site of the OmpF signal peptide, and the N-terminal amino acid sequence of the product was found to be identical to that of native human prokallikrein. However, the prokallikrein produced by E. coli formed insoluble aggregates and was always collected in the insoluble fraction. An electron micrograph of prokallikrein-producing cells indicated that the prokallikrein was secreted into the periplasmic space and formed insoluble inclusion bodies there. By treating the insoluble inclusion bodies with oxidized and reduced glutathione in 1 M guanidine-HCl solution, a portion of them could be solubilized in water and showed kallikrein activity of 8 units (approx. 264 μg kallikrein) per litre of culture by trypsin activation.

cloning, sequencing and expression in Escherichia coli of cDNA for a non-A, non-B hepatitis-associated microtubular aggregates protein

A 1.7 kb cDNA encoding a novel antigen (p44; apparent Mr 44K) associated with non-A, non-B (NANB) hepatitis, was isolated from the hepatic cDNA library of a chimpanzee infected with NANB hepatitis. The library was screened with a monoclonal antibody against this antigen. The cDNA cloned contained an open reading frame encoding a 444 amino acid protein with an Mr calculated to be 50,468. The cDNA hybridized to a 1.9 kb mRNA obtained from chimpanzee hepatocytes infected with either the NANB or hepatitis delta viruses. It hybridized weakly to mRNA from hepatitis B virus-infected hepatocytes, and not at all to mRNA from normal chimpanzee hepatocytes. Southern blot analysis revealed that p44 is a host protein in chimpanzees, and that an identical gene exists in the human genome.

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.