"Bioscie,ces I.xtbcmaar#., Re.s'e,rcl~ Cet~ter. M ilsubishi Kosei Corporation. Fakohamt~ 227. duIum and ~Department of Neuro, ohurma. cololly. Brui#t Resettrch [initiate. NiiRula U, iver~'io'. Niigar~ 951. Japan Received 18 Sane t992: revi~d version received 6 July 1992The tel;ion prcc~in8 putati~ trammemh~m¢ segment MI of the glutamate r¢ccptor (GluR) channd is well consc~eM amon8 subunits and has hta:n propo~d to constitute a p:irt of tee agonist binding site. Th¢ funct tonal signif~t n~: of this region was ¢xamined by hat rodmin8 point muttttions into eharlle, d residues of th~ ctl subtinit of th¢ mouse ~.amir:u-3.hydroxy.5.methyl-4.isoxaz~lc propionic acid (AMPA).s¢lectiv,s GIuR channel, The do~-re~pons¢ relationships of the mutant rcc¢pto~ w¢rc studied after cxpr~sion in .Ve~,pus oocyt¢~ by injo:tion of the mutant ¢tl subunit. sp~iflc mRNA togcth¢r with the wild.typ¢ ct2.subunit-spccific mRNA. Variable chang~ in the ECru valu¢~ for diff¢rent agonists wcr¢ found for the replacement of 81atomic acid 398 by lysin¢ and for th¢ replacement of lysin¢ 445 by Illutamic acid. These residucl may ~ involv¢d in sml¢ctivc interaction or the GIuR channd with alonists.
Genes 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 degrees C and the inducer (isopropyl-beta-D-thiogalactopyranoside, IPTG) concentration was 1 mM (standard conditions), production of functional Fab was very low (medium; 200 ng/l culture and periplasm; less than 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 degrees 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.
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