Human monoclonal antibodies recognize early and late viral proteins of human cytomegalovirus

High-throughput Sorting of an Anticalin Library via EspP-mediated Functional Display on the Escherichia coli Cell Surface

“…Bacterial production and purification of Anticalins in E. coli JM83 were performed as described. 31,61,96 The recombinant Fab fragment of the monoclonal antibody A3C5, 44,45 carrying a human C κ instead of the original C λ domain, was produced in a bench-top fermenter using the vector pASK85-A3C5 97 in E. coli K12 strain KS272 98 transformed with the helper plasmid pTUM4 99 and purified via the His 6 tag as described. 100 Protein purity was confirmed by SDS-PAGE using standard slab gel methodology 101 followed by staining with Coomassie brilliant blue R250.…”

“…1b) using a cognate high-affinity recombinant Fab fragment. 44,45 Finally, all fusion proteins were equipped with an N-terminal OmpA signal peptide 46 to effect initial translocation into the bacterial periplasm. The entire gene cassette was always placed under transcriptional control of the tightly regulated tetracycline promoter/operator.…”

Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors

“…A novel constitutively active and IRF-1 inducible (CIR) promoter was composed from constitutive and IRF-1 inducible elements for high-level constitutive expression and enhanced productivity in proliferation controlled cells. As a relevant product a heterodimeric IgG antibody molecule (Alexander et al, 1994;Haase, 1995) was expressed to investigate the capacity of the cells for the regulated synthesis of relevant pharmaceutical products. The stability of the IRF-1-mediated growth regulation for over 7 weeks in a continuously perfused bioreactor and the capacity to synthesize a secreted glycoprotein shows the potential for the development of an improved production process.…”

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“…One of the methods to solve this problem is establishment of efficient in vitro immunization methods in which primary B cells are activated to produce antigen-specific B lymphocytes (Koda and Glassy, 1990). Recently, a number of human MAbs have been produced against a variety of antigen using in vitro immunized lymphocytes (Power et al, 1990;Hoffmann et al, 1990;Gupta et al, 1992;Alexander et al, 1994). However, poor re-producibility was an problem in conventional in vitro immunization methods.…”