Regulated secretion and purification of recombinant antibodies in E. coli.

A plasmid for optimized protein expression of recombinant Fv antibodies (pOPE) in E. coli was used to express the variable domains of the murine monoclonal antibody HD39 specific for the human B-cell surface antigen CD22. The production of Fv antibodies by pOPE can be regulated over a wide range by varying the IPTG concentration. Antibodies that can discriminate between secreted and nonsecreted Fv antibody fragments were used to show that secretion is the limiting step for the production of functional Fv antibodies. IPTG concentrations above 20 microM increased the total antibody production, but did not yield larger amounts of secreted Fv antibodies. The addition of five histidines to the C terminus facilitates an easy single-step enrichment procedure based on immobilized metal affinity chromatography.     

Expression and secretion of interferon-alpha 1 by Streptomyces lividans: use of staphylokinase signals and amplification of a neo gene

A gene coding for mature human interferon, IFN-alpha 1, fused to the expression and secretion signals of a staphylokinase gene (sak) derived from Staphylococcus aureus phage 42D, was inserted into the Streptomyces promoter probe vector pIJ487. Streptomyces lividans transformed with the recombinant plasmid (pMG341) secreted biologically active IFN-alpha 1 into the culture medium. Expression of the IFN-alpha 1 gene was at least on the translational level directed by the sak signals since numerous upstream stop codons would have prevented the formation of a fusion protein. Long-term continuous chemostat cultivation under various limitation conditions was used to select clones with an IFN-alpha 1 yield increased about 60-100-fold (1-2 x 10(5) IU/ml). The increase in IFN-alpha 1 formation was accompanied by spontaneous amplification of the adjacent neo gene, but not of the remaining plasmid DNA. Examination of the DNA sequence around the endpoints of the amplified region revealed almost identical stem-loop structures followed by an octanucleotide direct repeat.     

Measurement of the adhesive strength of a gingival cell line to agar

Summary A new technique is described for measuring the adhesive strength of a gingival cell line to an agar substratum by the modification of the original “blister” test for adhesives. A cell monolayer was developed on a Petri dish with a hole in the center of the growing surface, overlayed with agar, and the system pressurized to debond the cells from the agar surface. Pressure changes were measured by a capacitance pressure transducer the output of which was measured by a strip-chart recorder. The modulus (E) of the agar overlay was determined and used in the calculation of the adhesive-bond strength (γ_a). The (γ_a) yield for the gingival cell line (cell-agar debond) was 48.8 ergs per cm², and for the control (no cells) (agar-polystyrene debond) was 30.0 ergs per cm². This research was supported by National Institute of Dental Research Grand DE 03983-02