The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv.

A method was developed to demonstrate recA-dependent P22-repressor breakdown in vivo by SDS-polyacrylamide electrophoresis of unfractionated extracts of phage-infected, lysogenic Salmonella typhimurium strains TA1530 rec+ and TA1530 recA1-. The antirepressor of P22 is not cleaved by recA protein. Under conditions of unregulated ant-overproduction (Harvey et al. 1981) antirepressor protects c2-repressor in vivo against proteolytic cleavage by recA protein.

Analysis of phage-specified protein synthesis after phage infection of UV-irradiated cells shows a turn-off of early gene expression, a regulatory event that is independent of the known P22 regulatory functions. This supports the suggestion of a cro lambda-like function in P22. We have identified the products of genes 18 and int as contributing to the complex 40,000 dalton band in our SDS-polyacrylamide gels. Both gene products appear to be subject to regulation by the cro-like function of P22. Proteins of 33,000, 29,000, 27,000, 25,000, and 24,000 MW, specified by as yet unidentified P22 genes of the early leftward operon, are regulated by the same function. Our data suggest that the cro-like function is expressed from the early rightward operon.

SDS-polyacrylamide gel analysis of P22-infected Salmonella has allowed identification of the c2 repressor (MW 31,000) and study of repressor synthesis in regulatory mutants of P22. Repressor is synthesized in reduced amounts or is absent in infections with P22, clNo.7, P22, c2 am08, P22 c3 am03, and P22 c3 am012, but is synthesized in markedly increased amounts in the virulent mutant, P22 virB3, and its component mutants, vx and k5. Higher levels of repressor are also found in the P22 cly 17 mutant.

Using SDS-polyacrylamide gel electrophoresis to study the early expression of P22 genes we show that early expression of the ant-gene (imm I region) is turned off after 6-8 min, independent of the 'late' acting mnt-repressor. A semi-clear mutant called cir5 is defective for this early ant turn-off. The mutation cir5 maps in the imm I region of P22 between genes mnt and ant. P22 cir5 mutants are defective for a repressor which acts in trans to regulate early ant synthesis. There appears to be no absolute requirement of the cir5 allele for the establishment of lysogeny. The overproduction of ant in the P22 cir5 mutant leads to a marked increase in abortive infections, killing the infected cells. The cir5-phenotype can be suppressed by an ant- mutation.