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An efficient, scalable transient expression system has been developed and characterized for production of recombinant antibodies in lettuce. The transient expression system utilizes lettuce ( Lactuca sativa L.) vacuum-infiltrated with Agrobacterium tumefaciens bearing antibody genes cloned into a single or two separate expression vectors. The lettuce can be obtained commercially (grocery stores or directly from growers), so plant growth facilities are not required. Compared to previously described systems, this transient expression system is extensively modified to reduce cost and manipulations and provides for significantly increased levels of antibody production. The optimized agro-infiltration procedure produced as much as 20–80 mg of functional antibody per kilogram of fresh lettuce leaf tissue in less than a week. This procedure has been used to produce a humanized IgG1 κ anti-tissue factor antibody (hOAT) as well as other antibodies. The results demonstrate that transient expression of recombinant antibodies in lettuce is very efficient, inexpensive and readily scalable. Thus, it is a useful tool for small-scale production of functionally active antibodies and other pharmaceutically important proteins and can be used to facilitate humanization or optimization of antibodies or to produce quantities of antibody (in the range of 100 mg) sufficient for in vivo studies.

Thienamycin non-producing mutants of Streptomydes cattleya were identified that displayed a cross-feeding relationship. A diffusible product from one of these mutants (RK-11) resulted in restoration of thienamycin production when fed to cultures of another mutant (RK-4). In vivo radiolabeling experiments were conducted to test whether the RK-11 mutant produced a late biosynthetic intermediate which contained a carbapenem ring and a cysteaminyl and/or a hydroxyethyl side chain. Both [35S]cystine and [methyl-3H]methionine were used to label the RK-11 product which was then fed to RK-4 cultures. None of the thienamycin subsequently produced by RK-4 converter cells was labeled, implying the lack of either side chain of the thienamycin molecule in the RK-11 product. Further stability studies suggested that the RK-11 product does not contain a carbapenem ring. Additional feeding experiments with RK-4 cells also ruled out the possibility that the RK-11 product is a co-factor necessary for thienamycin production. It is concluded that the RK-11 product may regulate expression of the thienamycin gene cluster.

We have developed a new gene expression and secretion system for Streptomyces lividans and used it to produce soluble forms of a human T-cell receptor CD4 at levels greater than 300 mg/l. The system uses the transcription, translation and secretion signals of the serine protease inhibitor gene STI-II which is naturally produced by S. longisporus. Using these signals, soluble derivatives of CD4 were secreted directly into the culture supernatant as correctly processed soluble, biologically active proteins. High level expression of the CD4 proteins depended on the transcription initiation signal, the amino acid sequence surrounding the signal peptide cleavage site and temporally controlled protease activities. We discuss these results in the context of the potential of this system for producing other eukaryotic proteins in Streptomyces.

In contrast to the Gram-negative bacteria, Gram-positive bacteria such as Streptomyces lack a mucopolysaccharide cell wall which allows them to produce and secrete a variety of proteins directly into their environment. In an effort to understand and eventually exploit the synthesis and secretion of proteins by Streptomyces, we identified and characterized two naturally occurring abundantly produced proteins in culture supernatants of Streptomyces lividans and Streptomyces longisporus. We purified these 10-kDa proteins and obtained partial amino acid sequence information which was then used to design oligonucleotide probes in order to clone their genes. Analysis of the sequence data indicated that these proteins were related to each other and to several other previously characterized Streptomyces protein protease inhibitors. We demonstrate that both proteins are protein protease inhibitors with specificity for trypsin-like enzymes. The presumptive signal peptidase cleavage sites and subsequent aminopeptidase products of each protein are characterized. Finally, we show that the cloned genes contain all of the information necessary to direct synthesis and secretion of the proteins by Streptomyces spp. or Escherichia coli.

A plasmid was found in Nocardia orientalis (formerly Streptonryces orientalis). Physical characterization of the plasmid DNA indicates a size of 33.5 kb and a single cleavage site for EcoR I. The presence of plasmid, and variation in its copy member, did not directly affect vancomycin resistance or production levels. The plasmid represents the first to be isolated and characterized from a glycopeptide-producing nocardia.

The production of antibiotics by soil-borne micro-organisms, the actinomycetes, has considerable economic importance. The manipulation of antibiotic producers has become a prime target for the application of recombinant DNA technology. Certain technical requirements have had to be met for gene cloning to be successful in the actinomycetes. These requirements, including the development of cloning vectors and transformation procedures, have been satisfied, in part, for some members of the Streptomyces genus. Some problems including sequence rearrangement and stability of plasmid maintenance are now being recognized. A number of genes have been cloned in Streptomyces and some preliminary results characterizing the gene for a Streptomyces-derived beta-galactosidase-like activity were described.

A gene fusion system that uses the Escherichia coligalK gene has been developed to characterize Streptomyces transcriptional regulatory sequences. The system consists of galK-deficient Streptomyces lividans mutants and plasmids containing the E. coli galK gene with its natural ribosome-bindmg site and sites upstream of galK for insertion of transcription signals. Expression of the E. coli galK gene in S. lividans can be quantitated by either an enzymatic or immunoblot assay or detected by genetic complementation of an S. lividans galK− mutant. The utility of the plasmid to select, detect and assess promoter function was examined using the S. lividans XP55 and S. fradiae aph gene promoters. The potential use of the galK fusion system to isolate and characterize Streptomyces transcription signals is discussed.

Summary The effect of two different compounded diets on experimentally induced Endamoeba histolytica infection in two hosts, the rat and the guinea pig, has been studied and the results reported here. When the guinea pigs were maintained on the modified guinea pig (MGP) diet, almost 100 per cent of the animals died of the infection with a mean day of death of 8.4 days, and an average degree of infection of 3.9 out of a possible 4.0. When the rat breeder diet was fed to the guinea pigs only 78 per cent of the animals died of the infection with the mean day of death of 14.8 days and an average degree of infection of 3.3. The differences produced by the two diets were statistically significant. In rats both diets produced a high incidence of infection and a high average degree of infection. The data presented here, along with the work already reported from our laboratory, add further evidence to that reported by others, that diet influences the course of amebic infection in rats and guinea pigs.