Vectored recombinants

For small molecules, natural products, peptides, oligonucleotides, gene vectors, recombinant proteins and monoclonal antibodies ... 

GlucaGen (glucagon) [expressed in a Saccharomyces cerevisiae vector-recombinant hormone]... 

Gene cloning is a method that uses recombinant technology to insert a gene into a vector DNA (plasmid obtained from a bacterium). The modified vector is put back into the bacterium, which then reproduces endlessly (clones) the new gene as well as the others in the vector. 

Workers in the early 1970s recognized that restriction enzymes provided tools not only for DNA mapping but also for constmction of new DNA species not found in nature. A collection of recombinant DNA species consisting of many passenger sequences joined to identical vector molecules is called a hbrary. Individual recombinant DNAs are isolated from single clones of the Hbrary for detailed analysis and manipulation. 

Plasmid DNAs. Plasmids are nucleic acid molecules capable of intracellular extrachromosomal repHcation. Usually plasmids are circular DNA species, but linear and RNA plasmids are known. In nature, plasmids can assume a variety of lifestyles. Plasmids can recombine into the host chromosome, be packaged into vims particles, and repHcate at high or low copy number relative to the host chromosome. Additionally, their information can affect the host phenotype. Whereas no single plasmid is usually capable of all these behaviors, the properties of various plasmids have been used to constmct vectors for a variety of purposes. 

Plasmid Vectors for Facile Introduction of Passenger DNA and Selection of Recombinants. The map of a commonly used plasmid vector, pUC19 (7), is shown in Figure 2. Three parts of the vector are key to its utility. The origin sequence, oh, allows the repHcation of plasmid DNA in high copy number relative to the chromosome. A gene, amp, encoding the enzyme beta-lactamase, which hydrolyzes penicillin compounds, allows... 

Fig. 3. Constmetion of a recombinant DNA by joining vector and passenger fragments where (I I I I I I) represent sticky ends. Both A and B genes represent selectable traits, so that introduction of foreign DNA in B gene leads to the loss of an identifiable function. REP represents repHcation and...

Fig. 4. Construction of recombinant phage in vectors derived from bacteriophage lambda where E represents the enzyme EcoRl. Other terms are defined...

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

Gene Expression Systems. One of the potentials of genetic engineering of microbes is production of large amounts of recombinant proteias (12,13). This is not a trivial task. Each proteia is unique and the stabiUty of the proteia varies depending on the host. Thus it is not feasible to have a single omnipotent microbial host for the production of all recombinant proteias. Rather, several microbial hosts have to be studied. Expression vectors have to be tailored to the microbe of choice. 

---