Bacteriophage engineering

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage A. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. 

Lycopene is a carotenoid with anticancer properties. To improve the production of lycopene by increasing the IPP flux in an engineered E. coli, the dxs gene was overexpressed and enhanced lycopene production was obtained [45]. In another example, the native promoters of DXP pathway genes in the E. coli chromosome were replaced with the strong bacteriophage T5 promoter (PTs), and the increase in isoprenoid precursors resulted in improved /3-carotene production (with a titer of 6 mg/g dry cell weight) [44]. 

A sample of double-stranded DNA is denatured. One of the resulting single strands is used as a template to direct the synthesis of a complementary strand of radioactive DNA using a suitable DNA polymerase. The "Klenow fragment" of E. coli, DNA polymerase I, reverse transcriptase from a retrovirus, bacteriophage T7 DNA polymerase, Taq polymerase, and specially engineered enzymes produced from cloned genes have all been used. 

Some bacteriophage encode their own DNA polymerases. However, they usually rely on the host cell to provide accessory proteins. The sequence of the DNA polymerase from phage T7 is closely homologous to that of the Klenow fragment and the 3D structures are similar. The 80-kDa T7 polymerase requires the 12-kDa thioredoxin from the host cell as an additional subunit. It has been genetically engineered to improve its usefulness in DNA sequencing 278... 

Makowski, L. 1994. Phage display structure, assembly and engineering of filamentous bacteriophage. ( hit. Opin. Struct. Biol. 4, 225—230. 

Ramesh, V, Amitabha De, and Nagaraja, V. (1994) Engineering hyperexpression of bacteriophage Mu protein C by removal of secondary structure at the translation initiation region. Prot. Engineer. 7,1053-1057. 

The stability of the /3-barrel itself was demonstrated in engineering experiments with OmpA. The four external loops of OmpA were replaced by shortcuts in all possible combinations (Koebnik, 1999). The resulting deletion mutants lost their biological functions in bacterial / -conjugation and as bacteriophage receptors, but kept the transmembrane /1-barrel as demonstrated by their resistance to proteolysis and thermal denaturation. The results confirm the expectation that the large external loops do not contribute to /1-barrel folding and stability. 

Figure 3-16 Model of bacteriophage fd engineered to display peptides as inserts in the coat proteins of the virus. The native virus structure is shown in gray proteins not present in the native virus are shown black or green, inserted near the N-termini of some major coat proteins is a 6-residue peptide. To one of these peptides a specific Fab antibody fragment (green) has bound from solution, and a second Fab is shown nearby. The N-terminal region of a minor coat protein at the end of the virion has been engineered to display a (different) Fab fragment. Steric constraints are less stringent for inserts in the minor proteins, but fewer copies per virion are possible. Reprinted with permission from Barbas, et...

As shown in Chap. 8, WBZ 4 is a re-engineered version of imatinib with an enhanced specificity needed to curb imatinib s potential cardiotoxicity. The inhibitory impact of WBZ 4 is restricted to KIT, PDGFR, and JNK, all with nanomolar affinity, as corroborated by screening against a T7-bacteriophage library of kinase displays (Ambit Biosciences, CA) [13],... 

---