PhoA gene

Rao, N., Roberts, M.F., Torriani, A. and Yashphe, J. (1 993) Effect of gIpT and gIpD mutations on expression of the phoA gene in Escherichia coli. Journal of Bacteriology 1 75, 74-79. 

In mammals, APases are glycoproteins essential in bone metabolism and phosphate transport. They are present as isozymes in different tissues including bone, intestine, kidney, and placenta. In E. coli, APase is a periplasmic enzyme which is not essential for phosphate metabolism. Note that E. coli also possesses a periplasmic acid phosphatase, a monomeric 45-kDa enzyme with active-site histidines. In contrast to the APase which is encoded by the phoA gene, the acid phosphatase is encoded by the appA gene. 

E. coli, BAP is encoded by the phoA gene and is synthesized as a precursor carrying an N-terminal signal sequence which is processed upon secretion into the periplasmic space. 

A C-to-T transition phoA503 mutant) resulting in a substitution of Val for Ala-22 in the mature BAP gives a conditional phenotype it dramatically reduces the synthesis of phosphatase activity during P starvation (85). The mutation does not alter the phoA gene expression, the dimer formation, or the translocation to the periplasm. 

The fusion of a cellulose-binding domain of endoglucanase Cen A (of Cellulo-monas fimi) to the phoA gene results in a hybrid protein which can be efficiently purified by affinity chromatography on cellulose (94). 

The targeting of antibodies to the periplasm requires the use of signal peptides. The pelB leader of the pectate lyase gene of Erwinia carotovora (56) is commonly used. The gill leader (9), the phoA leader of the E. coli alkaline phosphatase, and the ompA leader of E. coli outer membrane protein OmpA have also been used, being common to many protein expression vectors (57,58). Further examples are the heat-stable enterotoxin II (stll) signal sequence (47) and the bacterial chloramphenicol acetyltransferase (cat) leader (59). 

A variety of different promoters have been used for the expression of antibody genes. Widely used is the lacZ promoter (lacZ) derived from the lactose operon (53). The gill promoter (gill) from the bacteriophage M13 (9), the tetracycline promoter (IX teto/p ref. 54) and the phoA promotor of the E. coli alkaline phosphatase (47) also have been used successfully. It appears that very strong promoters, for example, the synthetic promoter PAI/04/03 (55), are... 

The wild-type E. coli utilized PolyP with a chain length of 100 phosphate residues as a sole source of phosphate in the growth medium (Rao and Torriani, 1988). The mutation in the phoA (alkaline phosphatase) gene prevented growth on this medium, while the mutation in the gene encoding the periplasmic acid phosphatase did nor affect PolyP utilization (Rao and Torriani, 1988). 

Bacterial alkaline phosphatase is the gene product of phoA, a member of the pho regu-lon (Table 9.1). When the pho regulon is induced by low external quantities of phosphate, synthesis of this alkaline phosphatase can represent as much as 6 mole% of total protein synthesis, and enzyme activity per cell can increase 1000-fold (Coleman and Gettins, 1983). The enzyme is synthesized as 43,000 Da monomers, which are transported to the periplasmic space and become active only after dimerization. As with many alkaline phosphatases, this enzyme accepts a broad range of substrates, which it hydrolyses at similar rates (Fernley and Walker, 1967 Reid and Wilson, 1971). Substrates are compounds with the general formula... 

Shin, P.K. and Seo, J.-H. (1990) Analysis of coli phoA-lacZ fusion gene expression inserted into a multicopy plasmid and host cell s chromosome. Biotechnol Bioeng., 36, 1097—1104. 

The gene for BAP, phoA, has been cloned into pBR322, and correctly processed, periplasmic APase has been overproduced 10-fold on phosphate limitation (92). Some commercial BAP is prepared from E. coli K12 SW 1033 carrying a plasmid pKI-5. 

This chapter covers j8-galactosidase (jSGal or LacZ) and luciferase (Lux/Luc) gene systems as typical reporter systems. Note that alkaline phosphatase (PhoA) can also be used as an excellent reporter system and has been described in Chapter 5. As selection markers, the ampicillin resistance system (Bla or ApO will be described. The chloramphenicol acetyltransferase (CAT) gene system will also be discussed as a typical marker/reporter with dual functions. 

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