OmpA signal peptide

Overexpression of apoaequorin (Inouye et al., 1989, 1991). To produce a large quantity of apoaequorin, an apoaequorin expression plasmid piP-HE containing the signal peptide coding sequence of the outer membrane protein A (ompA) of E. coli (Fig. 4.1.12) was constructed and expressed in E. coli. The expressed apoaequorin was secreted into the periplasmic space of bacterial cells and culture medium. The cleaving of ompA took place during secretion thus the... 

E. coli, like other Gram-negative bacteria, has an outer membrane which hampers excretion of proteins to the culture media. Thus, expressed proteins can remain in the cytoplasm or can be directed into the periplasm employing the N-terminus fusion of a signal peptide (e.g. OmpA, pelB, OmpF, PhoA, Tat signal peptides) [30]. 

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). 

If export competence is associated with a loosely folded precursor, then parameters that accelerate folding or stabilize folded states should impede export. This relationship has also been explored in E. coli using a DHFR fusion protein. At low levels of synthesis, a hybrid protein consisting of the signal peptide and the first 153 amino acid residues of OmpA joined to DHFR is efficiently secreted. However, addition of trimethoprim imparts a kinetic defect in the secretion rate of the hybrid protein. The effect of trimethoprim is dependent on the presence of a full length, presumably active, DHFR moiety, indicating that secretion in vivo is inhibited by stabilization of the native DHFR structure (FreudI et ai, 1988). 

Since the first 370-fold purification of RNase Tl (1), a number of important improvements have been made to increase the yield, purity, and efficacy of purification (31). The improvements in separation techniques include various affinity chromatographies that rely on such adsorbents as 2, 3 -cGMP, aminophenylphos-phoryl-GMP (50), 5 -GMP (40), and 2, 5 -GpG (51). In addition, the RNase Tl gene cloned into an expression—secretion vector efficiently produces the enzyme from . coli hosts (yield of 20 mg enzyme per liter of culture) (49). In this system, the RNase Tl gene is expressed under the inducible control of the lac promoter-operator as a fusion protein with the signal peptide of OmpA, the major outer membrane protein of . coli. The in vivo cleavage of the fusion... 

The ability of the LamB synthetic signal sequences to inhibit protein translocation in vitro correlates with their activity in vivo (L. Chen and P. C. Tai, personal communication). The wild-type and mutant E. coli LamB signal sequences described above (Section III,H) were added to the cell-free translation/translocation system of Chen et al. (1985). The wild-type peptide blocks translocation of OmpA and alkaline phosphatase 50% inhibition is reached at a peptide concentration of 1-2 fiM. 

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