Export of a hyperexpressed mammalian globular cytochrome b5 precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond.
A chimeric mammalian globular cytochrome b(5) fused to Escherichia coli alkaline phosphatase signal sequence (SS) was used as a model probe to investigate the influence of substituting each one of the standard 20 amino acids at its N-terminus on the Sec-dependent export of the precursor to the periplasmic space of E. coli. Substituting the native Met(+1) of the passenger protein flanking the SS with any one of the remaining 19 amino acids introduced significant changes in the export of cytochrome b(5) without jamming the Sec-dependent translocon. Acidic and hydrophilic residues proved to be the most efficient promoters of export. Small, nonbulky and basic residues yielded intermediate levels of the hemoprotein export. Replacement with a Cys(+1) residue generated significant quantities of both monomeric and disulfide-linked dimeric forms. However, bulky, aromatic and hydrophobic residues caused a significant decline in the rates of secretion. In expectation with their absences in the natural periplasmically secreted proteins, Pro and Ile-tagged cytochrome b(5) precursors failed to generate any detectable secreted recombinant products. Although Ala, amongst the native E. coli periplasmic proteins, is the preferred X(+1) residue with an occurrence of 50% frequency, it proved half as effective in promoting export when inserted proximally to the SS of cytochrome b(5). The mechanisms involved for these export variations are discussed. The findings will prove beneficial for high-level generation of recombinant proteins by secretory means for pharmaceutical and related biotechnological applications.
Kaderbhai
Naheed N
NN
William Davies Laboratories, Institute of Biological, Environmental and Rural Sciences, Plas Gogerddan Campus, Aberystwyth University Aberystwyth, Ceredigion SY23 3EB, United Kingdom. nnk@aber.ac.uk
Ahmed
Khalil
K
Kaderbhai
Mustak A
MA
D000818:Animals; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport / Q000235:genetics / Q000502:physiology
D011498:Protein Precursors; D021382:Protein Sorting Signals; D015786:Cytochromes b5