Cytoplasmic and periplasmic production of human apolipoprotein E in Escherichia coli using natural and bacterial signal peptides.
To understand the toxicity of high levels of heterologous human serum apolipoprotein E (ApoE) in Escherichia coli, as well as to prepare a system for producing the structural domains of this protein, plasmids were constructed in which the coding sequence of the N-terminal domain or all of ApoE followed E. coli or human apolipoprotein signal peptides (SP) or the N-terminal eleven amino acids (f10) of the gene 10-encoded protein of phage T7. High levels of production of the 22-kDa N-terminal domain (22K) of ApoE were obtained either as an f10::22K fusion protein, or using the natural SP, or SP derived from the periplasmic protein, alkaline phosphatase (PhoA), or from the outer membrane protein A (OmpA). Microsequencing showed that the SP of sPhoA::22K and sOmpA::22K, but not sApoE::22K, were correctly processed and, in the former cases, the protein could be released from the cells by osmotic shock. The extent of maturation of sPhoA::22K depended upon the host strain; with JM109, about 50% of the protein was not processed. Microsequencing of the f10::22K fusion protein, which could easily be purified following lysis of the cells, showed that the N-terminal methionine had been removed in agreement with the length parameter rule. Although considerable levels of the f10::ApoE fusion protein could be produced in the cytoplasm, production was markedly less using the PhoA signal peptide and the protein was not easily isolated following osmotic shock. The recombinant protein was biologically active after reconstitution with lipids in spite of the N-terminal modifications introduced.(ABSTRACT TRUNCATED AT 250 WORDS)
Monteilhet
C
C
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Lachacinski
N
N
Aggerbeck
L P
LP
D001057:Apolipoproteins E / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001057:Apolipoproteins E; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004247:DNA