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)

Isolation and characterization of three monoclonal antibodies to human serum low density lipoprotein apoprotein B.

Human serum low density lipoprotein (LDL) is a large (Mr = 2-3 X 10(6), complex particle composed of lipid, protein and carbohydrate. We obtained about 40 mouse spleen-myeloma hybrid cell lines which produce antibodies against LDL. Three of them, SC2, SC3 and SC10, have been cloned and subcloned and their antibody products characterized. They recognize three non-overlapping epitopes in native LDL. Two of them, SC3 and SC10, also are capable of recognizing very low density lipoprotein, (VLDL), whereas SC2 reacts only weakly with VLDL. All three antigenic determinants remain intact, and accessible to antibodies on the LDL protein apo B, prepared by delipidation in a 'non-denaturing' detergent, sodium deoxycholate. However, apo B prepared by organic solvent, ether-ethanol, or sodium dodecyl sulfate (SDS) delipidation, while reacting strongly with SC10, is only poorly recognized by SC2 or SC3. Proteolysis of LDL with trypsin, chymotrypsin, Staphylococcus aureus protease, papain or thermolysin gives, in each case, several non-identical protein fragments which are separable by SDS-polyacrylamide gel electrophoresis. Upon immunoblotting, some of these fragments are now recognized by either SC3 or SC10 but not SC2, some are recognized by both SC3 and SC10, and others are immunologically unreactive. The protein bands that are separated by SDS gel electrophoresis are composed of several non-identical fragments and contain the antigenic sites to differing degrees. Some of the immunologically reactive fragments do not appear to contain carbohydrate. Reduction and carboxymethylation do not destroy the immunoreactivity of LDL toward any of the antibodies; however, modification of lysine residues by citraconic anhydride markedly diminishes the reactivity of LDL toward SC3. It is likely that the two antibodies SC3 and SC10 are directed against different linear amino acid sequences or very stable domains, whereas the third, SC2, is directed against a more fragile conformational domain of apo B.