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Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1-183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60-80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scale are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. Purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Apolipoprotein (apo) A-I is a 28-kDa exchangeable apolipoprotein that plays a key role in lipoprotein metabolism. It is widely distributed among animal species and is rich in alpha-helical secondary structure. Unlike human apoA-I, which aggregates in the absence of lipid, chicken apoA-I is monomeric in the lipid-free state. To take advantage of this physical characteristic, a bacterial expression system for production of recombinant chicken apoA-I has been developed. The cDNA-encoding chicken apoA-I was cloned into the pET expression vector under the regulation of the lac operon and transformed into Escherichia coli. Recombinant apoA-I protein recovered from the soluble fraction of the bacterial cell pellet was purified to greater than 95% homogeneity by reversed-phase high-performance liquid chromatography. Although immunoblot analysis confirmed the identity of the overexpressed protein, its migration on denaturing polyacrylamide gel electrophoresis was slower than its natural counterpart. To determine if the vector-encoded 18 residue pelB N-terminal leader sequence was not cleaved by the bacterial leader peptidase, isolated recombinant chicken apoA-I was incubated with exogenous leader peptidase. This treatment resulted in an increased electrophoretic mobility, with migration to a position corresponding to plasma-derived chicken apoA-I. Electrospray mass spectrometry indicated a mass of 27,961 +/- 4 Da, in agreement with that predicted for natural chicken apoA-I. Far-UV circular dichroism spectroscopy indicated an alpha-helical content similar to apoA-I isolated from chicken plasma, suggesting that the protein is folded in solution. Fluorescence studies showed that the wavelength of maximum fluorescence emission of the two tryptophan residues in the protein was 331 nm, with no shift occurring following complexation with lipid. Recombinant apoA-I was shown to be functional in lipoprotein binding as well as to possess an ability to transform bilayer vesicles of dimyristoylphosphatidylcholine into discoidal complexes. This is the first report of bacterial expression of an avian apoA-I. Increased availability and the potential for site-directed mutagenesis of this protein will aid in further characterization of apoA-I and the mechanism whereby it functions in cholesterol transport., (Copyright 1998 Academic Press.)