Abstract A method of producing a purified lipid-binding peptide which can bind to phospholipids at one or more amphipatic alpha-helical peptide regions. The method includes providing a gene coding for the peptide, and introducing the gene in expressible, heterologous form in a suitable expression system capable of synthesizing a mixture of peptides which includes the lipid-binding peptide. Addition of either endogenous or exogenous lipids to the peptide mixture forms a low-density lipopeptide complex composed of lipid and the lipid-binding peptide, and this complex can be separated easily from nonlipid-binding peptides in the peptide on the basis of its size and/or density. The method is intended particularly for scaled-up production of purified human apolipoproteins and their alpha-helical lipid-binding regions. Also disclosed are related methods for producing recombinant apolipoproteins, therapeutic lipopeptide compositions, and a stabilized lipid emulsion for nutritional therapy. Further disclosed are methods for expressing apolipoproteins or lipid-binding segments thereof in bacterial, yeast and mammalian cell expression systems, and methods for purifying lipid binding proteins, including fused recombinant proteins.
A method of producing purified human apoAI comprising: providing a Chinese hamster ovary (CHO) cell transformed with an expression vector comprising a regulatable human metallothionein II gene promoter, and an expressible, heterologous gene coding for prepro apoAI, culturing the cell in a serum-free medium under promoter-induction conditions wherein a mixture of extracellular proteins containing mature apoAI is produced, forming a lipoprotein complex composed of endogenous CHO cell lipid particles and associated apoAI, and separating the lipoprotein complex from nonlipid-binding proteins in the extracellular medium by floatation.
Claim 2
Independent Claim
86 words
A method of producing purified human apoAI comprising: providing a Chinese hamster ovary (CHO) cell transformed with an expression vector comprising a regulatable human metallothionein II gene promoter, and an expressible, heterologous gene coding for prepro apoAI, culturing the cell in a serum-free medium under promoter-induction conditions wherein a mixture of extracellular proteins containing mature apoAI is produced, forming a lipoprotein complex composed of endogenous CHO cell lipid particles and associated apoAI, and separating the lipoprotein complex from nonlipid-binding proteins in the extracellular medium by floatation.
Claim 3
Independent Claim
67 words
A method of producing human apoAI comprising: providing a suitable mammalian expression system capable of synthesizing a protein mixture which includes an expressed heterologous gene product wherein the expression system comprises a Chinese hamster ovary (CHO) cell transformed with an expressible, heterologous gene coding for prepro apoAI, and culturing the CHO cell in a serum-free medium under conditions which permit the production of mature human apoAI protein.
Claim 4
Independent Claim
67 words
A method of producing human apoAI comprising: providing a suitable mammalian expression system capable of synthesizing a protein mixture which includes an expressed heterologous gene product wherein the expression system comprises a Chinese hamster ovary (CHO) cell transformed with an expressible, heterologous gene coding for prepro apoAI, and culturing the CHO cell in a serum-free medium under conditions which permit the production of mature human apoAI protein.
Claim 5
Dependent Claim
25 words
The method of claim 2, wherein the gene is introduced in an expression vector under the control therein of a regulatable human metallothionein-II gene promoter.
Claim 6
Dependent Claim
24 words
The method of claim 3, wherein the CHO cells include a clonal strain of cells which are selected for high-level expression of the gene.