Overproduction and immuno-affinity purification of myelin proteolipid protein (PLP), an inositol hexakisphosphate-binding protein, in a baculovirus expression system.

Myelin proteolipid protein (PLP) is a major integral membrane protein of central nervous system myelin and is considered to play a significant role in myelination. PLP has a four-transmembrane structure, judging from the hydropathy profile. In addition, it has InsP6 binding activity. Here, we have succeeded in producing PLP in large quantities of 3.9 pg/cell (6 mg/L) by using a baculovirus expression system and developing an efficient purification method, maintaining InsP6 binding activity. The recombinant PLP (rPLP) was purified by ion-exchange and immunoaffinity chromatography in a nonorganic solvent. The final yield of purified rPLP was 36%. The Kd and Bmax values for the InsP6-PLP binding were 55 nM and 33 pmol/microgram protein, respectively. The Kd value of purified rPLP is equal to that of mouse brain PLP. These results indicate that purified rPLP keeps its native conformation and binds InsP6 in an almost one-to-one ratio.

Biological activity of brain-derived neurotrophic factor with mismatched disulfide linkages produced by Escherichia coli.

E. coli cells harboring an expression plasmid with a brain-derived neurotrophic factor (BDNF) gene obtained from rat BDNF cDNA were sonicated and centrifuged to obtain a precipitate containing BDNF. Ten different proteins of BDNF were purified from the precipitate and the three disulfide linkages of six proteins were identified. Those disulfide structures were different from that of authentic BDNF and the biological activities of BDNF with mismatched disulfide linkages (EC50 of 2 to 15 ng/ml) were much lower than that of authentic BDNF (EC50 of 30 pg/ml). The BDNF with mismatched disulfide linkages inhibited the biological activity of authentic BDNF.

Production of biologically active mature brain-derived neurotrophic factor in Escherichia coli.

A gene coding for a brain-derived neurotrophic factor (BDNF) obtained from rat BDNF cDNA was used to construct an expression plasmid, pTRSBDNF. It contained a BDNF gene that was fused, in frame, to a region encoding the beta-lactamase signal peptide. The E. coli HB101 harboring pTRSBDNF produced 18 mg/liter of mature protein. The E. coli cells produced a larger amount of BDNF than that of human nerve growth factor (hNGF), which was produced using the same expression system. The E. coli cells harboring pTRSBDNF were sonicated and centrifuged to obtain a supernatant and precipitate. The BDNF purified from the supernatant containing 20% of the total BDNF had high biological activity (EC50 of 30 pg/ml) against neurons of chick dorsal root ganglia. On the other hand, the BDNF purified from the precipitate had low biological activity (EC50 of 2 ng/ml) and incorrect disulfide bonds.

Overproduction of biologically-active human nerve growth factor in Escherichia coli.

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.

Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli.

Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.