ABSTRACT
For the first time, a fungal production system is described for expression and secretion of the medically important human protein G-CSF, in Aspergillus niger. A reliable strategy was chosen with in-frame fusion of G-CSF behind a KEX2 cleavage site downstream of the coding region of the highly secreted homologous glucoamylase. This provided secretion levels of 5-10 mg/l culture medium of correctly processed G-CSF, although the majority of the protein (>90%) was biologically inactive. Following denaturation/ concentration and chromatographic separation/ renaturation, the G-CSF proliferation activity increased considerably, and analytical immobilised metal affinity chromatography confirmed the monomeric and correctly folded protein. These data suggest that this human secretory protein secreted into the medium of A. niger was not correctly folded, and that it escaped the endoplasmic reticulum folding control systems. This is compared to the folding of G-CSF produced in bacteria and yeast.
Subject(s)
Aspergillus niger/metabolism , Granulocyte Colony-Stimulating Factor/biosynthesis , Animals , Binding Sites , Cell Line, Tumor , Chromatography , Chromatography, Affinity , Endoplasmic Reticulum/metabolism , Escherichia coli/metabolism , Glycosylation , Humans , Hydrogen-Ion Concentration , Metals/chemistry , Mice , Pichia/metabolism , Plasmids/metabolism , Protein Folding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesisABSTRACT
Trends in preparation of PEGylated protein drugs strive for simple, fast, and cheap processes, resulting in well-defined homogeneous products. We investigated the on-column PEGylation of tumor necrosis factor alpha (TNF-α), where purification and conjugation were performed in one step by using immobilized metal affinity chromatography (IMAC). The same quality of the PEGylated product was obtained by the on-column approach starting from either the crude Escherichia coli protein extract or the purified protein. In comparison with the PEGylation in solution, the on-column approach resulted in more homogeneous PEGylated product. The on-column PEGylation reduces the number of production steps, costs, and preparation time.
Subject(s)
Chromatography, Affinity , Polyethylene Glycols/chemistry , Tumor Necrosis Factor-alpha/isolation & purification , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Immobilized Proteins/chemistry , Immobilized Proteins/isolation & purification , Immobilized Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/metabolismABSTRACT
G-CSF successfully prevents chemotherapy-induced neutropenia. Two second-generation drugs with improved therapeutic properties are already available and the development of new forms is still ongoing. For an efficient receptor dimerization two G-CSF molecules have to bind. Development of G-CSF dimers acting as receptor dimerizers was explored and their potential use evaluated. The in vitro biological activities of the prepared dimers were lower than G-CSF monomer activity, presumably due to non-optimal spatial orientation of the molecules. Most likely two dimers had to bind to trigger receptor dimerization instead of one dimer acting as a dimerizer. Although significantly lower in the residual in vitro biological activity, the diPEG-Fdim conjugate exhibited pharmacokinetical (PK) and pharmacodynamical (PD) properties comparable to pegfilgrastim or even better. An interesting PD profile with the second maximum in absolute neutrophil count (ANC) and a balanced elevated ANC profile over the longer time interval was namely observed.
