ABSTRACT
Here we describe the synthesis of the gene encoding human keratinocyte growth factor (hKGF) and the construction of vectors for cytoplasmic production of hKGF in Escherichia coli cells. The level of recombinant protein expression was 1-1.5% of the total cell protein. hKGF was purified to homogeneity by three-step ion-exchange and affinity chromatography. The protein is biologically active: it stimulates dose-dependent protein synthesis in cultured human epidermal keratinocytes.
Subject(s)
Escherichia coli/metabolism , Fibroblast Growth Factors , Growth Substances/biosynthesis , Keratinocytes/metabolism , Amino Acid Sequence , Base Sequence , Cells, Cultured , Chromatography, Affinity , Chromatography, Ion Exchange , Escherichia coli/genetics , Fibroblast Growth Factor 10 , Fibroblast Growth Factor 7 , Gene Expression , Genetic Vectors , Growth Substances/genetics , Growth Substances/pharmacology , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacologyABSTRACT
The human interleukin-10 gene was obtained by chemico-enzymatic synthesis, and vectors for cytoplasmic and periplasmic expression of the recombinant IL-10 gene in Escherichia coli cells were constructed. Mutant IL-10 genes bearing substitutions in a region upstream of the ATG codon and in the triplet coding for the second amino acid residue in the protein were obtained by in vitro mutagenesis. High levels of expression were observed for the fusion protein composed of IL-10 and an N-terminal fragment of IL-3 and for the mutant IL-10 containing cysteine as the second amino acid residue.
