RESUMO
We have investigated large scale production processes (up to 2 liters) of recombinant proteins using the baculovirus expression system in order to optimize the product yields. Experiments using cell lines of Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-Mb0503) were performed to show the different production capacities of the cell lines. The influence of the infection at different cell densities is described. Beyond that, TC100-, IPL41- and serum-free IPL41-medium were compared to demonstrate their different capabilities of supporting cell growth and protein expression. Additionally, the inhibitory effect of FCS on the protease activity of kallikrein, which is produced in its zymogenic form, is discussed Improved production parameters are described, which enabled us to produce up to 8000 units of activated pro-kallikrein within 14 days using perfusion cultivation.
Assuntos
Baculoviridae/metabolismo , Biotecnologia , Precursores Enzimáticos/biossíntese , Calicreínas/biossíntese , Animais , Bovinos , Contagem de Células , Células Cultivadas , Meios de Cultura , Precursores Enzimáticos/metabolismo , Humanos , Calicreínas/metabolismo , Mariposas/citologia , Mariposas/metabolismo , Perfusão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , TripsinaRESUMO
A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.
Assuntos
Baculoviridae/genética , Precursores Enzimáticos/genética , Pré-Calicreína/genética , Glândulas Salivares/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Cromatografia DEAE-Celulose , Cromatografia em Gel , DNA/genética , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Humanos , Cinética , Dados de Sequência Molecular , Peso Molecular , Mariposas , Plasmídeos , Pré-Calicreína/isolamento & purificação , Pré-Calicreína/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , TransfecçãoRESUMO
A cDNA fragment encoding human salivary-gland kallikrein, including the kallikrein-owned signal peptide, was inserted into a baculovirus vector adjacent to the polyhedrin promoter and expressed in transfected insect cells. Biologically active kallikrein was isolated to homogeneity from serum-free culture supernatant using a four-step protocol. The N-terminal amino acid sequence of the insect-derived kallikrein was identical to that of the natural proteinase, thus indicating the proper removal of the mammalian signal peptide.
Assuntos
Calicreínas/genética , Glândulas Salivares/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Linhagem Celular , Expressão Gênica , Vetores Genéticos , Insetos , Calicreínas/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
CDNA clones for human kallikrein have been identified in a cDNA library constructed from mRNA of human salivary gland. The entire coding sequence for preprokallikrein and for the 5'- and 3'-untranslated regions were isolated by using a mixture of oligonucleotides corresponding to amino acids 51-56 of human urinary kallikrein and one oligonucleotide corresponding to amino acids 233-238 of human pancreatic kallikrein. The DNA sequence proved that, with the exception of two amino acid exchanges, kallikrein of the human salivary gland is identical with pancreatic kallikrein. Salivary gland and renal kallikrein was expressed in Escherichia coli from plasmid pKK223-3 under the control of the tac promoter. The protein was identified by Western-blot analysis and by demonstration of its specific proteolytic activity.
