ABSTRACT
Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.
Subject(s)
Baculoviridae/genetics , Bombyx/virology , Larva/virology , Pupa/virology , Recombinant Proteins/biosynthesis , Spodoptera/virology , Animals , Baculoviridae/chemistry , Biotechnology/methods , Bombyx/genetics , Bombyx/metabolism , Cell Line , Chimerism , Electrophoresis, Polyacrylamide Gel , Female , Genetic Engineering/methods , Humans , Immunoblotting , Larva/genetics , Larva/metabolism , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Pupa/genetics , Pupa/metabolism , Recombinant Proteins/genetics , Spodoptera/cytology , Spodoptera/geneticsABSTRACT
Highly specific dockerin-cohesin interaction intrinsically involved in the cellulosome formation in Clostridium josui was applied for the construction of an affinity tag purification system. Amino acid substitutions were introduced into the dockerin domain of C. josui Cel8A at positions 11, 12, 44, and 45 and mutant dockerin domains were examined for their ability as an affinity tag: mutant dockerin-tagged proteins were adsorbed onto a cohesin (Coh2)-coupled Sepharose in the presence of Ca(2+) and desorbed from the protein and Coh2-Sepharose complex by the addition of a chelating agent, EGTA. Single-step purification tests showed that substitution of glycine or serine for isoleucine at position 45 markedly improved the recovery of the recombinant proteins from the proteins and Coh2-Sepharose complex. Surface plasmon resonance analysis of the interaction between the I45G mutant and Coh2 indicated that the mutation decreased binding rate and increased dissociation rate, resulting in decrease in dissociation constant. When model proteins such as JNK3, MAP2K3, IL-8, and pro-IL-18 were expressed as I45G dockerin-tagged proteins in the baculovirus expression system and purified by the single-step purification, purity of all the I45G dockerin-tagged proteins tested was higher than 90%. Furthermore, insertion of a thrombin cleavage site between the dockerin tag and target proteins enabled rapid removal of the tag from the target proteins by thrombin protease. This system, named the Dock tag purification system, can be widely utilized and contributes to various fields in academic and application researches.
Subject(s)
Bacterial Proteins/chemistry , Cell Cycle Proteins/chemistry , Chromatography, Affinity/methods , Chromosomal Proteins, Non-Histone/chemistry , Clostridium/metabolism , Recombinant Proteins/isolation & purification , Bacterial Proteins/metabolism , Binding Sites , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Mutagenesis, Site-Directed , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Recombinant Proteins/metabolism , Substrate Specificity , CohesinsABSTRACT
A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.
Subject(s)
Baculoviridae/genetics , Bombyx/genetics , Bombyx/metabolism , Interleukins/biosynthesis , Interleukins/pharmacology , Killer Cells, Lymphokine-Activated/drug effects , Killer Cells, Natural/drug effects , Transfection/methods , Animals , Cattle , Cell Proliferation/drug effects , Cells, Cultured , Humans , Interleukins/isolation & purification , Protein Engineering/methodsABSTRACT
Recombinant porcine mature interleukin-18 (rPomIL-18) has been purified from the haemolymph of silkworms. After co-infection of two recombinant baculoviruses (BmAcpVL1392-IL-18-His and BmAcpVL1392-casp-1) into the silkworm, rPomIL-18 was produced and secreted into the haemolymph. Polyethylene glycol (PEG) 6000 was added to the haemolymph at 8% (v/w) to precipitate storage proteins which would otherwise bind non-specifically to the metal chelating column and the supernatant then was applied to Sepharose bonded with Ni2+. rPomIL-18 was eluted from the column using 100 mM imidazole buffer at pH 8 with a purity of 93.6%. Approximately 5.3 mg purified rPomIL-18 was obtained from 22 ml haemolymph. It could induce interferon-gamma formation from porcine peripheral blood mononuclear cells.
