Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae. Proc Natl Acad Sci U S A 1990 Apr;87(7):2760-4
Date
04/01/1990Pubmed ID
2181448Pubmed Central ID
PMC53770DOI
10.1073/pnas.87.7.2760Scopus ID
2-s2.0-0025349629 (requires institutional sign-in at Scopus site) 72 CitationsAbstract
Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.
Author List
Medin JA, Hunt L, Gathy K, Evans RK, Coleman MSAuthor
Jeffrey A. Medin PhD Professor in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Adenosine DeaminaseAnimals
Chromatography, Affinity
Chromatography, DEAE-Cellulose
Cloning, Molecular
Genetic Engineering
Genetic Vectors
Humans
Insect Viruses
Insecta
Larva
Nucleoside Deaminases
Recombinant Proteins
Restriction Mapping
