Improved recovery of a fusion protein containing the antigenic domain 1 of the human cytomegalovirus glycoprotein B.
Heterologous expression in Escherichia coli often leads to production of the expressed proteins as insoluble and inactive inclusion bodies. The general strategy for protein recovery includes isolation and washing of inclusion bodies, solubilization of aggregated protein and refolding of solubilized protein. The process of refolding, as well as the other steps involved in inclusion body recovery, must be optimized according to the characteristics of each protein. For the development of reliable and inexpensive serodiagnostic tests, the antigenic domain 1 (AD-1) of human cytomegalovirus glycoprotein B was expressed in E. coli and a process was developed to increase recovery of the fusion protein containing AD-1. A comparison of disruption methods and different conditions involved in recovery of this fusion protein from inclusion bodies is presented. The developed method gives a high yield of the fusion protein with a purity sufficient for use in diagnostic tests.
Ferreira
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Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001, Covilhã, Portugal.
Sousa
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Queiroz
J A
JA
Domingues
F C
FC
D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013010:Sonication; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism
D000956:Antigens, Viral; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus