Production of serpins using baculovirus expression systems.
Headpin is a novel serine proteinase inhibitor (serpin) that is downregulated in many established HNSCC tumor cell lines and human oral SCC specimens. The use of the bacterial and yeast expression systems for headpin resulted in poor yields and proteins with low inhibitory activity. To circumvent these problems, we have developed a baculovirus-insect cell system for high-yield expression as well as fully functional protein. Here, we describe the strategies and methods used to express headpin in an insect cell heterologous system. In addition, procedures to purify the recombinant proteins are described. A metal affinity column followed by a gel-filtration chromatography provides a rapid and efficient method for large quantity preparation of headpin. This method should be useful as an alternative expression system for those serpins that are not purifiable when expressed using the Escherichia coli or yeast expression system.
Jayakumar
Arumugam
A
Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Box 0441, Houston, TX 77030-4009, USA.
Cataltepe
Sule
S
Kang
Ya'an
Y
Frederick
Mitchell J
MJ
Mitsudo
Kenji
K
Henderson
Ying
Y
Crawford
Sue E
SE
Silverman
Gary A
GA
Clayman
Gary L
GL
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D007313:Insecta / Q000166:cytology / Q000821:virology; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015843:Serpins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection / Q000379:methods
D011994:Recombinant Proteins; D015843:Serpins