Baculovirus expression and bioactivity of a soluble 140 kDa extracellular cleavage fragment of L1 neural cell adhesion molecule.
L1 neural cell adhesion molecule is the founding member of the L1 subfamily of the immunoglobulin superfamily and plays an important role in the overall development of both the central and peripheral nervous systems, making it an attractive candidate for promoting neural regeneration following injury. Currently, L1 used for experimental studies is primarily mammalian-derived; however, the insect cell expression system described here provides an alternative source of recombinant L1 with equivalent bioactivity. A 140 kDa L1 fragment based on a physiological plasmin cleavage site in the extracellular domain was cloned and expressed with a C-terminal 6x histidine tag. Recombinant insect cell-derived L1 was analyzed by Western blot using an antibody to human L1 to confirm immunogenicity and to optimize infection conditions for recombinant L1 production. The recombinant protein was secreted by insect cells, efficiently purified under non-denaturing conditions using dialysis followed by metal affinity chromatography, and analyzed by SDS-PAGE to produce a single band of the expected approximate 140 kDa size. The bioactivity of insect cell-derived L1 was compared to mammalian-derived L1-Fc and poly-L-lysine (PLL) using chick embryonic forebrain neurons. The results show comparable, robust neurite outgrowth at 24h on insect cell-derived L1 and mammalian-derived L1-Fc, with significantly longer neurites than those observed on PLL. Future studies will examine the immobilization of L1 to biomaterial surfaces in physiologically appropriate orientation via the C-terminal 6x histidine tag and will investigate their application in promoting axonal regeneration in the injured nervous system.
Cribb
Rebecca C
RC
Department of Bioengineering, Micro-Environmental Engineering Laboratory, Clemson University, 501 Rhodes Research Center, Clemson, SC 29634, USA.
Haddadin
Fuad T
FT
Lee
Jeoung Soo
JS
Webb
Ken
K
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002645:Chickens; D003720:Densitometry; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008970:Molecular Weight; D039842:Neural Cell Adhesion Molecule L1 / Q000302:isolation & purification / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism; D009474:Neurons / Q000166:cytology; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility
D039842:Neural Cell Adhesion Molecule L1; D010446:Peptide Fragments; D011994:Recombinant Proteins