Purification and characterization of a recombinant human testican-2 expressed in baculovirus-infected Sf9 insect cells.
Testican-2 is a member of the testican group of brain extracellular proteoglycans where a 45 kDa modular protein core is composed of a follistatin-like domain, a calcium-binding domain, a thyroglobulin type-1 (Tg1) domain and an acid C-terminal region with glycosaminoglycan attachment sites. The modular structure suggests that it could participate in various interactions. The aim of the present study was to express and characterize a recombinant human testican-2 in quantities sufficient for structural and functional studies. Human cDNA coding for a 422 amino acid testican-2 protein was cloned into the pFastBac1 vector and expressed in the Spodoptera frugiperda (Sf9) insect cell expression system. The protein was purified to homogeneity by three chromatographic steps using the His(6) tag in the first two steps and ion exchange chromatography as last one. The final yield of purified recombinant testican-2 was up to 3.5 mg/L culture medium and its molecular mass determined by SDS-PAGE was approximately 55 kDa. Analysis by enzymatic deglycosylation revealed presence of N-linked sugars with a total mass of 4 kDa. In contrast to the Tg1 domain of testican-1, which acts as an inhibitor of the lysosomal cysteine peptidase cathepsin L, the Tg1 domain of testican-2 did not inhibit cathepsins L, B, K and S. We identified the C1q subcomponent of complement component C1 as a potential interacting partner of testican-2. The C1q subcomponent is a recognition molecule which acts in concert with other C1 subcomponents to activate the classical pathway of complement activation. The reported new interaction could be of importance in various complement-mediated inflammatory and other immune processes.
Pavsic
Miha
M
Department of Biochemistry, Molecular and Structural Biology, J. Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Vito
Turk
T
Lenarcic
Brigita
B
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology; D002403:Cathepsins / Q000378:metabolism; D002460:Cell Line; D002942:Circular Dichroism; D015922:Complement C1q / Q000378:metabolism*; D006801:Humans; D011509:Proteoglycans / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D011509:Proteoglycans; D011994:Recombinant Proteins; C504404:SPOCK2 protein, human; D015922:Complement C1q; D002403:Cathepsins