Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1.
Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.
End
Caroline
C
Institute of Molecular Biology and Cell Culture Technology, University of Applied Sciences Mannheim, Windeckstrasse 98, 68163 Mannheim, Germany.
Lyer
Stefan
S
Renner
Marcus
M
Stahl
Cordula
C
Ditzer
Jutta
J
Holloschi
Andreas
A
Kuhn
Hella-M
HM
Flammann
Heiko T
HT
Poustka
Annemarie
A
Hafner
Mathias
M
Mollenhauer
Jan
J
Kioschis
Petra
P
D000373:Agglutinins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000737:chemistry; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D014644:Genetic Variation; D005822:Genetic Vectors / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D000373:Agglutinins; D002135:Calcium-Binding Proteins; D017077:Culture Media, Conditioned; C108174:DMBT1 protein, human; D004268:DNA-Binding Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins