Table 1.
Abbreviation List.
Fig 1.
Expression analysis in HEK293 cells of commonly used expression tags.
(A-D) The expression levels were compared by western blot analysis detecting the double Strep II tag, which is present as an N-terminal tag on all proteins. (A) Different short arm laminin chains and netrins were transiently expressed and analyzed by western blot. (B) Different expression tags were transiently expressed and visualized by western blot. (C) Netrin-1 delta was fused to different expression tags. (D) Expression of netrin-4 delta in different vector systems was analyzed by western blot analysis. (Isom.: modified SlyD; mFc: Fc part from the mouse IgG protein; mFc-opt: codon optimized Fc part from the mouse IgG protein; ser alb: serum albumin; MBP: maltose binding protein; basic: without fusion protein).
Fig 2.
Global application of the MBP tag as an expression enhancing tag.
(A) Protein expression levels in HEK293 cells of intracellular proteins (HSP90α and Gli1) with and without MBP. (B) Western blot analysis of transmembrane proteins (MEGF9 and MMP14) cloned into the basic and the MBP vector system. (C) Comparison of the expression levels of netrin-4 delta in the basic and the MBP vector transfected into COS-7, CHO-K1, and HEK293 cells. (D) The N-terminal laminin β2LN-LEa1-4 fragment and the respective R246Q mutant version causing congenital nephrotic syndrome were expressed and western blot analysis was performed.
Fig 3.
HEK293 cells were transiently transfected with eGFP alone (basic) or fused to different expression tags. (A) All proteins contained a signal peptide sequence for secretion. The eGFP signals were visualized by fluorescence microscopy. (B) To quantify the secreted eGFP, the supernatants from the HEK293 cells were excited with a laser at 488 nm and the emission signal was detected at 509 nm (top). Densitometric analysis of secreted eGFP from HEK293 cells by western blot analysis using a Strep-Tactin®-HRP conjugate to detect Strep II tagged eGFP fusion proteins (bottom). (C) Without signal peptide, eGFP was transiently expressed intracellularly with or without fused MBP. eGFP signals were visualized by fluorescence microscopy. (a.u.: arbitrary unit).
Fig 4.
Analysis of apoptosis upon transient transfection of netrin-1 delta and netrin-1 delta tethered to MBP.
(A) Apoptosis analysis by PI and annexin A5 co-staining. FACS analysis of transient transfected HEK293 cells with either netrin-4 delta or MBP-netrin-4 delta. (B) Graph shows the proportion of PI+/AnxA5+ cells in cells transfected with the empty basic vector, netrin-1 delta in the basic as well as the MBP vector (mean ± s.d.; n = 3; **P < 0.01). P values, Student’s t-test.
Fig 5.
Biophysical analysis of MBP as well as MBP fusion proteins and cell attachment studies.
(A) Light scattering profile obtained for a 1.80 mg/mL solution of MBP (dotted line) and for a 3.28 mg/mL solution of MBP-netrin-4 delta (solid line) in the TBS buffer display the respective hydrodynamic radius. (B) Concentration dependence of the hydrodynamic radius of MBP (dotted line) and MBP-netrin-4 delta (solid line), respectively, deduced from the peaks of DLS profiles. (C-D) B16-F1 cells were allowed to adhere to surface coated with MBP-netrin-4 delta. (C) Dependence of cell attachment on the coating concentration of MBP-netrin-4 delta. (D) Cell attachment at E50 coating concentration (MBP-netrin-4 delta) for different netrin-4 delta proteins (with and without tag).