Use of Staphylococcus aureus 6-P-beta-galactosidase and GFP as fusion partners for lactose-specific IIC domain from Staphylococcus aureus.

The hydrophilic part of membrane proteins plays an important role in the formation of 3D crystals. The construction of fusion proteins using well crystallizing proteins as fusion partners is a possibility to increase the hydrophilic part of membrane proteins lacking large hydrophilic domains. These fusion proteins might be easier to crystallize. Two bifunctional fusion proteins containing the membrane-bound, lactose-specific enzyme IIC domain of the lactose transporter (IICB(lac)) from S. aureus as N-terminal fusion partner were constructed by gene fusion. The C-terminal fusion partners were S. aureus 6-P-beta-Galactosidase and GFP, respectively. Both proteins were overexpressed in E. coli, purified to homogeneity and kinetically characterized: In the presence of the components of the lactose phosphotransferase system of S. aureus, the hybrid proteins phosphorylated their substrates, indicating that the fusion partners are sufficiently flexibly linked to allow the interaction of the IIC(lac) domain with the IIB(lac) domain of the lactose transporter. The activity of the 6-P-beta-Galactosidase as well as the fluorescence of GFP were preserved in the fusion proteins. The Vmax values determined for the IIC domain in the fusion proteins were dramatically reduced compared with the values determined for the separate IIC(lac) domain and the complete lactose transporter (IICB(lac)). The Km values were only slightly increased indicating that the Vmax values are much more influenced by the fusion than the substrate affinities. The substrate affinity and the Vmax value determined for the GFP-fused IIC(lac) domain are higher than for the 6-P-beta-Galactosidase-fused IIC(lac). The results suggest that the fusion with GFP enables a better interaction with the IIB(lac) domain than the fusion with 6-P-beta-Galactosidase. Moreover, the GFP-fused IIC(lac) domain proved to be more stable than the 6-P-beta-Galactosidase fusion protein.

Locus control region of the human CD2 gene in a lentivirus vector confers position-independent transgene expression.

Vectors derived from murine leukemia virus (MLV) have been used in many human gene therapy clinical trials. However, insertion of the locus control regions (LCRs) derived from the beta-globin gene locus or the CD2 gene into MLV vectors frequently led to vector rearrangement. Since the human immunodeficiency virus (HIV) sequence diverges significantly from the MLV sequence, we tested whether the LCR sequence is more stable in the context of an HIV vector. Clones derived from human fibrosarcoma line HT1080 cells transduced with an HIV vector containing the T-cell-specific CD2 LCR exhibit the same wide range of transgene expression as clones lacking the LCR. In contrast, Jurkat and primary T-cell clones derived from the transduction of the LCR-containing vector show, on average, a three- to fourfold increase in transgene expression relative to that of the control vector. This is consistent with previous observations that the CD2 LCR contains a T-cell-specific enhancer. In addition, the clones derived from the LCR-containing vector have a much lower clonal variation in transgene expression than those derived from the control vector. We also demonstrate that the level of transgene expression is proportional to the vector copy number. These results suggest that the human CD2 LCR sequence is compatible with HIV vector sequences and confers enhanced integration site-independent and copy number-dependent expression of the transgene. Thus, HIV vectors may represent the ideal vehicle to deliver genes controlled by various cis-acting elements such as LCRs.

The lactose transporter of Staphylococcus aureus--overexpression, purification and characterization of the histidine-tagged domains IIC and IIB.

The lactose-specific enzyme II (IICBlac) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) of Staphylococcus aureus couples translocation to phosphorylation of the transported lactose. It is composed of the N-terminal membrane-bound IIC domain, which includes the sugar-binding site, and the C-terminal IIB domain, which contains the phosphorylation site at Cys476. IIC (residues 1-461) fused with a C-terminal affinity tag of six histidine residues and IIB (residues 461-570) fused with an N-terminal histidine tag were overexpressed in Escherichia coli and purified by Ni2+ chelate affinity chromatography. 2 mg of IIClac-His6 obtained from 10 g of cells and 12 mg of His6-IIBlac obtained from 8 g of wet cells were purified to homogeneity. 56% of the total IIClac-His6 activity present in the membranes could be recovered. Purification by affinity chromatography yields the opportunity to exchange the detergent. The Km determined in an activity assay for IIClac-His6 in the presence of the histidine-tagged IIBlac domain (His6-IIBlac) was similar to the Km determined for histidine-tagged IICBlac-His [Peters, D. & Hengstenberg, W. (1995) Eur. J. Biochem. 228, 798-804], suggesting that substrate affinity is barely influenced by the expression of the domains as separate proteins. The Vmax is reduced by a factor of 25 compared with IICBlac-His. His6-IIBlac also complements the activity of the IICBlac mutant C476S, which possesses an inactive IIB domain. This result indicates that IIC and IIB are flexibly linked in such a way that free His6-IIBlac can displace the inactive IIB domain fron its contact site on the IIC domain. His6-IIBlac is shorter and more stable than a previously constructed IIB domain (IIBlac-His) [Peters, D. & Hengstenberg, W. (1995) Eur. J. Biochem. 228, 798-804)], which contained a C-terminal histidine tag. The Km values for phosphoenolpyruvate-dependent phosphorylation of His6-IIBlac and IIBlac-His are nearly indistinguishable, suggesting that the location of the affinity tag either at the N-terminal or at the C-terminal end of the domain does not influence the substrate affinity.