Microfluidic flow cell for sequential digestion of immobilized proteoliposomes.

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 µg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 µg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.

Surface interactions of monoclonal antibodies characterized by quartz crystal microbalance with dissipation: impact of hydrophobicity and protein self-interactions.

Surface adsorption of two monoclonal antibodies (mAb1 and mAb2), with widely different hydrophobicity and self-association behavior in solution, was examined by quartz crystal microbalance with dissipation monitoring to understand how adsorption and protein self-interactions near the surface are impacted by their intrinsic properties. The dependence of mass and viscoelastic properties of the adsorbed protein layer on the type of surface, presence of a surfactant, protein concentration, and pH were examined. Adsorption was significantly reduced in the presence of surfactant for both proteins, but for the more hydrophobic mAb2, residual adsorption remained on polystyrene (PS) and Teflon surfaces. Protein concentration had little impact on the adsorbed protein mass for silicon dioxide surface but had a significant impact for PS and Teflon surfaces. At high protein concentrations, an irreversible layer formed first upon which a reversible layer builds. Reversible adsorption was significantly greater at higher protein concentrations and significantly higher for mAb2, consistent with its higher propensity to reversibly self-associate in solution. The viscoelastic properties suggest that adsorbed protein layer at high protein concentrations is more hydrated. The adsorbed protein layer at lower pH was more hydrated, and possibly more unfolded, consistent with the behavior of the antibody in bulk solution.

Exceptional overproduction of a functional human membrane protein.

Eukaryotic--especially human--membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization.