Translation Levels Control Multi-Spanning Membrane Protein Expression

Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane

Topology diagram of model human proteins as expressed in a mammalian cellular plasma membrane.

<p>The predicted molecular weights for these proteins without post-translational modification are: CD20, 33.0 kDa; EG-VEGFR1, 44.8 kDa; RA1c, 35.5 kDa and Patched 1, 160.5 kDa.</p

Improved cell growth and general accumulation of integral membrane proteins using a dually regulated promoter.

<p>(<b>A</b>) Restricted <i>E. coli</i> growth in LB with the <i>phoA</i>-RA1c construct is relieved by using the <i>tphac</i> promoter, which reduces basal level expression. A 24-hour growth curve shows the empty pBR322 vector control (blue triangles), <i>phoA</i>-RA1c expression construct (green diamonds), <i>tphac</i>-RA1c expression construct (red circles) and <i>phoA</i>-EGFL7 as a non-membrane protein control (brown squares). (<b>B</b>) A representative western blot of RA1c expression from the <i>phoA</i> promoter is shown following induction by phosphate depletion when the cells reach approximately 2 OD₆₀₀ (time 0). Maximum expression is reached within two hours post induction. By 6 hours, aggregation has begun and by twelve hours almost all the protein has moved from the monomer band to high molecular weight aggregate. Basal expression is shown after overnight growth in LB medium (LBON). The western blot was probed with an HRP coupled anti-his antibody. (<b>C</b>) A comparison of basal expression in LB of the GPCR proteins, RA1c and EG-VEGFR1, from the <i>phoA</i> and <i>tphac</i> promoters by western blot analysis. The <i>phoA</i> constructs show significant accumulation levels of the membrane proteins while the <i>tphac</i> constructs have reduced the accumulation to background levels. The arrow points to the monomer protein band.</p

Ligand binding to the GPCR, LE-EG-VEGFR1.

<p><i>E. coli</i> membrane proteoliposomes were treated with thrombin to remove the LE-leader and incubated overnight at 4°C with EG-VEGF in PBS. Pelleted membranes were separated by SDS-PAGE and developed by immuno-blot using an anti-EG VEGF antibody. Samples are: lane 1) pBR322 negative control; 2) LE-EG-VEGFR1, N-terminal FLAG; 3) LE-EG-VEGFR1, C-terminal FLAG. The location of EG-VEGF (molecular weight 9 kDa) is indicated by an arrow.</p

Expression and folding of CD20 in the <i>E. coli</i> inner membrane.

<p>Cell surface expression and orientation of CD20 was assessed from spheroplasts of <i>E. coli</i> cells expressing either Uni-CD20 (blue), LE-CD20 (green) or an empty control vector (red) treated with Alexa-488 conjugated anti-CD20 antibody to the extracellular loop of CD20 and analyzed by flow cytometry.</p

Leader dependent accumulation of CD20 and EG-VEGFR1 following induction of the <i>tphac</i> promoter.

<p>(<b>A</b>) Protein accumulation maximizes within thirty minutes of induction with the Uni leader. (<b>B</b>) Protein accumulation continues over 20 hours after induction with the LE leader. (<b>C</b>) The effect of C-terminal truncations on expression from the LE leader. The full 79 amino acid LE leader was truncated from the C-terminus to observe the importance of the translation initiation rate as compared to the length of the LE leader. Truncated leaders were fused to CD20 and the whole cell lysates were immunoblotted with HRP conjugated anti-His antibody. Two film exposures are shown. (<b>D</b>) Reduced promoter activity results in reversal of the relative expression levels from the Uni and the LE leaders fused to CD20. Cultures were grown under partial promoter induction and the whole cell lysates were probed with HRP conjugated anti-His antibody for detection.</p

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab′ (G10rabFab′) fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab′ free cysteine with linear methoxy-polyethylene glycol­(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab′. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (<i>R</i>_H). A linear dependence of vitreal half-life on <i>R</i>_H suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from <i>R</i>_H measurements is proposed

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin