Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein production-a dominating theme in biotechnology for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome binding sites and terminators, which control expression in a cell factory. A major bottleneck during production is translational initiation. Previously we identified more effective translation initiation regions (TIRs) by creating sequence libraries and then selecting for a TIR that drives high-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange of different sequence elements in this device allows for different coupling efficiencies, therefore giving the devices a tunable nature. Sandwiching these devices between the cds and an antibiotic selection marker that functions over a broad dynamic range of antibiotic concentrations adds to the tunability and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the respective antibiotic. The power of the approach is demonstrated by substantially increasing production of two commercially interesting proteins, a Nanobody and an Affibody. The method is a simple and inexpensive alternative to advanced screening techniques that can be carried out in any laboratory.
A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. Methods: A HER2-binding Affibody molecule, Z HER2:342 , was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either 11 C or 68 Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the 11 C-labeled Affibody molecule. Results: Both the 11 C-and 68 Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4-5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (.5-fold) for the 11 C-labeled protein, and its overall absorbed dose was considerably lower. 11 C-Z HER2:342 showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P 5 0.002), comparable to uptake in tumors with low HER2 expression. Conclusion: To our knowledge, the Sel-tagging technique is the first that enables sitespecific 11 C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, 11 C-labeled Sel-tagged Z HER2:342 can successfully be used for rapid and repeated PET studies of HER2 expression in tumors. Overexpressi on of the human epidermal growth factor receptor 2 (HER2), found in about 20%-30% of breast cancers, is associated with poor prognosis, and HER2 is the molecular target for effective but expensive monoclonal antibody drugs such as trastuzumab (Herceptin; Roche) (1). Determination of the expression levels of HER2 is therefore important for predicting prognosis and for patient stratification for treatment. Currently, expression levels are mainly determined using biopsies for molecular assays such as in situ hybridization (2). These procedures are, however, invasive, are difficult to perform repeatedly, and risk missing overexpressing tissue due to sampling errors. Therefore, the radionuclide-based in vivo molecular imaging of HER2, primarily using polypeptide probes, is rapidly expanding, aiming for personalized medicine with image-and-treat strategies (3-6). Speed, sensitivity, and specificity are necessary for implementation in clinical routines.Comparing nuclear medicine imaging techniques, PET has better spatial resolution, higher sensitivity, and more accurate quantification than SPECT. With recent radiolabeling advances, the feasibility of using polypeptides...
Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.
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