Recombinant proteins are becoming increasingly important as biopharmaceutical products. Beyond the fundamental sequence and structure of the protein itself, small molecular weight additions of 1-2 kD molecules can have a profound affect on the activity and function of the desired protein. One such natural modification to secreted proteins is N-linked glycosylation. Traditional analysis techniques to determine glycan core structures include HPLC and mass spectrometry. Given the limitations and advantages of different techniques, an improved methodology was created by combining an HPLC column with automated sample spotting onto plates for conventional MALDI-MS analysis. These modifications allowed analysis of more samples in parallel over two to three days starting with as little as 100 μg of protein. The method was used to identify N-glycan structures of an IgG antibody from human sera samples. Analysis of tissue plasminogen activator (t-PA) from CHO cell cultures under culture conditions in the presence of different media environments illustrated how the method can identify changes in oligosaccharide structure.
Another small molecular weight addition to recombinant proteins is the design of small cell-penetrating peptides to add or improve function to the protein of interest. This strategy can be used to enhance delivery of proteins to intracellular targets. One application of protein delivery is to increase intracellular concentrations of transcription factors to alter cell function. The transcription factor chosen was the basic helix-loop-helix factor NeuroD2 expressed as a fusion protein linked to the TAT(47-57) cell penetrating peptide from the HIV-transactivator protein. TAT-NeuroD2 was expressed and partially purified using mammalian transient expression. The protein was shown to be functionally active by electrophoretic mobility shift assay and nuclear localization after extracellular incubation with living cells. Though TAT-NeuroD2 was primarily endosomally localized, it was preferentially transported to the nucleus upon release from endosomes. TAT-NeuroD2 transduction was shown to increase the percent of neuron-specific class II beta-tubulin (TuJ1) positive human neural progenitor cells (hNPCs) and drive neuronal protein expression including TuJ1 in hNPCs and synaptotagmin in hNPCS and neuroblastoma cells. These methodologies demonstrate how different modifications and additions to proteins can alter properties of the protein such as delivery efficiency for various biotechnology applications.
|Advisor:||Betenbaugh, Michael J.|
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 71/03, Dissertation Abstracts International|
|Subjects:||Neurobiology, Biochemistry, Chemical engineering|
|Keywords:||Cell-penetrating peptides, Glycans, Protein transduction, Recombinant proteins|
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