Detection of the Recombinant Proteins in Single Transgenic Microbial Cell Using Laser Tweezers and Raman Spectroscopy
2007-12-15T00:00:00Z (GMT) by
Laser tweezers Raman spectroscopy (LTRS) has been used for the rapid detection of recombinant somatolactin protein produced in single <i>Escherichia coli</i> bacteria and <i>Pichia pastoris</i> yeast cell in the current study. A cDNA sequence encoding mature peptide of zebrafish somatolactin β was inserted into two different expression vectors and transfected into <i>E. coli</i> or <i>P. pastoris</i> yeast cells. We measured Raman spectra of single <i>E. coli</i> cells at different culture times following the induction with isopropyl β-d-1-thiogalactopyranoside, from which the amount of the generated somatolactin proteins was obtained by the projection of the entire cell's spectrum onto the spectrum of the pure somatolactin proteins or the dot product between these two spectral vectors. We found that the intensity of the somatolactin β protein-associated spectra from single <i>E. coli</i> cells increased as the function of the culture time, which correlates with the accumulation of recombinant proteins inside the cells. This spectral observation was supported by evidence obtained by conventional methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses. The increased intensities of recombinant protein-associated Raman bands were also observed in another expression system, <i>P. pastoris</i> yeast cells. These findings demonstrate that the LTRS is a useful method for rapid sensing of recombination production in single host microorganism in vivo.
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