Detection of the Recombinant Proteins in Single Transgenic Microbial Cell Using Laser Tweezers and Raman Spectroscopy

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• 作者：Changan Xie ; Nhu Nguyen ; Yong Zhu ; Yong-qing Li
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：December 15, 2007
• 年：2007
• 卷：79
• 期：24
• 页码：9269 - 9275
• 全文大小：280K
• 年卷期：v.79,no.24(December 15, 2007)
• ISSN：1520-6882

文摘

Laser tweezers Raman spectroscopy (LTRS) has beenused for the rapid detection of recombinant somatolactinprotein produced in single Escherichia coli bacteria andPichia pastoris yeast cell in the current study. A cDNAsequence encoding mature peptide of zebrafish somatolactin was inserted into two different expression vectorsand transfected into E. coli or P. pastoris yeast cells. Wemeasured Raman spectra of single E. coli cells at differentculture times following the induction with isopropyl -D-1-thiogalactopyranoside, from which the amount of thegenerated somatolactin proteins was obtained by theprojection of the entire cell's spectrum onto the spectrumof the pure somatolactin proteins or the dot productbetween these two spectral vectors. We found that theintensity of the somatolactin protein-associated spectrafrom single E. coli cells increased as the function of theculture time, which correlates with the accumulation ofrecombinant proteins inside the cells. This spectralobservation was supported by evidence obtained byconventional 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, P. pastoris yeast cells. These findingsdemonstrate that the LTRS is a useful method for rapidsensing of recombination production in single host microorganism in vivo.