Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner.

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.

[1]  N. D. Da Silva,et al.  Enhanced Productivity through Gratuitous Induction in Recombinant Yeast Fermentations , 1994, Biotechnology progress.

[2]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[3]  Bernard,et al.  The green fluorescent protein targets secretory proteins to the yeast vacuole , 1999, Biochimica et biophysica acta.

[4]  W. Bentley,et al.  A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase , 2000, Applied Microbiology and Biotechnology.

[5]  J J Valdes,et al.  Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility. , 2000, Biotechnology and bioengineering.

[6]  M. J. Cormier,et al.  Primary structure of the Aequorea victoria green-fluorescent protein. , 1992, Gene.

[7]  B. Chung,et al.  Highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides. , 2000, Biotechnology and bioengineering.

[8]  F. Srienc,et al.  Quantitative analysis of transient gene expression in mammalian cells using the green fluorescent protein. , 1996, Journal of biotechnology.

[9]  H. Kennedy,et al.  Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase. , 1998, The Biochemical journal.

[10]  K. Keinänen,et al.  Secretion of green fluorescent protein from recombinant baculovirus-infected insect cells. , 1996, Biochemical and biophysical research communications.

[11]  J. D. de Winde,et al.  Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression. , 1999, The Biochemical journal.

[12]  C. Albano,et al.  Green Fluorescent Protein as a Real Time Quantitative Reporter of Heterologous Protein Production , 1998, Biotechnology progress.

[13]  J. Wahlfors,et al.  Green fluorescent protein (GFP) fusion constructs in gene therapy research , 2001, Histochemistry and Cell Biology.

[14]  K. Entian.  Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast , 2004, Molecular and General Genetics MGG.

[15]  S. Kohlwein.  The beauty of the yeast: Live cell microscopy at the limits of optical resolution , 2000, Microscopy research and technique.

[16]  M. Johnston,et al.  Regulated nuclear translocation of the Mig1 glucose repressor. , 1997, Molecular biology of the cell.

[17]  T. Steitz,et al.  High resolution crystal structures of yeast hexokinase complexes with substrates, activators, and inhibitors. Evidence for an allosteric control site. , 1977, The Journal of biological chemistry.

[18]  J. Gancedo.  Carbon catabolite repression in yeast. , 1992, European journal of biochemistry.

[19]  F Moreno,et al.  Hexokinase PII has a double cytosolic‐nuclear localisation in Saccharomyces cerevisiae , 1998, FEBS letters.

[20]  D. Piston,et al.  Quantitative imaging of green fluorescent protein in cultured cells: Comparison of microscopic techniques, use in fusion proteins and detection limits , 1995, Journal of microscopy.

[21]  Canghai Lu,et al.  High Throughput Studies of Gene Expression Using Green Fluorescent Protein-Oxidative Stress Promoter Probe Constructs The Potential for Living Chips , 2001, Journal of biomolecular screening.

[22]  J. Rine,et al.  In vivo examination of membrane protein localization and degradation with green fluorescent protein. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[23]  D. Botstein,et al.  Identification, cloning and sequence determination of the genes specifying hexokinase A and B from yeast. , 1986, Nucleic acids research.

[24]  S. Kain,et al.  Green fluorescent protein (GFP): applications in cell-based assays for drug discovery. , 1999, Drug discovery today.

[25]  W A Weigand,et al.  Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor. , 1999, Biotechnology and bioengineering.

[26]  T. M. Horiagon,et al.  Deletion mapping of the Aequorea victoria green fluorescent protein. , 1996, Gene.

[27]  J. Vandenhaute,et al.  Cloning and sequencing of the inulinase gene of Kluyveromyces marxianus var. marxianus ATCC 12424 , 1991, FEBS letters.

[28]  K. Entian,et al.  Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae , 2004, Molecular and General Genetics MGG.

[29]  W. Bentley,et al.  Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter. , 2000, Biotechnology and bioengineering.

[30]  W W Ward,et al.  Green fluorescent protein as a reporter of gene expression and protein localization. , 1995, BioTechniques.

[31]  S. Daunert,et al.  Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis , 2001, Fresenius' journal of analytical chemistry.

[32]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations , 1983 .

[33]  P. Herrero,et al.  Autophosphorylation of yeast hexokinase PII. , 1988, Journal of general microbiology.

[34]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[35]  W. Bentley,et al.  Expression of green fluorescent protein in insect larvae and its application for heterologous protein production. , 1997, Biotechnology and bioengineering.