Genetic Structures and Conditions of Their Expression, Which Allow Receiving Native Recombinant Proteins with High Output
¹Federal State Budgetary Institution «Research Institute of Experimental Medicine», North Western Branch of the RAMSs, Saint Petersburg, Russian Federation
²2Hannover Medical School, Hannover, Germany
*Corresponding author: Prof Michael M. Shavlovsky, PhD, ScD, Head of Human Molecular Genetics Laboratory, Department of Molecular Genetics, Federal State Budgetary Institution «Research Institute of Experimental Medicine», North Western Branch of the RAM S, 12, Acad. Pavlov str., 197376, Saint Petersburg, Russian Federation. Tel: 7-812-2343356 Fax: 7-812-3205849. E-Mail:firstname.lastname@example.org
We investigated the possibility of obtaining native recombinant amyloidogenic proteins by creating genetic constructs encoding fusion proteins of target proteins with Super Folder Green Fluorescent Protein (sfGFP). In this study, we show that the structures, containing the sfGFP gene, provide a synthesis, within a bacterial system, of fusion proteins with minimal formation of inclusion bodies. Constructs containing genes of the target proteins in the 3'-terminal region of the sfGFP gene followed by a polynucleotide sequence, which allows for affinity purification fusion proteins, are optimal. Heating bacterial cultures before the induction of the expression of recombinant genes in 42°C for 30 min (heat shock) was found to increase the output of the desired products, thus practically avoiding the formation of insoluble aggregates.
1. Shavlovsky MM. Molecular and genetic basis of pathogenesis of amyloidosis. Academic Medical Journal 2010; 10(4):63-81.
2. Buxbaum JN. Animal models of human amyloidoses: Are transgenic mice worth the time and trouble? FEBS Letters 2009; 583:2663–2673.
3. Sipe JD, Benson MD, Buxbaum JN, Ikeda S-I, Merlini G, Saraiva MJM, Westermark P. Amyloid fibril protein nomenclature: 2010 recomendations commttee of the International Society of Amyloidosis. Amyloid 2010; 17(3-4):101-104.
4. Solovyov KV, Gasteva AA, Egorov VV, Aleinikova TD, Sirotkin AK, Schwarzman AL, Shavlovsky MM. The role of the C-terminal fragment of human transthyretin in abnormal fibrillogenesis. Biochemistry 2006; 71:672 – 680.
5. Egorov VV, Solovyov KV, Grudinina NA, Lebedev DV, Isaev-Ivanov VV, Kiselev OI, Shavlovsky M.M. Atomic force study of peptides homologous to beta-domain of alpha-lactalbuins. Protein and Peptide Letters 2007; 14:471-474.
6. Polyakov DS, Grudinina NA, Solovyov K, Egorov VV, Sirotkin AK, Aleinikova TD, Totolyan Areg A. Shavlovsky MM. β2- microglobulin amyloidosis: fibrillogenesis of natural and recombinant human β2-microglobulins. Academic Medical Journal 2010; 10(2):40-49.
7. Solovyov KV, Polyakov DS, Grudinina NA, Egorov VV, Morozova IV, Aleynikova TD, Shavlovsky MM. Expression in e.coli and purification of the fibrillogenic fusion proteins ttr-sfgfp and β2m-sfgfp, Preparative biochemistry and biotechnology 2011; 41(4):337-349.
8. Polyakov DS, Grudinina NA, Solovyov K, Egorov VV, Sirotkin AK, Aleinikova TD, Totolyan Areg A, Shavlovsky MM. Production of recombinant human β2-microglobulin and its fibrillogenesis at low and neutral pH. Molecular Medicine 2011; 2:36-39.
9. Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 2006; 24: 79-88.
10. Sambrook J, Fritsch EF, Maniatis T, Molecular cloning, USA, Cold Spring Harbor Laboratory Press 1989; 174–175.
11. Macrides SC. Strategies for achieving high-level expression of genes in Escherihia coli. Microbiological Reviews 1996; 60(3):512-538.
The fully formatted PDF version is available.
Int J Biomed. 2012; 2(1):45-49. © 2012 International Medical Research and Development Corporation. All rights reserved.