Production of recombinant proteins in mammalian cells.
(Curr Protoc Protein Sci. 2001 May;Chapter 5:Unit5.10.)
Chen S, Gray D, Ma J, Subramanian S.
Chiron Corporation, Emeryville, California, USA.
The best strategy for consistent production of larger quantities of pure protein is stable expression. Popular hosts for stable expression are Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK-21) cells, myeloma cells, and the transformed kidney cell line 293. Protocols for stable production in CHO cells are described in this unit. Typical methods for transfection using commercially available plasmid expression vectors are described, along with methods to select for stable expression and methods for amplifying the expression level in the transfected cell. Following this, procedures are presented for efficient cell growth to obtain significant amounts of protein product. Support protocols describe freezing of cells, determination of growth rates, determination of specific productivity of cells, preparing samples for assay, and setting up a 10-day shaker-flask growth curve.
(Protein expression reviews)
1: Forstner M, Leder L, Mayr LM.
Optimization of protein expression systems for modern drug discovery.
Expert Rev Proteomics. 2007 Feb;4(1):67-78.
2: Hartley JL.
Cloning technologies for protein expression and purification.
Curr Opin Biotechnol. 2006 Aug;17(4):359-66. Epub 2006 Jul 12.
3: Barnes LM, Dickson AJ.
Mammalian cell factories for efficient and stable protein expression.
Curr Opin Biotechnol. 2006 Aug;17(4):381-6. Epub 2006 Jun 27.
4: Esposito D, Chatterjee DK.
Enhancement of soluble protein expression through the use of fusion tags.
Curr Opin Biotechnol. 2006 Aug;17(4):353-8. Epub 2006 Jun 15.
5: Cox B, Kislinger T, Emili A.
Integrating gene and protein expression data: pattern analysis and profile
Methods. 2005 Mar;35(3):303-14. Epub 2005 Jan 12.
6: Hunt I.
From gene to protein: a review of new and enabling technologies for
multi-parallel protein expression.
Protein Expr Purif. 2005 Mar;40(1):1-22.
7: Cabrita LD, Bottomley SP.
Protein expression and refolding--a practical guide to getting the most out of
Biotechnol Annu Rev. 2004;10:31-50.
8: Greene JJ.
Host cell compatibility in protein expression.
Methods Mol Biol. 2004;267:3-14.
9: Freeman WM, Hemby SE.
Proteomics for protein expression profiling in neuroscience.
Neurochem Res. 2004 Jun;29(6):1065-81.
10: Pearlberg J, LaBaer J.
Protein expression clone repositories for functional proteomics.
Curr Opin Chem Biol. 2004 Feb;8(1):98-102.
11: Yokoyama S.
Protein expression systems for structural genomics and proteomics.
Curr Opin Chem Biol. 2003 Feb;7(1):39-43.
12: Chambers SP.
High-throughput protein expression for the post-genomic era.
Drug Discov Today. 2002 Jul 15;7(14):759-65.
13: Andersen DC, Krummen L.
Recombinant protein expression for therapeutic applications.
Curr Opin Biotechnol. 2002 Apr;13(2):117-23.
14: Lesley SA.
High-throughput proteomics: protein expression and purification in the
Protein Expr Purif. 2001 Jul;22(2):159-64.
15: Heifetz PB, Tuttle AM.
Protein expression in plastids.
Curr Opin Plant Biol. 2001 Apr;4(2):157-61.
16: Konz JO, King J, Cooney CL.
Effects of oxygen on recombinant protein expression.
Biotechnol Prog. 1998 May-Jun;14(3):393-409.
17: Mastrangelo AJ, Betenbaugh MJ.
Overcoming apoptosis: new methods for improving protein-expression systems.
Trends Biotechnol. 1998 Feb;16(2):88-95.
18: Baker RT.
Protein expression using ubiquitin fusion and cleavage.
Curr Opin Biotechnol. 1996 Oct;7(5):541-6.
19: Kurland C, Gallant J.
Errors of heterologous protein expression.
Curr Opin Biotechnol. 1996 Oct;7(5):489-93.
20: Cole PA.
Chaperone-assisted protein expression.
Structure. 1996 Mar 15;4(3):239-42. No abstract available.
(Protein purification reviews)
1: Roy I, Mondal K, Gupta MN.
Leveraging protein purification strategies in proteomics.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Nov 30; [Epub ahead of
2: Franzreb M, Siemann-Herzberg M, Hobley TJ, Thomas OR.
Protein purification using magnetic adsorbent particles.
Appl Microbiol Biotechnol. 2006 May;70(5):505-16. Epub 2006 Feb 23.
3: Banki MR, Wood DW.
Inteins and affinity resin substitutes for protein purification and scale up.
Microb Cell Fact. 2005 Nov 11;4:32.
4: Linhult M, Gulich S, Hober S.
Affinity ligands for industrial protein purification.
Protein Pept Lett. 2005 May;12(4):305-10.
5: Hilbrig F, Freitag R.
Protein purification by affinity precipitation.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jun 25;790(1-2):79-90.
6: Anspach FB, Curbelo D, Hartmann R, Garke G, Deckwer WD.
Expanded-bed chromatography in primary protein purification.
J Chromatogr A. 1999 Dec 31;865(1-2):129-44.
7: Galaev IY.
New methods of protein purification. Affinity ultrafiltration.
Biochemistry (Mosc). 1999 Aug;64(8):849-56.
8: Galaev IY.
New methods of protein purification. Displacement chromatography.
Biochemistry (Mosc). 1998 Nov;63(11):1258-65.
9: Galaev IY.
New methods of protein purification. Expanded bed chromatography.
Biochemistry (Mosc). 1998 Jun;63(6):619-24.
10: Irwin JA, Tipton KF.
Affinity precipitation: a novel approach to protein purification.
Essays Biochem. 1995;29:137-56.
11: Gatlin LA, Nail SL.
Protein purification process engineering. Freeze drying: A practical overview.
Bioprocess Technol. 1994;18:317-67.
Protein expression technique books (Biowww bookshelf)
Protein expression technique books
Protein purification technique books (Biowww bookshelf)
Benchtop books on protein purification.
Plasmid Expression Vectors Carrying the T7 Late Promoter (Molecular Cell Biology)
7. Recombinant DNA and Genomics
Engineered Protein Expression (Cancer Medicine)
Part II Scientific Foundation, Section 1: Cancer Biology 2. Molecular Biology, Genomics, and Proteomics
Protein expression in e.coli problem (Forum)
Suggestion of codon optimization
protein purification and antimicrobial assay (Forum)
problem elution and purification of proteins
protein precipitation after IMAC (Forum)
35kDa protein with His-tag N-terminal precipitation
protein concentration (Forum)
Protein concentration measurement
Endogenous protein and overexpressed protein detection (Forum)
Troubleshooting undetectable endogenous protein
protein elution from gel (Forum)
elute proteins from electrophoretic gel either SDS-PAGE or native
Myofibrillar extraction from mouse heart (Forum)
protocol for the extraction of myofibrillar proteins from atria and ventricles of mouse heart
Protein overexpression with IPTG (Forum)
problem to get a kinase domain overexpressed with 0,5 mM IPTG for 2h at 37 C in supernatant.
GST purification probelms (Forum)
GST fusion protein expression and purification troubleshooting
problem in cleavage of fusion protein (Forum)
cleavage of maltose-binding protein
Purification of enzymatically active protein from PAGE (Forum)
purify the digested product of His-tagged fusion proteins pET System
Protein solubility (Forum)
E.coli expression protein solubility problem
Protein refolding problems (Forum)
refold a purified cysteine protease via dialyis of 6M urea
How to make the soluble protein from the insoluble protein (Forum)
how to purify the soluble protein from the insoluble protein