EXPRESSION OF RECOMBINANT PROTEIN IN E. coli

EXPRESSION OF RECOMBINANT PROTEIN IN E coli EXPRESSION OF RECOMBINANT PROTEIN Bacterial expression system Escherichia coli is the most commonly used bacterial expression system for expression of the heterologous proteins - Genetic manipulation is easy, - Its culturing is inexpensive, - Expression is fast, - In many cases, the level of expression is high, thus produces large amounts of a specific protein - Majority of foreign proteins are well tolerated, - Permits studies of the structure and function of proteins - Can be useful when proteins are rare cellular components or difficult to isolate Bacterial expression system What type of protein is expressed ? How to get soluble protein expressed in E coli ? Does protein need post-translational modifications for structure/activity? What is the codon usage in expressed protein? • Interrupted translation, which leads to a variety of truncated protein products • Misincorporation of amino acids For instance, lysine for arginine as a result of the AGA codon This can be detected by mass spectroscopy since it causes a decrease in the molecular mass of the protein of 28 Da • Inhibition of protein synthesis and cell growth Disadvantages Inclusion bodies Incapable of producing eukaryotic post-translation modifications – glycosylation – phosphorylation Certain proteins can not fold and form inclusion bodies Deficient in certain tRNAs commonly found in eukaryotic genes Common problems with bacterial expression systems • • • • • Low expression levels: ▪ change promoter ▪ change plasmid ▪ change cell type ▪ add rare tRNAs for rare codons on second plasmid Severe protein degradation: – use proteasome inhibitors and other protease inhibitors – try induction at lower temperature Missing post-translational modification:  co-express with kinases, etc Glycosylation will not be carried out: – use yeast or mammalian expression system Misfolded protein (inclusion bodies): – co-express with a chaperone – try refolding buffers Codon Plus Cell Lines Especially useful for the expression of eukaryotic/human proteins Elements of expression vector MCS: Multi cloning sites, Selectable Marker gene: Antibiotic Resistant gene ORI: Origin Replication Promoter, Repressor, Terminator, Translational initiator, Affinity Tag Co-expression Co-expression from different vectors To ensure plasmid stability, the vectors should have: different selectable markers, usually antibiotic resistance markers different origins of replication Often the copy numbers for the vectors will not be the same This could affect the expression levels of the proteins Therefore, the genes should be cloned into the different vectors (if possible under the control of different promoters) in order to optimize the expression levels Co-expression from one vector The genes are cloned into the same vector and could be expressed from one or more promoters If cloned under the regulation of one promoter (di- or multi-cistronic), the order in which the genes are cloned usually affects the expression levels of the proteins Therefore, several constructs should be made in which the genes are cloned in a different order to optimize the expression of the proteins The pET Expression System One expression system was developed in 1986 by W F Studier and B A Moffatt: creating an RNA polymerase expression system which was highly selective for bacteriophage T7 RNA polymerase www.novagen.com BL21 cell lines Champion pET302/NT-His and pET303/CT-His vectors ROP ORF: Interacts with the pBR322 origin to facilitate low-copy replication in E coli Common Protein Fusion Tags TAG SIZE COMMENT , 2011 Thrombin: recognized Leu-Val-Pro-Arg/Gly-Ser Enterokinase: recognized Asp-Asp-Asp-Asp/Lys Factor Xa: recognized Ile-(Glu or Asp)/Gly-Arg TEV protease: recognized Glu-Asn-Leu-Tyr-PheGln/Gly PreScission Protease is a fusion protein of GST and HRV 3C protease, recognized Leu-Glu-Val-Leu-PheGln/Gly-Pro pET vector system signal seq = signal sequence for potential periplasmic localization I = internal tag N = N-terminal tag C = optional C-terminal tag protease cleavage sites: T = thrombin; E = enterokinase; X = Factor Xa LIC = ligation-independent cloning www.novagen.com The pGEX Expression System (Glutathione S-transferase -GST) The pGEX Expression System Thrombin (serine protease) recognized (LVPR/GS) The pGEX Expression System Factor Xa: A blood clotting protease recognized IEGR/ The pGEX Expression System PreScission Protease recognized Leu-Glu-ValLeu-Phe-Gln/Gly-Pro The pGEX-6P Human rhinovirus (HRV) 3C protease is a cysteine protease, commonly referred to as the PreScission Site TM that recognizes the cleavage site of Leu-Glu-Val-Leu-Phe-GlnGly-Pro, and cleaves between Gln and Gly PreScission Protease is a fusion protein of GST and HRV 3C protease Since the protease is fused to GST, it is easily removed from cleavage reactions using Glutathione Sepharose™ 4B Cleavage of GST tag using thrombin or Factor Xa Prepare pGEX Vector pGEX Cloning Insert into pGEX Vector Separation techniques for proteins ... target protein from proteolytic degradation · Some of the affinity tags (e.g maltose binding protein or MBP) help in the solubilization of the target protein, hence target proteins remain in the... co -expression the protein that is expressed in insoluble aggregates (inclusion bodies) with one or more other proteins •The co -expression of proteins that play a role in regulation of expression, such.. .EXPRESSION OF RECOMBINANT PROTEIN Bacterial expression system Escherichia coli is the most commonly used bacterial expression system for expression of the heterologous proteins - Genetic