The Protein Production Platform
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 From cDNA to milligram amounts of protein in as little as four weeks.
 Contact us at mwchen@ntu.edu.sg for more information.


About   Working with PPP   Cloning   Large-scale expression   BEVS   Mammalian expression   Expression vectors   FAQ   



Cloning

The main advantage of having a capacity of this magnitude is the ability to generate multiple constructs of the target proteins rather than focusing only on the full-length versions. A “multi-construct approach” based on protein domains of interest dramatically increases the likelihood of obtaining soluble and correctly folded protein at the end of the pipeline1. Although originally from a structural biology setting, we have found that this approach is amenable to other fields of research as well.


  • In-silico to in-vitro
    Use the LIMS to create and manage your protein constructs
  • High-throughput cloning
    The platform will clone your protein constructs into E. coli
  • High-throughput protein purification
    Your protein constructs will be purified using IMAC and SEC
  • Purification using affinity chromatography
    All constructs will be fused to a N-terminal cleavable HIS6-tag
  • Expression in insect cells for difficult proteins
    We are currently implementing high-throughput cloning using baculovirus
  • Detergent screening
    Transmembrane proteins will be screened for optimal detergent conditions
  • Multiple quality control steps
    All protein batches will be verified using mass spectrometry and/or sequencing
  • We deliver!
    Receive plasmids, expression strain glycerol stocks and protein batches

All cloning is performed in 96-well format using ligation-independent cloning (LIC) technology in Escherichia coli 2,3. Usually, all targets are fused with an N-terminal hexa-histidine tag to aid in downstream purification and a TEV-protease cleavage site for optional tag removal. The main vector in use is pNIC28-Bsa4 (GenBank accession EF198106), developed by Opher Gileadi at SGC Oxford. We use a RecA- strain of E. coli for the initial cloning work and plasmid production followed by a re-transformation of the construct into an expression strain (typically, a T1-phage resistant BL21-DE3 Rosetta strain developed in house). All clones will be put through a small-scale analytical expression screen to assess the likelihood of successful large-scale protein expression and purification. Basically, we use 96-deep well blocks to grow all cultures at a volume of 1 mL Terrific Broth. Once the optical density at 600nm reaches 2-3, the temperature will be lowered from 37ºC to 18ºC and protein production induced with IPTG. After over-night cultivation, we take out a small sample from the liquid culture and verify the presence of target protein using SDS-PAGE. We also continue to perform cell lysis, clarification and micro-scale immobilized metal-ion affinity chromatography (IMAC) to assess the levels of soluble and purifiable target protein from each clone.


  1. The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins
    Gräslund S, Sagemark J, Berglund H, Dahlgren LG, Flores A, Hammarström M, Johansson I, Kotenyova T, Nilsson M, Nordlund P, Weigelt J
    Protein Expr Purif. 2008 Apr;58(2):210-21
  2. Protein production and purification
    Nat Methods, 2008 Feb;5(2):135-46
  3. High-throughput production of human proteins for crystallization: The SGC experience
    Pavel Savitsky,a James Bray,a Christopher D.O. Cooper,a Brian D. Marsden,a,b Pravin Mahajan,a Nicola A. Burgess-Brown,a and Opher Gileadia
    J Struct Biol. 2010 October; 172(1): 3–13.