The PEF production pipeline provides a quick snapshot of the services we offer to help fast-track your research.  From the Gene-to-Protein complete package to any stage in-between, we can customise to suit your project. Contact us to discuss how we can help support your research. Download the complete list as a pdf here.

Jump to Molecular Cloning Jump to Small-Scale Expression Jump to Large-Scale Expression Jump to Purification Jump to Characterisation and Analysis

 Gene to Protein

E. coli, P. pastoris, Insect cell/baculovirus, Mammalian cell

  • Clone the gene of interest into chosen expression vector
  • Small-scale expression screening
  • Large-scale (≥1 L) expression culture
  • Primary purification

 Molecular Cloning
 Gene synthesis

Generate the DNA sequence of a gene of interest. Codon optimisation (optional) for the host expression system.

 Expression construct

Clone the gene of interest into chosen expression vector. DNA sequence verification of cloned construct.

 Mutagenesis

Single or multiple nucleotide base change e.g. amino acid substitution, insert/delete restriction enzyme site, stop codon and small tag. DNA sequence verification of cloned construct.

 Plasmid production

Production and purification of plasmid DNA from mini- to mega-prep scale.

 Small-scale Expression Screening

 E. coli

Expression screen in multiple conditions.  Each construct tested in two different expression strains, two temperatures and two media. Analyse expression and Ni-NTA binding by SDS-PAGE. Resin-binding assay for polyhistidine-tagged constructs only. Determine optimal expression condition.

 Pichia pastoris

Expression screen of 10 yeast clones per construct. Analyse expression and Ni-NTA binding by SDS-PAGE. Resin-binding assay for polyhistidine-tagged constructs only. Determine optimal expression condition.

 Insect cell/baculovirus

Expression screen in multiple conditions. Each construct tested in two cell lines and at two temperatures. Analyse expression and Ni-NTA binding by SDS-PAGE and/or Western blot. Resin-binding assay for polyhistidine-tagged constructs only. Determine optimal expression condition.

 Mammalian cell

Transient expression screen in two cell lines. Analyse expression and Ni-NTA binding by SDS-PAGE and/or Western blot. Resin-binding assay for polyhistidine-tagged constructs only. Determine optimal expression condition

 In vitro cell-free

Expression screen using Leishmania tarentolae lysate. Analyse expression by SDS-PAGE.

 Large-scale Protein Expression

E. coli or Pichia pastoris expression in shake flask

Expression culture at ≥ 1 L in shake flasks. Cells and/or supernatant collected at optimal time of harvest. Analysis by SDS-PAGE.

Insect cell/baculovirus expression in shake flask

Scale-up of virus stock. Expression culture at ≥ 1 L in shake flasks.  Cells and/or supernatant collected at optimal time of harvest. Analysis by SDS-PAGE and/or Western blot.

Mammalian cell transient expression in shake flask

Maxi-prep plasmid production. Expression culture at ≥ 1 L in shake flasks. Cells and/or supernatant collected at optimal time of harvest. Analysis by SDS-PAGE and/or Western blot.

E. coli or Pichia pastoris fermentation in bioreactor

Fermentation in a stirred tank bioreactor, from 2 L to 20 L. Analysis by SDS-PAGE.

Insect or mammalian cell expression in bioreactor

Expression in a WAVE bioreactor, from 1 L to 25 L. Analysis by SDS-PAGE and/or Western blot.

 Protein Purification

Primary purification from cell pellet

Sample extraction followed by affinity chromatography (e.g. IMAC, GST, MBP, Strep(ll), heparin). Analysis by SDS-PAGE.

Primary purification from supernatant

Tangential flow ultrafiltration for sample concentration and buffer exchange.  Primary purification by affinity chromatography (e.g. IMAC, GST, MBP, Strep(ll), heparin, Protein A). Analysis by SDS-PAGE.

Secondary or polishing purification

Secondary or polishing purification to improve product purity by chromatography techniques such as affinity, ion exchange, hydrophobic interaction or gel filtration.  Analysis by SDS PAGE.

Fusion tag removal

Scout optimal protease cleavage condition to remove fusion tag from protein. Proteases used are Enterokinase, Factor Xa, HRV-3C, SUMO, TEV or Thrombin. Chromatography to recover untagged protein. Analysis by SDS PAGE.

Endotoxin removal

Removal of endotoxin from purified protein. Analysis by SDS-PAGE and endotoxin determination by FDA-licensed LAL cartridges.

 Protein Characterisation and Analysis

Endotoxin measurement

Endotoxin level determination by FDA-licensed LAL cartridges (for samples prior to animal studies)

Mass spectrometry - intact protein analysis

Accurately determine native protein molecular weight of purified sample

Mass spectrometry - peptide mass fingerprint

Identification of purified protein or excised band from polyacrylamide gel

Analytical size exclusion chromatography

Estimate native protein molecular weight, determine multimeric structure and degree of aggregation

Dynamic light scattering

Determine size distribution profile of proteins and protein complexes. Suitable for high throughput detection of protein aggregation, thermal stability and storage conditions

Asymmetric flow-field flow-fractionation

Accurately determine size distribution profile of virus-like particles and protein complexes

Transmission electron microscopy

Imaging and morphological characterisation of virus-like particles and protein complexes