Recombinant Protein Production in Prokaryotic Systems

Recombinant Protein Production in Prokaryotic Systems

Use of prokaryotic expression systems offers an economical method to achieve production of large amounts of recombinant protein. The Facility provides technical support for production of recombinant protein in bacteria.

The core maintains a repository of inducible bacterial expression vector technologies. (View a list of available expression vectors.) This collection of expression vectors allows for fusion of epitope tags (alone or in combination) to the protein of interest (GOI), including GST (Glutathione-S-transferase), 6 histidines (6His), SUMO, FLAG, or maltose binding protein (MBP) at the NH3- or COOH-terminus of a protein. High level protein expression (analytical or preparative scale) is achieved by IPTG induction of T7/T5 RNA polymerases in high-performance Epicuran Coli (e.g. Rosetta (DE3)).

Available services

Optimization of soluble protein expression

The purpose of this service is to optimize the expression of soluble protein. Because some proteins fold poorly or incompletely when over-expressed in E. coli, the Facility uses several strategies to adjust variables that account for inclusion body formation such as N-terminal protein fusion to soluble tags (e.g. GST, SUMO, MBP), culture medium and aeration, growth temperature (18°C-37°C), expression vector characteristics including promoter activity, and use of bacterial strains engineered to express underrepresented tRNA species during production.

Bacterial expression vectors containing a gene of interest are transformed into a strain of bacteria (e.g. BL21) optimized for recombinant protein expression. An analytical scale (100 ml) induction of protein expression is used to determine the extent of the recombinant protein’s solubility under the defined experimental conditions by SDS-PAGE and Coomassie stain.
Investigators have the option to provide the facility with previously constructed baculovirus transfer vectors, have facility staff engineer the plasmid DNA construct, or to provide the facility with small volumes of previously prepared virus stock.

Timeline:

<1 week

Required Materials:

1-2 µg of bacterial expression plasmid containing gene of interest.

Deliverables:

  • Cryopreserved glycerol stocks of transformed bacterial strain(s)
  • Experimental reports and QC data

 

Preparative production

The laboratory has the capacity to express recombinant proteins in bacteria upto 12 L per a day.

Timeline:

<1 week

Required Materials:

1-2 µg of bacterial expression plasmid containing gene of interest or, cryopreserved glycerol stock.
Induction instructions (e.g. temp., time, IPTG conc.)

Deliverables:

  • Induced cell pellets
  • Experimental reports and QC data