Protein Expression

1. Bacteria expression service

I. Subcloning

• Amplification/Isolation of gene out of customer supplied vector and subcloning into bacterial expression vector.
• Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.

II. Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.

• Transformation of recombinant constructs into high-efficiency expression bacterial strain.
• Expression optimization: induction time, temperature and IPTG concentration.
• SDS-PAGE, Coomassie blue staining and Western blot with tag-specific or custom-supplied antibody.

III. Large-Scale Culture

• One liter of bacterial culture will be induced with IPTG and harvested for protein purification.

IV. Purification

• Protein purification using a Ni-NTA (for 6 x HIS-tagged proteins) or glutathione (for GST-tagged proteins) column at either native or denatured condition.

2. Baculoviral expression service

I. Clone gene of interest into baculoviral transfer vector¹

• Amplification/Isolation of gene out of a customer supplied construct and subclone into baculoviral transfer vector.
• Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.
• Maxi-prep of the recombinant vector DNA.

II. Generation, identification, and plaque purification of baculoviral colonies expressing target proteins

• Transfection of insect cells (SF9) with the recombinant transfer vector (gene of interest cloned in the vector) and baculoviral DNA.
• Isolation and plaque-purification of ten baculoviral recombinant clones.
• Test for expression of the recombinant protein by Western blot (customer must provide appropriate antibodies) if desired.

III. Amplification of recombinant baculovirus for high titer stock

• Generation of 500 ml of high-titer viral stock from low-titer stock solution (approximately 10 ml is required) of a single recombinant.
• Additional rounds of amplification may be needed if initial virus titer is too low.
• Determination of the titer by end-point dilution assay.

IV. Large-scale culture of recombinant baculovirus

• One liter of insect cells will be infected with high-titer virus stocks (25-30 ml of high titer virus required).

V. Purification²

• Protein purification using a Ni-NTA (for 6 × HIS-tagged proteins) or glutathione (for GST-tagged proteins) column at either native or denatured condition.

Note:

1. If the gene is not provided, Abgent will charge an additional $ 1,000 (cloning and sequencing included), and 1-2 additional weeks are needed to amplify the gene from the library and confirm sequences.
2. Abgent does not guarantee the yield of purified protein due to protein to protein variabilities. Additional large-scale culture may be needed to obtain the desired amount of protein for antibody production.

3. Mammalian expression service

I. Generation of recombinant expression construct

• Amplification/isolation of the gene of interest out of the customersupplied templates and subcloning it into a eukaryotic expression vector.
• Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.

II. Maxi-prep of endo-free plasmid

• Transformation of recombinant constructs into E.coli bacterial strain.
• Amplification/purification of the recombinant vector DNA.

III. Transient protein expression

• Transfection of target cells.
• Validation of expression (i.e., Western blot, Elisa or FACS).

V. Small scale expression

• Pilot expression (1-3L) and optimization.

VII. Purification and characterization

• Affinity, ion exchange, gel filtration or hydrophobic chromatography (alone or in combination).
• Characterization by Western blot or customer specified assay.