Phase

Time

I. Plasmid Construction

3 weeks

-- Gene Analysis/Amplification

-- Subclone Design: non-excrete/excrete plasmid, tag selection

-- Integration: Target gene into the vector

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

-- Maxi-prep of recombinant vector DNA

II. Transient Gene Expression

1 week

-- Transfection of host cells with the recombinant plasmid.

-- Culture condition optimized: Induce time, Medium.

III. Stable Expression Cell Line (option)

3-6 weeks

-- Transfect cells with plasmid and select living cells from a screening condition

-- Single-cell cloning: Dilute cells into single ones and culture them in a 96-well plate

-- Fermentation or cell factories culture

IV. QC Verification & Small-scale Amplification

1 week

-- Validation for expression of the recombinant protein by SDS-PAGE (Customer must provide appropriate antibodies, if desired WB)

-- Generation of 500 ml of high-density cell harvest

V. Purification

1 week

-- Protein purification using one-step method: affinity chromatography (Ni-NTA/glutathione column)

-- Validation for expression of the recombinant protein by SDS-PAGE

Total

9-13 weeks