Alpha Lifetech Inc. focuses on recombinant proteins' expression in different Biosystems for many years, including prokaryotic and eukaryotic organisms, as well as development of novel high-potency recombinant proteins to meet customers' requirements.
Mammalian cell expression has become the dominant recombinant protein production system for clinical applications because of its capacity for post-translational modification and human protein-like molecular structure assembly. The proteins produced in the mammalian system have excellent structural and functional features that are usually most close to their cognate native form.
The basic procedures included in the process of protein expression are as follows:
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Custom Service & Procedures:
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Phase |
Time |
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I. Plasmid Construction |
3 weeks |
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-- Gene Analysis/Amplification
-- Subclone Design: non-excrete/excrete plasmid, tag selection
-- Integration: Target gene into vector
-- Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing
-- Maxi-prep of recombinant vector DNA |
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II. Transient Gene Expression |
1 week |
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-- Transfection of CHO cells with the recombinant plasmid.
-- Host cells: HEK293, Per.C6 (Option)
-- Culture condition optimized: MOI, time course, cell lines and so on. |
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III. Stable Expression Cell Line (option) |
4-8 weeks |
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-- 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 |
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IV. QC Verification & Small-scale Amplification |
1 week
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-- 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 |
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V. Purification |
1 week |
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-- Protein purification by one-step method: affinity chromatography (Ni-NTA/glutathione column) -- Validation for expression of the recombinant protein by SDS-PAGE （Customer must provide appropriate antibodies if WB is desired) |
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Total |
10-14 weeks |
Service Features:
-- High Success Rate: more than 95%
-- Fusion-Tag Selection: Single Tag or Combined Tags
-- Traceability: Clear Genetic Information of Host Cells
-- Serum Free Medium: BSA Free
-- Low Level of Endotoxin: <0.1 EU/100μg (LAL)
-- Low Level of Host DNA: < 1 ng/g
-- Fermentation Advantage: Various Scales (1, 5, 10, 30, 80L fermenter)
-- Time & Products: Short Time Cycle; High purity (no less than 95%)
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