Membrane Protein Constitutive Overexpression Cell Line Development

Creative Biolabs offers membrane protein constitutive overexpression cell line development service for membrane protein expression, membrane protein antibody discovery, membrane protein functional studies, membrane protein drug screening, etc.

Membrane protein constitutive overexpression cell line is a time-consuming technique that usually necessitates the stable integration of recombinant DNA into the host cell genome. Antibiotic selection can identify stable integrants since the expression vector contains an antibiotic resistance gene. Integration of the transgene into the host cell genome may either be random or the host cell may be engineered to contain a specific sequence recognized by a recombinase that allows targeted integration. The expression levels are highly dependent on where the transgene integrates when random integration is employed. After that, selection of clonal cells is required to find highly expressing cell lines that are stable over time in culture.

Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) and for gene up-regulation (by using ORF of gene of interest). We offer 3^rd generation lentivirus to get a stable expression for most mammalian cell lines. The advantage of using the 3^rd generation lentivirus is that is very safe and they are replication incompetent.

Fig.1. Lentivirus expression system.

Cell Line Selection

All mammalian cell lines should provide a near-native environment for overexpressed mammalian membrane proteins, with the appropriate post-translational modifications and lipid environment. Human embryonic kidney cells (HEK293), baby hamster kidney cells (BHK-21), monkey kidney fibroblast cells (COS-7), and Chinese hamster ovary cells (CHO) are the most commonly utilized cell lines in the context of mammalian membrane protein production. The homogeneity of the final purified protein is another significant feature to consider when choosing a mammalian cell line. N-glycosylation may be necessary for optimal mammalian membrane protein expression in some cases, but this results in a very diverse protein population with 10-20 kDa of flexible sugar chains.

• Vector Construction

With the lentivirus system, we generated a stable cell line overexpressing the target protein of interest (X). We provide sequence analysis, construct design, gene synthesis, vector construction, plasmid quality control, and sequencing alignment.

Fig.2. Vector construction.

• Lentivirus Packaging

With the lentivirus packaging system, we produced the lentivirus in high-quality titer. Titer determination, sterility testing, mycoplasma free testing will be conducted to ensure the quality of lentivirus.

Fig.3. Lentivirus Packaging.

• Stable Cell Line Quality Control

After lentivirus transfection, we conducted puromycin screening to kill the un-transfected cells. FACS was used to validate the membrane protein expression. Then we use limited dilution method to select the single clone. Membrane protein expression was validated after single clone expansion. Quality controls include membrane protein expression, cell viability after recovery, mycoplasma free testing.

Fig.4. Stable Cell Line Quality Control.

With extensive experience, Creative Biolabs offers a wide range of services for membrane protein stable cell line development. We are committed to your most trusted partner to meet all your needs. For more information, please contact us and our team will get back to you as soon as possible.

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