Overexpression of Membrane Proteins

A good beginning is half the success - the membrane protein expression strategy

Design of expression constructs

The location of the N-terminus of a transmembrane protein on either the intracellular side or the extracellular side determines the topology, and folding of the membrane protein. We first analyze the topology of a membrane protein and determine if a signal peptide should be added to the N-terminus of a transmembrane protein based on its topology. The synthesis, folding, and maturation of a membrane protein are the products of the cooperative work of ribosomes and endoplasmic reticulum, in which the genetic coding of a protein plays a significant role in deciding the yield and fate of a membrane protein. We use state-of-art artificial intelligence to optimize the nucleic acid sequence of a membrane protein, to ensure a deliberate balance of protein yield, correct folding and maturation for a membrane protein. The AlpHelix scientists have expertise in designing the right expression constructs for membrane proteins and making high-purity protein products, thus enabling our customers to get out of the tedious and challenging work of producing membrane proteins for drug discovery.

Selection of expression systems

We have a wealth of protein overexpression systems, including E. coli, yeast, insect and mammalian cell expression systems, to provide customers with high-quality protein products. In addition, we optimize expression constructs according to customer requests, so as to save customers’ time and cost on membrane protein expression and purification.

A Comparison of Four Protein Expression Systems
Expression systems Advantages Disadvantages
E. coli expression system Clear expression background, high expression level, simple operation, short culture time, and resistant to contamination by other microorganisms No post-translational modifications; endotoxin contamination (see AlpHelix’ solutions)
Yeast expression system Simple operation, high expression level, low cost, and having post-translational modifications for a protein Protease contamination could be serious; N-glycosylation and O-glycosylation are distinct from those of insect cells and mammalian cells (see AlpHelix’ solutions)
Insect cell expression system Efficient expression, complete post-translational modifications, easy purification of proteins from serum-free medium, no endotoxins Baculovirus needs to be prepared. The insect cells, after virus infection, are eventually all dead. This makes continuous cell culture and protein expression impossible. N-glycosylation and O-glycosylation of insect cells are not identical to those of mammalian cells. More costly than prokaryotic and yeast expression systems
Mammalian expression system Having complete post-translational modifications of a protein, including N-glycosylation and O-glycosylation. The overexpressed proteins are mostly similar to those from natural sources Low yield; some glycosylation is unstable. The most expensive expression system

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