Common Problems and Solutions for Prokaryotic Expression Experiments of Recombinant Antibodies-Special topic on antibody modification-Tekbiotech-Yeast Display Service,Phage display technology

Common Problems and Solutions for Prokaryotic Expression Experiments of Recombinant Antibodies

I. What are the hosts for prokaryotic expression of recombinant antibodies?

Escherichia coli is the most commonly used host mainly because of its simple and inexpensive culture method, rich supporting vectors and strains, clear screening platform, fast production cycle, and simple requirements for culture medium. In addition to E. coli, Pseudomonas halogenus and Pseudomonas putida can also be used for antibody production, with higher production efficiency than E. coli. Gram-negative bacteria secrete proteins in the periplasm, which are restricted by space and cellular mechanisms. rAb expression in Gram-positive bacteria (such as Bacillus megaterium, Bacillus subtilis, Bifidobacterium longum, Lactobacillus paracasei, Vibrio choleraesuis, etc.) that lack an outer membrane overcomes this limitation, allowing proteins to be expressed extracellularly, showing high-yield antibody expression and secretion.

II. What is the effect of codon optimization on prokaryotic expression of antibodies?

Codon shift occurs when the frequency of the optimal codon in the expressed target gene is significantly different from that of the expression host, and the host does not match the tRNA that regulates the translation process. Codon shift affects polypeptide folding, mRNA sequence and stability, and the accuracy of amino acid insertion. The mRNA sequence determines the binding of ribosomes and proteins, thereby regulating the overall splicing and translation machinery that affects protein synthesis. Codon deviation can be addressed by codon optimization of the target gene to adapt to the host's tRNA pool (codon optimization can be performed using online software such as Optimizer, Java Codon Adaptation Tool, or GeneOptimizer) or by modifying the host to increase the availability of underrepresented tRNAs.

III. What is the impact of gene optimization on prokaryotic expression of antibodies?

The variable regions in antibodies contain highly diverse sequences from subsequent VDJ recombination and somatic hypermutation, which endow the antibody molecule with specificity and affinity. These diverse sequences can sometimes compromise the stability of rAbs, making them prone to aggregation and reducing expression. Certain amino acids in the antibody structure (framework region or complementary determining region (CDR)) are not conducive to its expression in rAb form, resulting in low yields, especially in prokaryotic hosts. Replacing these amino acids with favorable amino acids can improve antibody expression.

IV. What is the impact of vector components on prokaryotic expression of recombinant antibodies?

The ideal vector system should enable efficient transcription, mRNA processing, RNA transport, and translation of the primary mRNA transcript, thus enabling stable protein production. An efficient vector system can not only increase yields, but also simplify antibody processing and purification. The expression of scFv-Fc was increased by optimizing the components of the vector, such as promoters, leader sequences, fusion proteins, and termination signals. Commonly used promoters are T7 promoter, lac promoter, synthetic trp promoter, and trc promoter. Position effects can be overcome by using insulator elements, ubiquitous chromatin opening elements (UCOEs), scaffold or nuclear matrix attachment regions (A/MARs), and stabilizing and anti-repressing elements (STARs), thereby quickly establishing stable high-yield clones.

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