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Learn about How to Design Antigen?

Abstract:       In the research work of protein antibody, it's necessary to carefully design antigen to obtain the best effect from the produced antigen. During the immune process, the antigen not only doesn't strongly respond to the immune reaction, but also produces the specific antibody bound with the target protein. We need to analyse the nature and characteristics of the protein, and then formulate the plan for antigen preparation.

Keywords:    Antigen Design, Antigen Preparation, Antigen Analysis

1. Introduction

First, we shall analyse basic properties of the studied antigen. We can access some databases to check the modification, structural domain features, and splicing of the protein. If the protein has post processing, we shall choose a complete and adult segment for analysis. Take PYGL_HUMAN(UniProt ID: P06737) for example to illustrate:

Amino Acid Sequence of PYGL

Figure 1.  Full Amino Acid Sequence of PYGL

2. Antigen Analysis

2.1. Analysis for Basic Properties

The figure below shows some modifications of the protein. Prokaryotic expression system doesn't have the function of modification and should be avoided. Some proteins have the signal peptide. There are segment reports for transmembrane domain, intracellular domain and extracellular domain in some databases. The signal peptide of eukaryotic protein can't be processed in prokaryotic system and make the protein expression impossible. Thus, it should be removed during prokaryotic expression. For antibody preparation, extracellular domain is preferentially selected. The transmembrane domain doesn't contribute to antibody preparation and is hardly expressed in prokaryotic system. Hence, signal peptide and transmembrane domain should be removed in antigen preparation using prokaryotic expression.

Molecule Processing and Amino Acid Modifications

Figure 2.  Molecule Processing and Amino Acid Modifications

2.2.  Analysis for Transmembrane Domain

There are many databases for analysing transmembrane domain. During analysis for transmembrane domain, the result can be checked by copying the whole protein sequence into the text box. Detailed comments and illustrations have been specified for some most studied proteins in some web databases.

TMHMM Posterior Probabilities

Figure 3.  TMHMM posterior probabilities for WEBSEQUENCE

2.3.  Analysis for Homology

The principle of specificity refers to the analysis for homology with other proteins in the same specie by the database. Meanwhile, the homology with immunized host should be indexed to avoid the immunologic tolerance of the host. We choose human and rabbit for BLASTP. The specificity is very high.

BLASTP Program

Figure 4.  BLASTP Program

2.4.  Analysis for Hydrophilicity, Immunogenicity, Exposure

The hydrophilicity, immunogenicity and exposure of 745-847 aa at the end of C-terminal is very high.

Analysis for Hydrophilicity, Immunogenicity, Exposure

Figure 5.  Analysis for Hydrophilicity, Immunogenicity, Exposure

Finally, analysis for rare codons: The result can be viewed by inputting the full gene sequence into the prokaryotic expression host codons analysis database. If two or above rare codons appears continuously, the expression will be very difficult.

As the recombinant protein used by antigen, the full-length of the protein is a little long. The expression of full length is difficult. We can select the segment with better antigenicity for partial recombinant expression. We determine the solution finally. Above all, it's more beneficial to antibody preparation, when we select the 745-847 aa segment to induce prokaryotic expression. Translate the segment into the codon gene preferred by Escherichia coli (Sequence is as follows); Insert the whole synthesized gene into prokaryotic expression vector; Transform Escherichia coli vector to induce the expression.

Codon Gene Sequence

Figure 6.  codon gene sequence preferred by Escherichia coli

There are 103 amino acids in the protein. The low molecular mass makes the purification difficult and doesn't meet the principle with high molecular mass and complex structure antigen. Thus, FineTest tries its best to choose high molecular mass fusion tags for expression. FineTest has patented fusion tags for your choice, and also offers high quality prokaryotic proteins and antigens to scientific researchers.