Purification of bioactive proteins by specific elution with antigen peptides

对于利用基因工程微生物或细胞产生的生物活性蛋白的纯化，免疫亲和层析是非常有效的。然而，在免疫亲和纯化中，由于抗体配体的高亲和性，有时需要从抗体配体中洗脱目标蛋白，这可能导致其变性。为了解决这个问题，我们研究了抗肽抗体的利用，这些抗体是在不使用靶蛋白作为抗原的情况下获得的。我们还尝试了用抗原肽作为抗肽抗体的洗脱液的特异性洗脱方法，以避免在洗脱步骤中目标蛋白的变性。本研究利用针对几种生物活性蛋白的c端和n端区域的抗肽抗体，研究了抗肽抗体作为免疫亲和纯化配体的可行性和特异性洗脱方法。利用嵌合α -淀粉酶C端和n端对应的肽段作为免疫原，获得了针对重组CD2和胰岛素b链C端和n端区域的抗肽抗体。这些抗肽抗体吸附天然蛋白，以及抗原肽。这些抗肽抗体的抗原识别特性是用取代氨基酸的肽来澄清的。利用抗肽抗体作为亲和配体，从发酵液中纯化出高纯度的蛋白。这些配体可以区分具有不同c端区域的靶蛋白。所吸附的蛋白被含有抗原肽的洗脱液在温和的pH和/或离子条件下特异性洗脱，而不使生物活性蛋白变性。低浓度抗原肽可有效洗脱蛋白质。

For purification of bioactive proteins produced by use of genetically engineered microorganisms or cells, immunoaffinity chromatography is very effective, However, in immunoaffinity purification, elution of the target proteins from antibody ligands sometimes requires extreme conditions which may cause their denaturation, because of the high affinity. To solve the problem, we studied utilization of anti-peptide antibodies, which were obtained without use of a target protein as antigen. We also tried specific-elution method using an antigen peptide as an eluent from anti -peptide antibodies to avoid denaturation of a target protein during the elution step.In this work, the feasibility of anti-peptide antibodies as ligands for immunoaffinity purification and the specific elution method were studied by use of anti-peptide antibodies against the C-terminal and N-terminal regions of several bioactive proteins. Anti-peptide antibodies against the C-terminal and N-terminal regions of chimeric alpha-amylase, recombinant CD2 and insulin B-chain were obtained by using peptides corresponding to the C- and N-terminal regions as immunogens. These anti-peptide antibodies adsorbed the native proteins, as well as the antigen peptides. The characteristics of antigen recognition of these anti-peptide antibodies were clarified by use of peptides having replaced amino acids. The proteins were purified from fermentation broth to high purity by use of the anti-peptide antibodies as affinity ligands. These ligands could discriminate the target proteins having different C-terminal regions. The adsorbed proteins were specifically eluted by the eluents containing the antigen peptides under mild pH and/or ionic conditions without denaturation of bioactive proteins. Low concentrations of antigen peptides could effectively elute the proteins.