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，但在免疫亲和纯化中，由于亲和力高，从抗体配体上洗脱目标蛋白有时需要极端的条件，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使用，这些抗体是在不使用目标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洗脱剂从抗肽抗体中洗脱的方法，以避免洗脱过程中目的蛋白的变性。在本工作中，我们研究了抗肽抗体作为免疫亲和纯化配基的可行性，以及利用抗肽抗体针对几种生物活性蛋白的C-端和N-端区进行特异性洗脱的方法。以嵌合α-淀粉酶、重组CD2和胰岛素B链的C-末端和N-末端的多肽为免疫原，制备了抗人CD2和胰岛素B链的抗肽抗体。这些抗肽抗体吸附天然蛋白质和抗原肽。用取代氨基酸的多肽阐明了这些抗肽抗体的抗原识别特性。以抗肽抗体为亲和配基，从发酵液中纯化得到高纯度的蛋白质。这些配体可以区分具有不同C-末端区域的靶蛋白。吸附的蛋白质在温和的pH和/或离子条件下被含有抗原肽的洗脱液特异性洗脱，而不会使生物活性蛋白质变性。低浓度的抗原肽可以有效地洗脱蛋白质。

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.