Nakayama G R, Valkirs G, McGrath D, Huse W D
Ixsys, Inc., San Diego, CA 92121, USA.
Immunotechnology. 1996 Sep;2(3):197-207. doi: 10.1016/s1380-2933(96)00049-8.
Antibody fragments have been expressed on the major coat protein of filamentous phage using a gene VIII expression system, but with low copy numbers (averaging 0.2 Fab/phage).
As a general strategy to increase copy number, the phage vector was optimized by site directed mutagenesis.
One mutation was the introduction of a random six amino acid tether between the heavy chain and pseudo gene VIII to form a phage library. The other mutation was the removal of two cysteine residues which form a disulfide bond between the heavy and light chains. An assay was developed to measure Fab concentrations and used to calculate the average number of copies displayed on phage.
Phage libraries containing random tethers were panned, and clones containing a proline rich motif were extracted. Removing the interchain disulfide had a greater effect on copy number and soluble Fab concentrations in the periplasmic space of the bacterial cultures.
A tenfold increase in the copy number was achieved using the optimized vector. Incorporation of these vector mutations may be a general strategy for optimizing Fab display on the major coat protein of bacteriophage M13.
利用基因VIII表达系统，已在丝状噬菌体的主要外壳蛋白上表达抗体片段，但拷贝数较低（平均每个噬菌体0.2个Fab）。
作为增加拷贝数的通用策略，通过定点诱变对噬菌体载体进行优化。
一种突变是在重链和伪基因VIII之间引入一个随机的六个氨基酸的连接子，以形成一个噬菌体文库。另一种突变是去除在重链和轻链之间形成二硫键的两个半胱氨酸残基。开发了一种测定法来测量Fab浓度，并用于计算噬菌体上展示的平均拷贝数。
淘选含有随机连接子的噬菌体文库，并提取含有富含脯氨酸基序的克隆。去除链间二硫键对细菌培养物周质空间中的拷贝数和可溶性Fab浓度有更大影响。
使用优化后的载体，拷贝数增加了十倍。纳入这些载体突变可能是优化噬菌体M13主要外壳蛋白上Fab展示的通用策略。
