Cloning and expression of in vivo refolded antibody fragment

A process for expression and recovery of an in-vivo refolded recombinant antibody fragment in a soluble form comprising;

• a. providing a microbial host cell overexpressing disulfide isomerase with non-expression of enzymes for reducing ability of cytoplasm, said cell comprising a DNA construct encoding light and heavy chain of a recombinant antibody fragment;
• b. culturing said microbial host cell of step (a) in a complex nutrient medium comprising glycerol at 30° C., pH 7 to obtain a culture;
• c. reducing temperature of the culture and adding IPTG to said culture to induce expression to obtain a culture broth comprising in-vivo refolded recombinant antibody fragment;
• d. centrifuging the culture broth of step (c) to obtain a cell mass;
• e. disrupting the cell mass of step (d) to obtain a cell lysate;
• f. centrifuging the cell lysate of step (e) to obtain a first supernatant;
• g. precipitating the first supernatant of step (f) at pH 4.0 followed by centrifugation to obtain a second supernatant;
• h. subjecting the second supernatant of step (g) to ultrafiltration to obtain the in-vivo refolded recombinant antibody fragment in a retentate fraction;
• i. subjecting said retentate fraction of step (h) to a multimodal chromatography and an affinity chromatography to obtain the purified in-vivo refolded recombinant antibody fragment in a soluble form.