Affiliations: [a] Protein Research, Department Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, New Borg El-Arab, Alexandria, 71783, Egypt | [b] Bioprocess Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, New Borg El-Arab, Alexandria, 71783, Egypt
Correspondence:
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Corresponding author: E.M. Redwan, Protein Research Department, GEBRI, Mubarak City for Scientific Research and Technology Applications, New Borg El-Arab, Alexandria, 71783, Egypt. Tel.: +20 101 57 11 72, +203 45 93 422; Fax: +203 45 93 423; E-mail: [email protected]
Abstract: Polymeric immunoglobulin (dimeric IgA and pentameric IgM) molecules can assembly by using the immunoglobulin J (joining) chain and across the epithelial cell layers. Based on its amino acid and gene sequences data, disulfide bond (2 bonds) assignment secondary structure predictions, and chemical properties, a model for J-chain folding has been proposed. However, the crystal structure of the J-chain protein is still far from obtained, because the J-chain expression and its protein downstream has a permanent aggregation problems, due to its two free thiol groups. Our work focused on the chemical blocking of free cysteines-SH or to mutate these cysteines into serine residues. The chemical blocking yielded partially soluble proteins with new structures (carboxyamidomethyl cysteine and carboxyamidomethyl methionine) at cysteine and methionine residues. While mutate the cysteines15,69 into serine has been yielded a complete soluble (11.5 mg/l) J-chain protein which migrate (SDS-PAGE) at 27 KDa. We were used pET22b expression vector and E. coli BL21 (DE3) to produce the J-chain protein. Fed-batch strategy was employed to maximize the protein concentration by using the manually predetermined exponential feeding rate. The process was automated through a computer aided data bioprocessing system AFS-BioCommand multi-process management program to regulate the cell growth rate by controlling interactively both the nutrient feed rate, temperature, pH and agitation speed based on dissolved oxygen. Maximum dry cell weight concentration in the bioreactor was 78 g/l after induction.
Keywords: J chain, sulfahydryl group, blocking, idoacetamide, direct sit mutagenesis FPLC, MALDI-TOF-MS
