ABSTRACT
Human Interferon beta [INF-beta] is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-beta-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b[+] with T7 promoter. Recombinant human beta interferon [rhINF-beta] was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 [DE3]. The sugar, Isopropyl-beta-D-thiogalactopyranoside [IPTG] was used as a chemical inducer for rhINF-beta production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-beta protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD[600nm] = 3.42 was found to be 35% of the total protein content of the strain which translates to 0.32 g L[-1]. The constructed vector could efficiently overexpress the rhINF-beta into the periplasmic space of E. coli. The obtained yield of the produced rhINF-beta was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins
ABSTRACT
Human granulocyte colony-stimulating factor [hG-CSF] can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment
Subject(s)
Plastids , Lettuce , Gene ExpressionABSTRACT
Antibodies provide a suitable tool in fundamental research and their high affinity and specificity make them invaluable for diagnostic and therapeutic applications. A promising alternative to conventional antibodies are the heavy chain antibodies [VHH] of Camelidae having short length, high solubility and stability are preferred to other antibody derivatives. In this study, our goal was production of recombinant VHH antibody fragments [against cancer associated mucin, MUC1] in tobacco plants. The VHH gene cDNA was cloned in TA vector and then subcloned into a plant expression binary vector pBI 121. The VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. The presence of VHH gene in transformed plants was confirmed by PCR. Western blot analysis showed that the recombinant VHH protein was expressed in tobacco plant. ELISA results with MUC1 antigen confirmed that the biological activity and antigen-specific responses of the plant derived VHH protein compare favorably with that of the parent recombinant antibodies. This is the first report of production of camelied VHH antibody against tumor specific antigen from two-humped camel [Camelus bactrianus] in plants