ABSTRACT
Background: Biosynthetic teriparatide [1-34] [TPD] is a N-terminally truncated version of human parathyroid hormone [hPTH]. The recombinant form of this polypeptide has been expressed in Escherichia coli [E. coli] and approved as the first anabolic treatment of osteoporosis in the EU and the USA. Feasibility of expression and secretion of a tag- fused form of TPD into Bacillus subtilis [B. subtilis] was examined due to several advantages of B. subtilis over E. coli in production of recombinant proteins with pharmacological activities
Methods: A codon optimized gene containing TPD open reading frame carrying enterokinase site in its upstream was fully synthesized. According to our cloning scheme, this synthetic polynucleotide was used as a template for PCR amplification using engineered primers in such a way that a polyhistidin tag was added in frame to the upstream of the amplicon as well as two restriction sites at its ends. The resulted amplicon, a cassette containing His-tag, enterokinase site and TPD, from 5' to 3', was cloned into pTZ57R/T vector and subjected to sequencing.The cassette was then subcloned into pHT43 shuttle vector and transformed into B. subtilis. Expression of target protein was analyzed by SDS-PAGE and western blotting upon induction by IPTG
Results: The accuracy of construction of pHT43-TPD was confirmed by sequencing and restriction map analyses. SDS-PAGE and western blotting results showed that the recombinant fusion form of hPTH was successfully expressed and secreted into cytoplasm and extracellular medium
Conclusion: TPD may be successfully expressed and secreted in B. subtilis; however, optimization of expression conditions is required for enhancing target protein yield
ABSTRACT
Antibodies are used in many areas of diagnosis and treatment. Fully human monoclonal antibodies [mAb] have attracted high attention due to not stimulating immune response in the body. This study was carried out with the purpose of designing and optimizing multiplex polymerase chain reaction for amplification human anti-tetanus toxoid antibody genes. After collecting blood samples from a human immunized with tetanus toxoid and lymphocyte separation, total RNA was extracted and cDNA was synthesized. all varieties of light [Kapa] and heavy chains of antibody were amplified by two separated multiplex PCR reactions using 14 pair primers. PCR was performed to link VH and VL together and make a ScFv segment, and semi nested PCR was performed to add cutting sites of restriction enzymes at the two ends of the segment. In two multiplex PCR reactions, VH and VL segments were amplified with the length of 410 and 680 bp, respectively. After gel extraction, VH and VL were linked together as a 1070 bp ScFv segment. Restriction sites were added to the two ends of ScFv. By selection of an appropriate primer set and optimization of effective factors of multiplex PCR, all varieties of antibody genes derived from total human lymphocyte cDNA could be amplified and extracted using two multiplex PCR reactions instead of several uniplex ones. Therefore, using only two Multiplex PCR reactions, all genetic varieties of human antibody could be amplified as VH and VL segments from the blood of a subject immunized with tetanus toxoid