Biotechnology-2013: B-cert: A training program for producers of bioengineered plants - Ronnie W. Heiniger - North Carolina State University

 

Abstract

 The introduction of genetically-engineered (GE) plants that produce proteins that have pharmaceutical uses for improving human health or reducing diseases depends upon production systems capable of producing these GE plants in controlled conditions that prevent the movement of genetic materials from the target plant into the environment. As a result of public concern over the stewardship of biotechnology in the US, USDA-APHIS developed the Biotechnology Quality Management System (BQMS) program. BQMS is a federal program designed to improve the stewardship of GM organisms through intensive education, monitoring, and oversight of all aspects of process from laboratory to the field. The BQMS Program helps organizations involved in biotechnology research and development, including small businesses and academic researchers, analyze the critical control points within their management systems to better maintain compliance with the APHIS regulations (7 CFR part 340) for the import, interstate movement, and field release of regulated genetically engineered (GE) organisms. Unfortunately, the BQMS program does not address one of the most important links in the production process-the grower. To correct this deficiency North Carolina State University in cooperation with the North Carolina Biotechnology Center and the Northeastern Economic Development Corporation have developed a grower certification program called B-Cert. The B-Cert program brings together the procedures required to prevent loss of genetic materials from the target site, the standards for certification, and the requirements established by USDA-APHIS for growing transgenic crops to develop guidelines and standards for crop management, isolation requirements, equipment sanitation, environmental safeguards, record keeping requirements, and other critical processes.   we've got expressed full‐length viscum in in N. tabacum PCPs and N. benthamiana leaves at ranges of up to 7 mg/kg of purified product. Expressing the A and B chains become also possible in both systems, but the yield of heterodimeric product turned into decreased by as much as 0.97 g/g, while expressing a chain by myself did not produce quantifiable amounts of recombinant protein. The yield of full‐period viscumin became comparable with that of plant lectins expressed in yeast however ~6‐fold lower than refolded viscumin A and B chains expressed in E. coli. but the bacterial procedure has a low healing, calls for widespread dilution and is complex, while the plant‐based technique protected most effective half the wide variety of steps. in line with an immediate fee comparison among the two tactics, the plant expression gadget is presently 50% much less highly priced, however slight upgrades in yield can lessen production fees by way of even up to ~88%. In comparison with the native host V. album, the heterologous expression method reduced the lead‐time by means of several years, expanded containment in addition to area–time yield, and facilitated focused product modifications. consequently, generating recombinant viscumin in plant life is a beneficial opportunity to each microbial and native structures.  We transiently expressed complete‐duration viscumin (visFL), the usage of the local five′‐UTR and coding sequence, integrated in assemble pTRAc‐visFL. We additionally expressed the individual a series (visA) the usage of construct pTRAc‐CHSLPH‐visAA‐S, or the a series collectively with the separate B chain (visB) the usage of construct pTRAc‐CHSLPH‐visBA‐S in both N. tabacum by using‐2 PCPs (determine 2a) or within the leaves of intact N. benthamiana flowers (determine 2b) by infiltration with A. tumefaciens . single‐chain constructs visA and visB carried a recombinant signal peptide for secretion into the apoplast of intact plant life or into the extracellular area of through‐2 suspension cells to bypass immediately self‐intoxication of the respective host. We detected a band of ~30 kDa in all samples by means of western blot analysis 5 days postinfiltration (dpi) the usage of a sequence‐unique monoclonal antibody (mAb) TA‐five. This band migrated slightly slower than the bacterial nonglycosylated viscum in a chain well-known (~28 kDa). It corresponded to the expected length of the N ‐glycosylated viscumin a sequence (~30 kDa) which incorporates the mass of the polypeptide plus 1.9 kDa representing a easy N ‐glycan added to a unmarried expected acceptor website online (Chauhan, Rao, & Raghava, 2013). Such glycosylation turned into anticipated because of vacuolar focused on (Strasser, 2014) by way of an inner signal sequence of the seasoned toxin (Frigerio et al., 2001). the best yield of the viscumin a series, relative to the alternative infiltration units, changed into detected within the visFL samples. in the PCPs and intact leaves, the visFL:visA + visB:visA ratios for the viscumin a series yield have been 6.eight:four.0:1.0 and 35:three.five:1.0, respectively. in the visFL and visA + visB PCP samples, we also detected a ~65 kDa band corresponding to the expected size of an N ‐glycosylated viscumin heterodimer or a series homodimer, as formerly mentioned in E. coli (Kourmanova et al., 2004). other than the gene design (full‐period vs. separate chains), the distinction in signal peptides and 5′‐UTRs may have additionally affected the a series yields determined for the three expression setups (Jansing & Buyel, 2018; Meshcheriakova, Saxena, & Lomonossoff, 2014). Low absolute protein concentrations might also have averted dimer detection inside the visA samples.  Biography  Ronnie W. Heiniger is a Professor in the Crop Science Department at North Carolina State University. He received his Ph.D. in crop ecology from Kansas State University in 1994. Heiniger has worked for the past 15 years as a research and extension specialist at the Vernon G. James Research and Extension Center in Plymouth, NC. Heiniger is known for his applied research and has published over 30 journal articles and presented papers covering his work in precision agriculture and crop management. Heiniger has received the Gerold O Mott Award for outstanding research from the American Society of Agronomy and is a member of the Academy of Outstanding Extension Specialists at North Carolina State University.                                                                                                                                                                                                                                                             Ron_Heiniger@ncsu.edu