Core D provides technical expertise and state of the art molecular and cellular biology methodology for cloning and expression of the proteins described in the program project. This centralized operation coordinates all molecular biology work and ensures that all proteins are cloned and expressed in a uniform fashion. This is particularly important since several investigators plan to analyze different aspects of some of the same proteins. This uniformity greatly enhances accuracy of comparison between the proteins. Core D designs and clones expression vectors encoding wild type or mutated proteins as requested by the individual investigators. All wild type genes described in this program project are identified, sequenced and assembled in cloning vectors. Thus, vectors expressing wild type or mutated proteins can be cloned from cDNAs. Vectors expressing consensus sequence peptides will be constructed from synthetic oligonucleotides. Depending on the requirement for the analysis of the protein (amount and/or modification of protein), the expression vectors are designed for expression in prokaryotic or eukaryotic cell lines. Core D also establishes cell lines expressing the proteins, determines conditions for optimum protein expression and aids in the purification of the expressed protein. Thus the service of Core D includes: a) advice toward an optimal expression system, b) design methodologies to express desired proteins, c) confirm wild type DNA sequence by restriction and/or DNA sequence analysis, d) design primers for DNA sequence analysis, e) design primers for PCR amplification of wild type or mutant constructs, f) extract DNA, g) digest DNA with restriction enzymes, h) amplify DNA by PCR, i) ligate DNA (fragments) in cloning and expression vectors, j) transform/transfect ligated DNA into cell lines to express the proteins, k) select clones, I) amplify clones, m) isolate permanently transfected cell lines, n) maintain transformed cell lines, o) establish optimum protein expression conditions, p) assist in protein purification, q) maintain cell lines carrying wild type and expression vectors. All personnel in Core D trains new users associated with the projects in molecular biology and cell culture techniques and oversees proper use of cell culture facilities. This ensures safe maintenance of cell systems and guarantees compliance with NIH guidelines. Core D designs and clones expression vectors encoding proteins requested by the principal investigators of the individual projects. They establish cell lines, determine optimum conditions for protein expression and aid in the purification of the expressed protein.
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