DPPD is a Mycobacterium tuberculosis recombinant
antigen that elicits specific delayed type
hypersensitivity reactions similar in size and morphological aspects to that elicited by purified
protein derivative, in both guinea pigs and humans infected with M.
tuberculosis. In addition, earlier clinical studies with
DPPD suggested that this molecule could improve the specificity of the
tuberculin skin test, which is used as an important aid for the diagnosis of
tuberculosis. However, these studies could only be performed with
DPPD engineered as a fusion molecule with another Mycobacterium spp.
protein because no expression of
DPPD could be achieved as a single molecule or as a conventional fusion
protein in any commercial system. Although
recombinant fusion proteins are in general suitable for several
biological studies, they are by definition not ideal for studies involving highly purified and defined
polypeptide sequences. Here, we report two alternative approaches for the expression of immunologically reactive recombinant genuine
DPPD. The first approach used the rapidly growing, nonpathogenic Mycobacterium smegmatis as host cells transformed with the pSMT3 plasmid vector containing the full-length
DPPD gene. The second approach used Escherichia coli transformed with the pET-17b plasmid vector containing the
DPPD gene engineered in a three-copy fusion manner in tandem with itself. Though at low levels, expression and purification of immunologically reactive
DPPD in M. smegmatis could be achieved. More abundant expression and purification of
DPPD as a homo-trimer molecule was achieved in E. coli (> or =2 mg/L of bacterial broth cultures). Interestingly, expression could only be achieved in host cells transformed with the
DPPD gene containing its
leader peptide. However, the expressed
proteins lacked the leader sequence, which indicates that processing of the M.
tuberculosis DPPD gene was accurately achieved and necessary in both M. smegmatis and E. coli. More importantly, the delayed type
hypersensitivity reactions elicited by purified molecules in guinea pigs infected with M.
tuberculosis were indistinguishable from that elicited by purified
protein derivative. Because the
DPPD gene is present only in the
tuberculosis-complex organisms of the Mycobacterium genus, these highly purified molecules should be helpful in identifying individuals sensitized with tubercle bacilli.