Rational design of multiple TB antigens TB10.4 and TB10.4-Ag85B as subunit vaccine candidates against Mycobacterium tuberculosis.
OBJECTIVE
Rational design of recombinant antigens TB10.4 and TB10.4-Ag85B as subunit vaccine candidates against Mycobacterium tuberculosis. The main purpose is to obtain a large quantity of soluble antigens.


METHODS
Recombinant antigens were cloned in frame with the N-terminal thioredoxin and expressed in E. coli. The thioredoxin tag was removed by TEV protease. Nickel-affinity and size-exclusion chromatography were used to purify antigens to homogeneity. Antigen stability at different pH levels was studied by photon correlation spectrometry. Circular dichroism was used to probe antigen secondary structure and thermal stability.


RESULTS
N-terminal thioredoxin fusion dramatically increased antigen solubility. Soluble TB10.4 and TB10.4-Ag85B were purified to homogeneity and obtained in milligram quantity. Co-expression of bacteria chaperons increased the yield of TB10.4-Ag85B. Soluble TB10.4 and TB10.4-Ag85B purified from the inclusion body showed a reversible structure change. However, Ag85B and soluble TB10.4-Ag85B showed a clear melting temperature, above which the secondary structure was lost dramatically.


CONCLUSIONS
Soluble TB10.4 and TB10.4-Ag85B were purified from the E. coli in significant quantities. The methods to purify and characterize these recombinant antigens were established, which paved the way for further vaccine development based on these antigens.
Shi
Shuai
S
Molecular Pharmaceutics, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7360, USA.
Yu
Lan
L
Sun
Dengyun
D
Liu
Jian
J
Hickey
Anthony J
AJ
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D002942:Circular Dichroism; D015195:Drug Design; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D011489:Protein Denaturation; D020673:Protein Renaturation; D013816:Thermodynamics; D032581:Tuberculosis Vaccines / Q000138:chemical synthesis* / Q000235:genetics / Q000276:immunology; D022223:Vaccines, Subunit / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C501710:TB10.4 antigen, Mycobacterium tuberculosis; D032581:Tuberculosis Vaccines; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis