. (1999) 'The cardiomyopathy and lens cataract mutation in B-crystallin alters its protein structure, chaperone activity, and interaction withintermediate laments in vitro.', Journal of biological chemistry., 274 (47). pp. 33235-33243. Further information on publisher's website: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details.

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Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection. MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein aggregation in vitro, and it contains the core Ôa-crystallinÕ domain found in all sHsps. In this study we characterized the chaperone activity of recombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human aB-crystallin, a well characterized member of the sHsp family. Recombinant MTB HSP 16.3 was expressed in Escherichia coli and purified to apparent homogeneity. Similar to aB-crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 mM ATP the chaperone activity was enhanced by twofold. ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPcS, a nonhydrolyzable analog of ATP. Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E. coli at 48°C. While the sequence similarity between human aB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core Ôa-crystallinÕ domain are much closer.Keywords: ATP; human aB-crystallin; molecular chaperone; Mycobacterium tuberculosis HSP 16.3; small heat shock proteins.One-third of the world's population is infected with latent inactive tuberculosis and active tuberculosis is the leading cause of death due to an infectious disease [1]. Each year, new infections occur in 54 million people; 6.8 million people develop clinical disease, and 2.4 million cases result in death [2]. There is still limited knowledge of the molecular pathogenesis of the latent stage of this organism [3]. Individuals who have been infected with Mycobacterium tuberculosis can harbor stable dormant bacilli for decades before developing an active infection later in life [4]. Recent reports indicate an important role for M. tuberculosis (MTB) heat shock protein (HSP) 16.3 in the survival of MTB during prolonged periods of infection [5][6][7]. It was shown that MTB HSP 16.3, initially described as the immunodominant 14-or 16-kDa antigen [8][9][10][11], was a major component in tuberculosis infection in humans and played an important role in enhancing protein stability and survival [5]. Eighty-five percent of patients with active tuberculosis showed a positive reaction to this antigen, suggesting that this protein expressed in vivo had a key role in MTB infection [11,12]. The 14K antigen was later renamed MTB HSP 16.3 [13]. MTB HSP 16.3 accumulates to become the dominant protein in the latent stationary phase of M. tuberculosis infection [7]. Over-expression of HSP 16.3 in log phase growth of M. tuberculosis slowed the growth rate and protected against stationary phase autolysis in vitro [7]. MTB HSP 16.3 has been characterized as a membrane asso...

Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection. MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein aggregation in vitro, and it contains the core Ôa-crystallinÕ domain found in all sHsps. In this study we characterized the chaperone activity of recombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human aB-crystallin, a well characterized member of the sHsp family. Recombinant MTB HSP 16.3 was expressed in Escherichia coli and purified to apparent homogeneity. Similar to aB-crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 mM ATP the chaperone activity was enhanced by twofold. ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPcS, a nonhydrolyzable analog of ATP. Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E. coli at 48°C. While the sequence similarity between human aB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core Ôa-crystallinÕ domain are much closer.Keywords: ATP; human aB-crystallin; molecular chaperone; Mycobacterium tuberculosis HSP 16.3; small heat shock proteins.One-third of the world's population is infected with latent inactive tuberculosis and active tuberculosis is the leading cause of death due to an infectious disease [1]. Each year, new infections occur in 54 million people; 6.8 million people develop clinical disease, and 2.4 million cases result in death [2]. There is still limited knowledge of the molecular pathogenesis of the latent stage of this organism [3]. Individuals who have been infected with Mycobacterium tuberculosis can harbor stable dormant bacilli for decades before developing an active infection later in life [4]. Recent reports indicate an important role for M. tuberculosis (MTB) heat shock protein (HSP) 16.3 in the survival of MTB during prolonged periods of infection [5][6][7]. It was shown that MTB HSP 16.3, initially described as the immunodominant 14-or 16-kDa antigen [8][9][10][11], was a major component in tuberculosis infection in humans and played an important role in enhancing protein stability and survival [5]. Eighty-five percent of patients with active tuberculosis showed a positive reaction to this antigen, suggesting that this protein expressed in vivo had a key role in MTB infection [11,12]. The 14K antigen was later renamed MTB HSP 16.3 [13]. MTB HSP 16.3 accumulates to become the dominant protein in the latent stationary phase of M. tuberculosis infection [7]. Over-expression of HSP 16.3 in log phase growth of M. tuberculosis slowed the growth rate and protected against stationary phase autolysis in vitro [7]. MTB HSP 16.3 has been characterized as a membrane asso...

The L49 single-chain Fv fused to beta-lactamase (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen. However, large-scale production of L49-sFv-bL from refolded E. coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein. Sequence analysis of the L49-sFv framework regions revealed three residues in the framework regions at positions L2, H82B, and H91, which are not conserved for their position, occurring in <1% of sequences in Fv sequence databases. One further unusual residue, found in <3% of variable sequences, was observed at position H39. Each unusual residue was mutated to a conserved residue for its position and tested for refolding yield from inclusion bodies following expression in E. coli. The three V(H) single mutants showed improvement in the yield of active protein and were combined to form double and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein. In an attempt to further improve yield, the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resulting in a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein. The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations. Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts, respectively. These results show that a rational protein-engineering approach improved the yield, stability, and refolding characteristics of L49-sFv-bL while maintaining binding affinity and therapeutic efficacy.