Mutations in maltose-binding protein that alter affinity and solubility properties

Maltose-binding protein (MBP) from Escherichia coli has been shown to be a good substrate for protein engineering leading to altered binding (Marvin and Hellinga, Proc Natl Acad Sci U S A 98:4955–4960, 2001a) and increased affinity (Marvin and Hellinga, Nat Struct Biol 8:795–798, 2001b; Telmer and Shilton, J Biol Chem 278:34555–34567, 2003). It is also used in recombinant protein expression as both an affinity tag and a solubility tag. We isolated mutations in MBP that enhance binding to maltodextrins 1.3 to 15-fold, using random mutagenesis followed by screening for enhanced yield in a microplate-based affinity purification. We tested the mutations for their ability to enhance the yield of a fusion protein that binds poorly to immobilized amylose and their ability to enhance the solubility of one or more aggregation-prone recombinant proteins. We also measured dissociation constants of the mutant MBPs that retain the solubility-enhancing properties of MBP and combined two of the mutations to produce an MBP with a dissociation constant 10-fold tighter than wild-type MBP. Some of the mutations we obtained can be rationalized based on the previous work, while others indicate new ways in which the function of MBP can be modified

A Ribosomal Misincorporation of Lys for Arg in Human Triosephosphate Isomerase Expressed in Escherichia coli Gives Rise to Two Protein Populations

We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame

A novel method for treatment of Class III malocclusion in growing patients

Abstract Background Management of Class III malocclusion is one of the most challenging treatments in orthodontics, and several methods have been advocated for treatment of this condition. A new treatment protocol involves the use of an alternating rapid maxillary expansion and constriction (Alt-RAMEC) protocol, in conjunction with full-time Class III elastic wear and coupled with the use of temporary anchorage devices (TADs). The aim of this study was to evaluate the dento-skeletal and profile soft tissue effects of this novel protocol in growing participants with retrognathic maxilla. Methods Fourteen growing participants (7 males and 7 females; 12.05 ± 1.09 years), who displayed Class III malocclusions with retrognathic maxilla, were recruited. Pre-treatment records were taken before commencing treatment (T1). All participants had a hybrid mini-implant-supported rapid maxillary expansion (MARME) appliance that was activated by the Alt-RAMEC protocol for 9 weeks. Full-time bone-anchored Class III elastics, delivering 400 g/side, were then used for maxillary protraction. When positive overjet was achieved, protraction was ceased and post-treatment records were taken (T2). Linear and angular cephalometric variables were blindly measured by one investigator and repeated after 1 month. An error measurement (Dahlberg’s formula) study was performed to evaluate the intra-examiner reliability. A paired-sample t test (p < 0.05) was used to compare each variable from T1 to T2. Results Treatment objectives were achieved in all participants within 8.5 weeks of protraction. The maxilla significantly protracted (SNA 1.87°± 1.06°; Vert.T-A 3.29± 1.54 mm p < 0.001), while the mandibular base significantly redirected posteriorly (SNB −2.03° ± 0.85°, Vert.T-B − 3.43± 4.47 mm, p < 0.001 and p < 0.05 respectively), resulting in a significant improvement in the jaw relationship (ANB 3.95°± 0.57°, p < 0.001; Wits 5.15± 1.51 mm, p < 0.001). The Y-axis angle increased significantly (1.95° ± 1.11°, p < 0.001). The upper incisors were significantly proclined (+ 2.98°± 2.71°, p < 0.01), coupled with a significant retroclination of the lower incisors (− 3.2°± 3.4°, p < 0.05). The combined skeletal and dental effects significantly improved the overjet (5.62± 1.36 mm, p < 0.001) and the soft tissue Harmony angle (2.75° ± 1.8°, p < 0.001). Conclusions Class III elastics, combined with the Alt-RAMEC activation protocol of the MARPE appliance, is an efficient treatment method for mild/moderate Class III malocclusions. The long-term stability of these changes needs further evaluation