A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris

Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.Codon-optimized "Opti-Pgp" and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from T(m) ∼40 °C to 49 °C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids.The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins

Comparative study of thermostability and structure of close homologues - bamase and binase

Parameters of heat denaturation and intrinsic fluorescence of bamase and its close homologue, binase in the pH region 2-6 have been determined. The bamase heat denaturation (pH 2.85.5) proceeds according to the “all-or-none” principle. Bamase denaturation temperature is lower than that of binase and this difference increases from 2.5 °C at pH 5 to 7 °C at pH 3. Enthalpy values of bamase and binase denaturation coincide only at pH 4.5-5.5, but as far as pH decreases the bamase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the bamase Trp94 by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found. © 1993 Taylor & Francis Ltd

КОНСТРУКЦИЯ И АЛГОРИТМ РАБОТЫ ЛАЗЕРНОГО КОМБИНИРОВАННОГО ПОЖАРНОГО ИЗВЕЩАТЕЛЯ

The point optical smoke detector is currently the most effective means of fire detection at the early stage of its occurrence. The urgent task for this type of detector is to increase its sensitivity to «black» smoke and ensure resilience to the effects of electromagnetic interference and particle smoke origin.The objective of this work is to develop a structure and algorithm of point combined fire detector that provides high sensitivity to various types of fumes, detection rate and high noise immunity. The decision of the current objective is carried out using the proposed optical scheme of the dual-channel devices of smoke detection (for control of the radiation intensities scattered by smoke particles and passed through the smoke).The design and algorithm of the combined fire detector comprising a dual-channel laser device of smoke detection and carbon monoxide detector is developed.The results of the made detector tests indicate about its increased in comparison with the conventional single-channel point smoke fire detector sensitivity to various types of fumes and detection rate of fires. The high functional characteristics of the detector are provided with application additional channel of smoke detection (to intensity change of transmitted radiation through it) created by the requirements for unrestricted entry of smoke in the area of control and effective algorithm for processing of recorded signals. Точечный оптический дымовой пожарный извещатель остается в настоящее время наиболее эффективным средством обнаружения пожара на ранней стадии его появления. Актуальной задачей для данного типа извещателя является повышение его чувствительности к «черным» дымам и обеспечение устойчивости к воздействию электромагнитных помех и частиц не дымового происхождения. Целью настоящей работы являлась разработка конструкции и алгоритма работы точечного комбинированного пожарного извещателя, обеспечивающего высокую чувствительность к различным типам дымов, скорость их обнаружения и высокую помехоустойчивость.Решение поставленной цели осуществлено использованием предложенной оптической схемы двухканального устройства обнаружения дыма (по контролю интенсивностей излучения, рассеянного частицами дыма и прошедшего через дым).Разработана конструкция и алгоритм работы комбинированного пожарного извещателя, включающего двухканальное лазерное устройство обнаружения дыма и датчик угарного газа.Результаты тестовых испытаний изготовленного извещателя свидетельствуют о его повышенной в сравнении с обычным одноканальным точечным дымовым пожарным извещателем чувствительности к различным типам дымов и скорости обнаружения возгораний. Высокие функциональные характеристики извещателя обеспечиваются применением дополнительного канала обнаружения дыма (по изменению интенсивности проходящего через него излучения), созданными условиями беспрепятственного захода дыма в зону контроля и эффективным алгоритмом обработки регистрируемых сигналов.

Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence

Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein. © 2014 Xue et al

The Effect of Macromolecular Crowding, Ionic Strength and Calcium Binding on Calmodulin Dynamics

The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells.Comment: Accepted to PLoS Comp Biol, 201