A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression

MultiBac: expanding the research toolbox for multiprotein complexes

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms

The Generalized Montgomery Coordinate:A New Computational Tool for Isogeny-based Cryptography

Recently, some studies have constructed one-coordinate arithmetics on elliptic curves. For example, formulas of the 𝑥-coordinate of Montgomery curves, 𝑥-coordinate of Montgomery− curves, 𝑤-coordinate of Edwards curves, 𝑤-coordinate of Huff’s curves, 𝜔-coordinates of twisted Jacobi intersections have been proposed. These formulas are useful for isogeny-based cryptography because of their compactness and efficiency. In this paper, we define a novel function on elliptic curves called the generalized Montgomery coordinate that has the five coordinates described above as special cases. For a generalized Montgomery coordinate, we construct an explicit formula of scalar multiplication that includes the division polynomial, and both a formula of an image point under an isogeny and that of a coefficient of the codomain curve. Finally, we present two applications of the theory of a generalized Montgomery coordinate. The first one is the construction of a new efficient formula to compute isogenies on Montgomery curves. This formula is more efficient than the previous one for high degree isogenies as the√élu’s formula in our implementation. The second one is the construction of a new generalized Montgomery coordinate for Montgomery−curves used for CSURF

KINETIC ANALYSIS OF START MOTION ON STARTING BLOCK IN COMPETITIVE SWIMMING

The aim of this study was to investigate kinetic features of start motion with use of an instrumented starting block. This is the first study that quantified joint torques of the whole body during start motion. Six male swimmers dived from the instrumented starting block, which contains force plates and sensors. Four high-speed cameras were used to obtain kinematics data of the swimmers. Inverse dynamics calculation was carried out with use of the kinetics and kinematics data. The results showed that 1) the large pulling up forces exerted by both hands were generated by extension toques of the shoulder joints, 2) the rear side lower limb joints exerted large extension torque to obtain horizontal reaction force, and 3) the knee joint of the front side lower limb exerted large flexion torque to maintain the large vertical reaction force until 60% normalized start motion time

Fast Enumeration Algorithm for Multivariate Polynomials over General Finite Fields

The enumeration of all outputs of a given multivariate polynomial is a fundamental mathematical problem and is incorporated in some algebraic attacks on multivariate public key cryptosystems. For a degree-$d$ polynomial in $n$ variables over the finite field with $q$ elements, solving the enumeration problem classically requires $O\left(\tbinom{n+d}{d} \cdot q^n\right)$ operations. At CHES 2010, Bouillaguet et al. proposed a fast enumeration algorithm over the binary field $\mathbb{F}_2$. Their proposed algorithm covers all the inputs of a given polynomial following the order of Gray codes and is completed by $O(d\cdot2^n)$ bit operations. However, to the best of our knowledge, a result achieving the equivalent efficiency in general finite fields is yet to be proposed. In this study, we propose a novel algorithm that enumerates all the outputs of a degree-$d$ polynomial in $n$ variables over $\mathbb{F}_q$ with a prime number $q$ by $O(d\cdot q^n)$ operations. The proposed algorithm is constructed by using a lexicographic order instead of Gray codes to cover all the inputs. This result can be seen as an extension of the result of Bouillaguet et al. to general finite fields and is almost optimal in terms of time complexity. We can naturally apply the proposed algorithm to the case where $q$ is a prime power. Notably, our enumeration algorithm differs from the algorithm by Bouillaguet et al. even in the case of $q=2$

An Efficient Algorithm for Solving the MQ Problem using Hilbert Series

The security of multivariate polynomial cryptography depends on the computational complexity of solving a multivariate quadratic polynomial system (MQ problem). One of the fastest algorithms for solving the MQ problem is F4, which computes a Groebner basis but requires numerous calculations of zero reduction that do not affect the output.The Hilbert-driven algorithm evaluates the number of generators in the Groebner basis of degree $d$ using Hilbert series, then it reduces the number of zero reduction computations. In this paper, we propose a high-speed algorithm designed for randomly generated semi-regular MQ problems. Although the Hilbert-driven algorithm is limited to computing homogeneous ideals, we demonstrate its applicability to semi-regular non-homogeneous ideals in this work. Furthermore, when using the Hilbert-driven algorithm to solve non-homogeneous MQ problems with F4, we demonstrate the efficient achievement of reducing zero reduction for F4. We implemented the proposed algorithm in C++ and report successful decryption of a new record $m=21$ Type VI equations. This was achieved using an AMD EPYC 7742 processor and 2TB RAM, and the decryption process was completed within approximately 9 h