Preparation, Purification, and Secondary Structure Determination of Bacillus circulans Xylanase. A Modular Laboratory Incorporating Aspects of Molecular Biology, Biochemistry, and Biophysical Chemistry

New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation.

Section: Discussionmentioning

confidence: 99%

“…The biochemistry laboratory course consists of two 4‐hour laboratory meetings and a 1‐hour lecture meeting per week for the 10‐week quarter. The experiments described herein encompass 6 weeks of the quarter, and are an adaptation of a previously described laboratory module [1–4].…”

Section: Translation Factor Informationmentioning

confidence: 99%

Expression, purification, and analysis of unknown translation factors from Escherichia coli: A synthesis approach

Walter

Littlefield

Delbecq

et al. 2010

“…What is described herein are two labs, one on protein chemical denaturation and one on protein thermal denaturation, which are valuable in reinforcing lecture concepts. These labs fit well into a previously described module focused on protein overexpression, purification, and secondary structure determination [2] (see instructor's notes).…”

mentioning

confidence: 95%

“…Xylanolytic enzymes have many applications, including clarification of juices and wines, saccharification of agricultural and forestry wastes for fermentation to fuels, improvement of the nutritional value of agricultural silages, and conversion of xylans to simple xylose derivatives used, for example, as sweeteners. BCX was chosen for study because it is highly overexpressed (75 mg of pure protein from 1 L of growth), easily purified in a single chromatographic step, and very stable [2] (see instructor's notes in ref. [2] for each of the labs performed in the preparation of the pure BCX needed for these stability studies).…”

mentioning

confidence: 99%

“…BCX was chosen for study because it is highly overexpressed (75 mg of pure protein from 1 L of growth), easily purified in a single chromatographic step, and very stable [2] (see instructor's notes in ref. [2] for each of the labs performed in the preparation of the pure BCX needed for these stability studies). In addition, it has 11 tryptophan and 15 tyrosine residues, allowing for monitoring of its intrinsic fluorescence upon protein unfolding ( Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Thermal and chemical denaturation of Bacillus circulans xylanase

Raabe¹,

Gentile²

2008

Self Cite

A number of institutions have been, or are in the process of, modifying their biochemistry major to include some emphasis on the quantitative physical chemistry of biomolecules. Sometimes this is done as a replacement for part for the entire physical chemistry requirement, while at other institutions this is incorporated as a component into the traditional two-semester biochemistry series. The latter is the model used for biochemistry and molecular biology majors at the University of Richmond, whose second semester of biochemistry is a course entitled Proteins: Structure, Function, and Biophysics. What is described herein is a protein thermodynamics laboratory module, using the protein Bacillus circulans xylanase, which reinforces many lecture concepts, including: (i) the denatured (D) state ensemble of a protein can be different, depending on how it was populated; (ii) intermediate states may be detected by some spectroscopic techniques but not by others; (iii) the use and assumptions of the van't Hoff approach to calculate DH o , DS o , and DG o T for thermal protein unfolding transitions; and (iv) the use and assumptions of an approach that allows determination of the Gibb's free energy of a protein unfolding transition based on the linear dependence of DG o on the concentration of denaturant used. This module also requires students to design their own experimental protocols and spend time in the primary literature, both important parts of an upper division lab.Keywords: Protein thermal and chemical denaturation, van't Hoff analysis, biophysical methods, laboratory exercises, protein structure, function, folding.The University of Richmond is a private liberal arts college of 2,800 undergraduates, which offers both B.A. and B.S. degrees in biochemistry and molecular biology, an interdisciplinary program that brings together faculty from both biology and chemistry. Begun officially in 2003, this program continues to grow, and last year a high of 28 majors graduated. As part of the major, students take a two-semester sequence with lab in biochemistry, which is usually done in either their junior or senior years. The second semester course is Proteins: Structure, Function, and Biophysics, which has 150 min of lecture (2 3 75 min classes) and 180 min of lab (one meeting, capped at 16 students) per week. Multiple faculties rotate through this course, each providing a slightly different focus with different labs. In one version of this course, the focus is on protein folding, modeled after that originally described by Anthony-Cahill [1]. What is described herein are two labs, one on protein chemical denaturation and one on protein thermal denaturation, which are valuable in reinforcing lecture concepts. These labs fit well into a previously described module focused on protein overexpression, purification, and secondary structure determination [2] (see instructor's notes).The lecture portion of the proteins course is divided into three parts: protein spectroscopy, protein thermodynamics, and protein primary literatur...

A survey on faculty perspectives on the transition to a biochemistry course‐based undergraduate research experience laboratory

Craig

2017

It will always remain a goal of an undergraduate biochemistry laboratory course to engage students hands-on in a wide range of biochemistry laboratory experiences. In 2006, our research group initiated a project for in silico prediction of enzyme function based only on the 3D coordinates of the more than 3800 proteins "of unknown function" in the Protein Data Bank, many of which resulted from the Protein Structure Initiative. Students have used the ProMOL plugin to the PyMOL molecular graphics environment along with BLAST, Pfam, and Dali to predict protein functions. As young scientists, these undergraduate research students wanted to see if their predictions were correct and so they developed an approach for in vitro testing of predicted enzyme function that included literature exploration, selection of a suitable assay and the search for commercially available substrates. Over the past two years, a team of faculty members from seven different campuses (California Polytechnic San Luis Obispo, Hope College, Oral Roberts University, Rochester Institute of Technology, St. Mary's University, Ursinus College, and Purdue University) have transferred this approach to the undergraduate biochemistry teaching laboratory as a Course-based Undergraduate Research Experience. A series of ten course modules and eight instructional videos have been created (www.promol.org/home/basil-modules-1) and the group is now expanding these resources, creating assessments and evaluating how this approach helps student to grow as scientists. The focus of this manuscript will be the logistical implications of this transition on campuses that have different cultures, expectations, schedules, and student populations. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):426-436, 2017.