Description

This report examines the issues faced by scientists engaged in protein expression and purification. The pace at which protein research is currently being conducted indicates that more efficient and rapid expression of genes in homologous and heterologous expression systems and rapid purification must be developed. The objectives of this report are to examine the emerging procedures for expressing proteins and to explore the tools and techniques associated with the isolation of proteins. Efforts to understand the relationship between protein structure and biological function have intensified, and the huge number of candidate proteins generated by genomics programs has generated interest in all aspects of protein expression and purification. Proper expression and purification techniques are essential for the large-scale production of pure proteins, which can be used in subsequent analysis, i.e. high-throughput screening and 3-D structure determination. In addition, expression and purification systems that rapidly yield high levels of pure recombinant proteins are fundamental for the identification of target molecules for drug development. The keys to efficient, high production expression systems are good host strains, vectors, and growth conditions. Bacterial, yeast, insect, frog oocytes, and mammalian are the most common types of expression systems used by researchers today. Many research programs, however, are hampered by the impracticality and costs associated with expressing and purifying proteins in amounts large enough to allow for their proper characterization and evaluation. A particular protein of interest is often in low abundance in its natural source and can be difficult to purify and subject to proteolytic cleavage, unfolding, and non-specific refolding during extensive purification. Other scientists report that the examination of the involvement of specific residues in protein structure-function is hampered by the limitations of genetically modified proteins produced in bacterial systems. Consequently, a prerequisite to successful, detailed protein studies has, in many cases, necessitated the over-production of biologically functional proteins. In response to this phenomenon, well-established molecular biological techniques can use non-native eukaryotic systems for the synthesis of recombinant protein that exhibit all the three-dimensional, post-translational, and functional features of native proteins. Competitors are actively engaged in developing platform technologies that yield high-efficient, low background, high-throughput systems for protein analysis at lower cost and ease of scale-up. New improvements in protein purification include dual tag vectors that address the reduced yield and low purity often experienced when working with expression and purification systems. Also, new systems are emerging that offer rapid, high quality cloning and transfer of DNA segments between vector systems. In addition, future developments are expected to include automated platforms for cloning, protein expression, and assay development. The final report is based on the opinions of a worldwide panel of research scientists. It provides you with the answers needed to better serve the protein science market and is available at a fraction of what it would cost to conduct a custom study similar in size and scope. Protein Expression & Purification is the second installment in our series - which details all aspects of protein research - and focuses specifically on the protein expression and purification methods employed by protein scientists. As there are new demands for expression systems that address the issues surrounding protein tags, protein domain expression, and growth conditions that affect protein integrity and yield, new technologies will have to address these issues. Companies that seek to be successful in this market must understand the challenges scientists face today with their technology in order to improve it and make it more powerful. Report Highlights More than 450 researchers engaged in protein expression and/or purification participated in this survey between September 4 and November 23, 2001. The report details findings for each and every question in the survey. Below is a glimpse of some of the findings derived from different questions: • Study results indicate that most respondents use bacterial expression systems. • The "ability to express functional protein" is the primary reason researchers choose a particular expression system. • Rabbit reticulocyte lysate is the most common lysate preparation used by these scientists. • The majority of respondents prefer to use cell-free expression systems over in vivo expression methods "when the over-expressed protein is toxic to the host cell." • Most respondents use polyhistidine/metal affinity systems for purifying their proteins. • The principal reason most respondents use tagged, recombinant proteins is that they are easy to purify, though the fact that they can achieve high levels of purified protein(s) also contributes to this usage. (Electronic copy also includes 1 print copy)

Table of Contents

Section 1 Executive Overview and Introduction -Executive Overview -Key Findings -Introduction -Protein Expression & Purification -Protein Expression Systems -Overview of Protein Expression Systems -Cell-Free Expression Systems -Protein Production Innovations -Protein Purification Methods -Chromatography -Assessing Protein Purification Section 2 Study Methodology and Demographics -Study Methodology -Objectives -Comments -Demographics -Market Segment -Organization Size -Laboratory Size -Region -Job Position -Area(s) of Research Section 3 Significant Findings -Protein Expression -Number of times protein is typically expressed in a 12-month period -Percentage of protein expression performed using specific expression systems -Primary reason for choosing a particular expression system -Cell-free expression system used -Lysate preparation used for cell-free expression -Circumstances in which cell-free expression systems are used over in vivo expression methods -Interest in using the in vitro expression cloning technique to synthesize proteins encoded by cDNA inserts in a plasmid library -Reservations about using the in vitro expression cloning technique -Preference of incorporating affinity tags during protein expression -Techniques used to monitor the expression of protein Protein Purification -Number of protein purification preps typically performed in a 12-month period -Percentage of protein purifications used for specific purification systems -Average yield per prep -Tagged-protein purification methods used Application & Analysis -Type of protein primarily used -Reasons for using type of protein -Consideration of the recombinant protein's application when determining the method for its expression -Applications of expression systems used -Analytical techniques or assays used Technologies & Suppliers -Top-of-mind company for products and services for protein expression and purification -Future use of products and services for protein expression and purification -Companies that supply cell-free expression systems -Companies that supply tagged-protein vector systems -Percentage of time cleaving protein tags -Protease used to cleave protein tag -Techniques used to monitor success/completeness of cleavage -Companies that supply tagged-protein purification resin/columns -Primary antibodies used for ELISAs or blotting -Preferred suppliers of primary antibodies against your tag -Interest in protein purification automation -Ideal number of preps per run in an automated protein purification system -Minimum yield of protein required per prep -Interest in automating protein assay applications -Ideal number of preps per run -Minimal amount of protein required for assays -Familiarity with suppliers of products and services for protein expression and purification -Estimated percentage of total annual budget for products/services dedicated to protein expression and purification Section 4 Presentation of Survey Data -Over 75 pages of detailed tables, full-color analytical charts and graphs highlighting the responses and findings for each of the 39 questions in the survey instrument. Section 5 Cross Tabulations of Survey Data -Over 45 questions or answer choices tabulated against Market Segment, Geographic Region, Area of Research or other relevant questions. Section 6 Appendix -Related Reports -About BioInformatics -Our Valued Clients