Techniques in Protein Chemistry III

Ruth Hogue Angeletti

• Publisher: Academic Press
• Year: 2014
• Print ISBN: 9780120587568
• eBook ISBN: 9781483259468
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

Techniques in Protein Chemistry III compiles papers presented at the Fifth Protein Society Symposium in Baltimore on June 22-26, 1991. This book discusses the protein and peptide recovery from PVDF membranes; high-sensitivity peptide mapping utilizing reversed-phase microbore and microcolumn liquid chromatography; and capillary electrophoresis for preparation of peptides and direct determination of amino acids. The TFMSA/TFA cleavage in t-Boc peptide synthesis; applications of automatic PTC amino acid analysis; and identification of O-glycosylation sites with a gas phase sequencer are also elaborated. This text likewise covers the conformational stability of the molten globule of cytochrome c and role of aqueous solvation in protein folding. This publication is useful to students and researchers interested in methods and research approaches on protein chemistry.

Section VII

Protein Folding and Conformation

Outline

Chapter 33: Refolding of human recombinant insulin-like growth factor II (IGF-II) in vitro, using a solubilizing affinity handle

Chapter 34: Conformational Stability of the Molten Globule of Cytochrome c : Role of Electrostatic Repulsion

Chapter 35: Calcium-Induced Folding of Troponin-C: Formation of Homodimeric and Heterodimeric Two-Site Domains From Synthetic Peptides

Chapter 36: The Role of Aqueous Solvation in Protein Folding: Insights from Aqueous-Alkane Partitioning

Chapter 37: MOLECULAR CHARACTERIZATION OF AN AGGREGATE FORMED BY A BOVINE GROWTH HORMONE FOLDING INTERMEDIATE

Chapter 38: USE OF SEQUENCE HYDROPHOBIC MOMENT TO ANALYZE MEMBRANE INTERACTING DOMAINS OF BOTULINUM, TETANUS AND OTHER TOXINS

Chapter 39: Fourier Transform Infrared Spectroscopic Analysis of Proteins in Terms of Detectability, Conformation and Surface Adsorption Density

Chapter 40: RECOMBINANT HEMOGLOBINS FOR THE ELUCIDATION OF A NEW MECHANISM OF OXYGEN AFFINITY MODULATION BY CL− IONS

Chapter 41: AN IMPROVED STRATEGY FOR THE DETERMINATION OF THE ROLE IN CATALYSIS OF AMINO ACID RESIDUES IN THYMIDYLATE SYNTHASE

Chapter 42: PROTEIN CONFORMATIONAL CHANGES IN THE μs TIME REGION INVESTIGATED WITH A LASER PULSE PHOTOLYSIS TECHNIQUE

Chapter 43: Macromolecular Dynamics Observed by Intramolecular Energy Transfer Using Frequency-Domain Fluorometry

Chapter 44: STOPPED-FLOW CIRCULAR DICHROISM AND 19F NMR AS PROBES FOR THE FOLDING OF RAT INTESTINAL FATTY-ACID BINDING PROTEIN (IFABP)

Refolding of human recombinant insulin-like growth factor II (IGF-II) in vitro, using a solubilizing affinity handle

Göran Forsberg, Kabi Pharmacia KabiGen, S-112 87 Stockholm, Sweden

Elisabet Samuelsson¹, ¹Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44, Stockholm, Sweden

Henrik Wadensten, Kabi Pharmacia KabiGen, S-112 87 Stockholm, Sweden

Tomas Moks¹, ¹Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44, Stockholm, Sweden

Maris Hartmanis, Kabi Pharmacia KabiGen, S-112 87 Stockholm, Sweden

Publisher Summary

Insulin-like growth factor II (IGF-II) consists of 67 amino acid residues in a single chain. In analogy with IGF-I, there are 6 cysteine residues forming 3 disulfide bonds. The recombinant IGF-II was initially produced as a secreted fusion protein, ZZ-IGF-II, in E. coli. However, most full-length fusion protein was found in multimeric aggregates held together with disulfide bonds. A redox system, using the fusion partner as a solubilizer, was developed to transform multimeric, misfolded variants into the monomeric, correctly folded molecule. This chapter presents a comparison between IGF-II refolding as a fusion protein and as a native molecule. A hydrophilic fusion partner may act as a solubilizer of insulin-like growth factor II during refolding. The fusion partner apparently does not interfere with the refolding. Instead it seems to keep the more hydrophobic IGF-II moiety soluble, making denaturating agents or organic solvents unnecessary in the refolding buffers. The fusion partner can then subsequently be removed using site specific cleavage. This concept can be used to refold proteins that normally precipitate during refolding.

I Introduction

Recombinant proteins or peptides are presently being produced in a variety of expression systems, using several different host cells. Expression in Escherichia coli is frequently used, since very high expression levels can be obtained. However, recombinant proteins produced intracellularly in E. coli tend to precipitate, forming inclusion bodies. To dissolve these, strong denaturing agents must be used. The native product may then be obtained using a properly designed refolding procedure. Proteins secreted from E. coli normally accumulate in the periplasmic space, where they can aggregate. Recently, an E. coli system where proteins are secreted to the medium, was described (Moks et al, 1987; Nilsson et al, 1991). In this system, the product is initially expressed as a fusion protein, where a 14 kDa IgG-binding peptide derived from staphylococcal protein A is used as fusion partner. After site-specific cleavage and separation of the fusion partner, a correctly folded protein can be obtained.

The insulin-like growth factors are homologous to insulin and promote growth in a variety of cells. For a review of the insulin-like growth factors and their actions, see Humbel, 1990. So far, IGF-I is the best studied polypeptide among the IGF-s. It has been produced in several heterologous organisms such as yeast (Elliot et al, 1990) and E. coli (Moks et al, 1987). However, even when producing IGF-I as a secreted product from these organisms, most of the IGF-I is found in biologically inactive forms with mismatched disulfides. Both incorrect monomeric as well as multimeric forms have been identified. The misfolded forms can be converted into the native form using simple redox systems (Forsberg et al, 1990). However, even though the protein concentration in these systems has been low, 0.1 mg/ml or less, precipitation and aggregation can be observed during the redox procedures. Recently, it was ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Foreword
• Preface
• Acknowledgments
• Section I: Protein Microsequence Methods
• Section II: Protein Microsequencing Workshop
• Section III: Peptide/Protein Separation Techniques
• Section IV: Peptide Synthesis
• Section V: Amino Acid Analysis
• Section VI: Structural Analysis of Glycoproteins
• Section VII: Protein Folding and Conformation
• Section VIII: Mass Spectrometry
• Index