BiotechnologyProteins to PCR

Introductory biology texts often present the biologist as a naturalist, such as Darwin or Lamark, who through careful observations develops theories and draws conclusions about living organisms. Originally these scientists kept biology and its subtopics as pure areas of study and resisted the multidisciplinary nature of modern science. For instance, in the 1830s Cagniard de Latour and von Liébig argued that fermentation was a biological phenomenon, not chemical. At this time biochemistry had yet to evolve, and the notion that biology and chemistry overlapped had not been fully realized. As the biology of the cell was discovered, several different scientific disciplines found it necessary to communicate in order to answer questions. In 1953, for instance, the structure of DNA was elucidated only after Watson and Crick pooled the information produced by biologists, chemists, and physicists. Since that time, the various scientific disciplines have continued to actively interact. However, it wasn’t until the 1970s and 1980s that biology and business wholeheartedly converged to produce today’s biotechnology industry.

Although biotechnology has existed for many years (e.g., the baking and brewing industries, and the microbial production of enzymes and vitamins), the explosion in interest and investment seen during the 1980s was unparalleled.

Science has become highly interdisciplinary, and, consequently, scientists require a diverse array of skills to accomplish their research. Where once a biologist might have relied on visual observation of an organism for a behavioral study, today the same research could combine visual observations with molecular techniques. It is commonplace to see a biochemist relying on recombinant proteins for analysis, a molecular biologist on a computer for data analysis, and a microbiologist on a DNA sequence for the detection of a pathogenic microorganism. Unfortunately, these disciplines are often segregated in the classroom, and true integration does not occur until graduate research. Similarly, in industry the narrow focus of research technicians will often prevent their exposure to or participation in duties outside of their immediate job responsibilities.

Our goal is to remove the barriers between scientific disciplines and to demonstrate the diverse techniques and strategies used in the biotechnology laboratory. To accomplish this, you will weave through a series of interrelated experiments designed to mimic the discovery process. The discovery process in this manual will focus on purifying and characterizing a protein and then cloning its associated gene. This manual will not only act as a source of techniques and methods involved in protein and nucleic acid research, but it also will serve as a reference and describe the research process itself.

The initial experiments presented in this first chapter will involve the common techniques of media preparation, handling and observation of yeast and bacteria, and the culturing of yeast for protein production. These exercises will lead directly to subsequent experiments on the purification and characterization of the enzyme α-galactosidase.

The obvious first step in biochemical research is the discovery of some interesting biological activity followed by efforts to determine what type of biomolecule is responsible for that activity. The next few chapters in this book will deal with some of the issues that arise when the molecule of interest is a protein that must be purified and characterized prior to cloning the associated gene.

The initial step in protein isolation from its source is to physically or chemically disrupt the biological tissue or organism in order to release the protein into the extract. In some cases, the organism secretes the protein, and cell disruption is unnecessary. The protein is usually produced in dilute concentration so it is advantageous if the initial isolation also results in the concentration of the protein. An important feature in any isolation procedure is product yield with the goal being to extract as much of the desired protein as possible in a minimum amount of time.

Depending on the source of the protein, the means by which a crude extract is produced will differ. The background section of this chapter will cover the salient features of cell disruption and crude extract preparation, including maintenance of protein stability.

In Chapter 2, glucose and galactose were assessed for their ability to induce the synthesis of α-galactosidase. Galactose was clearly shown to induce enzyme synthesis to a greater extent than glucose. The batch cultivation of S. carlsbergensis on galactose based YPG will yield sufficient enzyme for its subsequent purification. The next step in the purification is to isolate the enzyme from its biological source and to stabilize the resulting crude extract.

The experiments for this chapter will be to determine the distribution of the α-galactosidase activity between the yeast culture supernatant, periplasm, and cytoplasm. The specific activity of the fraction with the highest enzyme activity will be determined and used as a measurement of enzyme purity.

A crude protein extract derived from some type of physical or chemical manipulation of a source will typically contain several types of contaminating biomolecules. These contaminants can include carbohydrates, lipids, nucleic acids, proteins, salts, and other cellular debris. The separation of the protein fraction of the extract from these contaminants is usually referred to as the capture step and is typically performed early in the purification process. The desired protein is then separated from the other captured proteins in subsequent steps using higher resolution purification techniques.

Two popular methods of protein capture are bulk precipitation and batch chromatography. Bulk precipitation methods typically involve salting out the protein fraction by the addition of chaotropic salts, such as ammonium sulfate. The protein fraction can also be precipitated by the addition of certain organic solvents or by long chain synthetic polymers. Batch chromatography involves the binding of the protein fraction to some type of chromatographic gel, followed by filtration to remove contaminants, and, finally, elution and collection of the captured proteins.

The crude α-galactosidase extract has now been prepared, and the conditions for maintaining activity have been investigated. α-Galactosidase will now be isolated, or captured, from the crude extract prior to its purification and analysis. This capture will effectively separate gross contaminants such as cellular debris, nucleic acids, and lipids from α-galactosidase. The experimental section in this chapter will consist of the capture of the α-galactosidase component from the crude extract by batch ion exchange chromatography.

Following the removal of the gross contaminants from the crude extract, the remaining protein components must be resolved. The most popular and ubiquitous technique for this separation is column chromatography. Similar to batch purification, column chromatography utilizes chemical and biological properties of the protein for its purification, but produces greater resolution.

The background section of this chapter will cover some of the routine column chromatographic techniques used in protein purification. This discussion will elaborate on the material presented previously in Chapter 4 concerning chromatographic theory and methods.

In the last experiment, α-galactosidase was captured from the crude extract and assessed for its purity. The increase in specific activity of the enzyme over the original supernatant demonstrates an initial purification. The enzyme must now be further purified to apparent homogeneity prior to its analysis. The experiment for this chapter will be to purify α-galactosidase by ion exchange column chromatography using a step gradient of sodium chloride to elute the enzyme. The α-galactosidase will be assessed for purity by determining its specific activity.

The analysis of a protein is usually an attempt to characterize its structure and/or determine its degree of purity. Proteins of unknown structure are purified to apparent homogeneity and then analyzed to determine structure. The success of the structural analysis is, of course, critically dependent on the level of purification achieved.

If the structure of the protein is already known, then the analysis is performed to determine the level of purity, and it is, therefore, simply a critique of the method(s) used for the purification. To give an example of this situation, when a company wishes to test an improved method it has developed for the production of a currently manufactured protein whose structure is well known, the purity of the protein is analyzed simply to evaluate the new purification process. The improved purification process could lead to increased profits either due to increased product purity or to improved process throughput.

The various methods employed for the analysis of proteins will be discussed in the background section along with an assessment of the relative importance of the various techniques. Overlap of some material previously discussed is unavoidable because methods used for preparative purification of proteins (e.g., chromatography) can also be used to analyze the protein for impurities.

The purification scheme for α-galactosidase has progressed through several steps, including its induced synthesis, batch capture, and column purification. This isolation and purification of α-galactosidase has been an attempt to simulate an actual purification because we have not assumed any prior knowledge of the enzyme structure. The exercises described in this chapter represent the last step in our purification process. The IEX purified α-galactosidase will be analyzed by native polyacrylamide gel electrophoresis. The purpose of the analysis is to evaluate the purity of the enzyme and also to be the final purification by extracting the enzyme from the gel (if the extraction step is determined to be necessary).

Cloning a gene is similar to finding a needle in a hay stack. A gene, even in the simplest of organisms, represents only a tiny fraction of the DNA in a genome (all the genetic information in the cell). A typical scheme for cloning involves removing the genomic DNA from a donor, breaking the DNA into many thousands of small pieces, and then searching those pieces for the desired gene. The search is accomplished by using molecular probes that adhere specifically to the targeted gene.

Strategies for cloning DNA differ markedly from those used for isolating and purifying proteins. Cloning relies on using nucleic acid probes to search for specific DNA or on introducing genome fragments into new hosts in which the targeted gene is expressed. Probes are used to locate DNA while expressed genes are usually identified by phenotypic changes in the host. Cloning does not always start with DNA. Messenger RNA (mRNA) can be isolated and converted into complementary DNA (cDNA). As we will see, cloning from either DNA or RNA each has merit depending on the circumstances.

The first half of this manual focused on the production, purification, and analysis of α-galactosidase. The final product of the purification, which was isolated by native PAGE, would have been subsequently sequenced. The resulting amino acid sequence is the data necessary to link the protein to the gene that codes for α-galactosidase.

This chapter will provide an overview to the strategies and approaches used in cloning. A major strategy for cloning applies protein sequence data, and this is the strategy demonstrated in this manual. The laboratory exercise will emphasize the most important aspect of experimentation, namely planning and strategy.

Before anything can be constructed, it is first necessary to gather the building materials, and, therefore, harvesting genomic DNA is a prerequisite for cloning. Normally two types of DNA are required for cloning, namely, the source DNA containing the targeted gene (that which is to be cloned), and the vector (a DNA molecule that carries the target). The source or genomic DNA can be from any organism or DNA virus. The vector, on the other hand, is a specially designed DNA molecule derived from either a bacteriophage, a plasmid, or some combination of both. As we shall see, the vector will serve as a carrier for the genomic DNA fragments.

Methods for isolating nucleic acids depend on its source. It generally involves collecting cells or tissues, disrupting the cells with enzymes and/or detergents, separating the nucleic acids from other biomolecules and cellular debris, and finally concentrating and/or drying the nucleic acid. The complexity of isolating DNA differs depending on the source. Viral DNA simply requires stripping away the protein coat with an organic solvent (e.g., phenol and chloroform), while plant cells first require removal of the thick cell wall by physically pulverizing the cells (Table 8.1).

Several different schemes can be used for the cloning of the MEL1 gene encoding α-galactosidase. If the complete gene is to be cloned, then the genomic DNA must be isolated. This is in contrast to isolating yeast mRNA and synthesizing and cloning cDNA. This chapter will examine the means of isolating nucleic acids, measuring the yield, and techniques for their storage. The experimental focus will be on the isolation of genomic DNA from the yeast Saccharomyces carlsbergensis (source) and plasmid DNA (vector) from Escherichia coli. The yeast is the source of the MELI gene that encodes α-galactosidase.

Cloning involves isolating, fragmenting, and combining genomic DNA with a vector, followed by introducing the recombinant molecule into a host where it is replicated. The genomic DNA used in cloning is predominantly chromosomal which is large and fragile (i.e., large polymers break). By necessity, the DNA must be broken into manageable pieces, a process normally accomplished by DNA cleaving enzymes called restriction endonucleases. After the DNA is fragmented, the pieces are linked to a vector to form recombined or recombinant molecules. This population of different genomic fragments linked to vector molecules is called a gene bank or gene library.

The typical method of preparing DNA for a gene bank requires that both the genomic and vector DNAs be cleaved with a restriction endonuclease. The restriction endonucleases used for cloning are site specific DNases that recognize four base or longer sequences and cleave the DNA in or near that sequence. Genomic DNA is cleaved randomly to yield an assorted collection of fragments, while the vector is cut only once in a predetermined location. The two sets of molecules are then combined and enzymatically coupled (ligation) by DNA ligase.

Thus far, you have developed a scheme for the cloning process and have isolated both the vector and genomic DNAs. The next step is to construct a gene bank which requires: (1) cleaving the vector site-specifically with a restriction endonuclease; (2) cleaving the genome randomly with a restriction endonuclease; and (3) linking genomic pieces with the vector. In the laboratory you will use restriction endonucleases for the cleavage of genomic and vector DNAs, electrophoretically examine the DNAs, and then recombine DNA molecules through ligation.

Many organisms can act as hosts for recombinant DNA, but E. coli is the most common. Chemically treated E. coli take up extracellular DNA in a process called transformation. Once the DNA is within the cell, the vector provides necessary information for the recombinant molecule to replicate and survive. As noted previously, a vector has an origin of replication (ori) and selectable markers, such as antibiotic resistance genes, both of which ensure the plasmid’s replication and survival.

In Chapter 9 you created a gene bank by linking fragmented yeast genomic DNA into linearized vectors. By adjusting the concentrations of the DNA within the ligation reaction, a percentage of the ligated products are recombinant molecules containing both plasmid and yeast DNA. However, this pool of ligated DNA molecules contains recircularized vectors in addition to other products.

In this experiment, you will introduce the ligated DNA into E. coli. This process first requires the preparation of competent E. coli to take up the DNA. Second, it requires performing the transformation, and, finally, selecting for transformed bacteria. Furthermore, a strategy will be employed that differentiates between cells harboring recircularized and recombinant plasmids.

Cloning strategies usually include the use of probes to search E. coli. These probes may be oligonucleotides derived from the amino acid sequence of a protein, a closely related gene cloned from a different species, or cDNA synthesized from the same species. Regardless of the origin, probes have the common feature of being homologous to, and thus being able to hybridize with, the targeted gene. Probes labelled with a radioactive tag or with an antigen can be used to rapidly screen thousands of clones to identify the desired gene.

The isolation, fragmentation, and ligation of DNA, and the subsequent transformation of E. coli were all steps necessary to generate a large population of random clones. The objective of this chapter is to provide you with the techniques needed to screen these random clones in order to find the MEL1 gene. The laboratory section of this chapter will involve synthesizing a labelled probe, testing the probe, and applying that probe to screen E. coli colonies harboring recombinant DNA.

It is necessary to clone DNA prior to its molecular analysis and subsequent manipulation and application. Once cloned, its characteristics such as fragment size, restriction sites, nucleotide sequence, and subsequence identification (biologically significant sequences) are forthcoming.

Clones are usually between 7–20 Kb, but the targeted sequence (i.e., the gene) is usually only a portion of that fragment. Gross characteristics, such as fragment size and restriction endonuclease sites, can be deduced by combining restriction digestion with agarose gel electrophoresis (Chapter 9) in a technique called restriction mapping. However, restriction mapping does not locate the target sequence. Combining restriction mapping with Southern blotting will determine the location of the target sequence on the clone. Determining the size of the gene (located within the fragment), the precise location of restriction sites, and the identification of subsequences (e.g., start codons, promoters, introns), require detailed data which is obtained by DNA sequencing. This technique combines DNA polymerization (similar to that used in a random probe synthesis) with Polyacrylamide gel electrophoresis.

In the previous laboratory experiments, you performed the steps needed to isolate DNA from yeast, fragment it, ligate the fragments to a vector, insert the recombinant molecules into E. coli, and then screen those transformed E. coli to locate a clone with the MELI gene. The next logical step in this cloning scheme is to characterize the isolated clone. The following laboratory exercises will focus on restriction mapping, Southern blotting, and techniques used for DNA sequencing. The analysis of the DNA sequence will be examined in Chapter 13.

The sequence of nucleotides within a DNA molecule can yield a wealth of information. Important biological features such as amino acid sequence, exact amino acid composition, and gene structure are contained within sequences. The data can be used for highly specific manipulations of cloned DNA, such as changing a single nucleotide, substituting promoters between genes, and fusing of genes to yield hybrid (heterologous) proteins. Probes can also be designed for a variety of uses, including the detection of genetic diseases and pathogenic microbes.

The nucleotide sequence of a clone is fundamental. From this data, all known restriction sites, subsequences, and coding regions can be determined. The comparison of sequences allows for the elucidation of important biological motifs, such as the Pribnow box, the alteration of sequences through site-directed mutagenesis, and gene fusion methodologies. The synthesis of homologous oligonucleotides allows for the design of highly specific probes (or primers). These oligonucleotides, or primers, have led to the development of an extremely powerful technique, the polymerase chain reaction (PCR). Based on the specificity of primers, PCR allows for the in vitro replication of small regions of DNA.

The objective of the preceding twelve chapters was to gather data from a protein and clone its associated gene. Whether you were successful in this endeavor or simply followed the process, that objective has been achieved. In reality, the means by which you cloned the MEL1 gene, however, is not important. It is the gene itself, including its nucleotide sequence, that is important.

Now that you have a gene and its sequence, what do you do with it? This chapter will focus on the analysis of DNA sequence data and on one method for its application.

The range of different techniques used in the biotechnology laboratory is vast, and as demonstrated by the experiments of the previous thirteen chapters, can cross through many disciplines. Any one researcher can easily be expected to perform every task presented in this manual, from making agar plates to analyzing sequence data on a computer. As a person becomes more experienced in a chosen area of research, his or her repertoire of skills increases far beyond those presented in this manual. However, the skills presented here are the base upon which other abilities are built.