Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements

Abstract

E. coli is most preferred system used for the production of recombinant proteins in bacteria and the availability of improved genetic tools/methods are making it more valuable than ever. Major challenges faced by this expression system are the expression of unusually difficult/complex proteins with rare codons or membrane and toxic proteins. The proteins expressed either in large amount or hydrophobic in nature tend to form insoluble mass. Despite the appropriate expression system, some proteins express at very low level or not at all. Choosing the correct expression system/protocols are obligatory for the substantial expression of protein in the native form. A number of vectors, their compatible hosts and culture conditions can be used to express recombinant proteins in large amounts and in native form. Also, vectors with the fusion tags/chaperons facilitate protein expression in soluble fraction and assist in proper protein folding besides restoring the native structure of protein. The recovery of native proteins from insoluble inclusion bodies can be achieved by optimization of refolding conditions. In the present review, we discussed recent updates on prokaryotic expression system for successful heterologous gene expression in E. coli and focused on strategies to maximize the yields of native recombinant proteins.

Introduction

Genetic engineering and recombinant nucleic acid (DNA or RNA) technologies are exploited to obtain new combinations of genetic material from different organisms. Genetic engineering was first accomplished by Herbert Boyer and Stanley Cohen in 1973, to create the functional organism that combined the genetic information from different species and replicated them very nicely [1]. They introduced genes from toad Xenopus laevis into bacteria, E. coli and demonstrated that these genes were actively expressed generation after generation [2].

There are many hosts used for the production of recombinant protein such as bacteria, yeast, insect, plants and animals. E. coli has been the most popular means of producing recombinant proteins for over two decades from not only prokaryotic but also eukaryotic origins, as it can grow on inexpensive media under well defined laboratory conditions and have very short doubling time i.e., 20 min. Due to its rapid growth, selection of mutants is easy and convenient. Moreover, E. coli cells are highly efficient to receive (incorporate) foreign DNA and express recombinant proteins at very high rate [3]. Various expression systems are available for various applications. Approximately, 80% proteins with solved three-dimensional structures submitted to the protein data bank (PDB) in 2003 were expressed in an E. coli expression system [4]. Genentech, was the first company to commercialize the human protein somatostatin produced by genetically modified E. coli [5] followed by human insulin in 1978 [6]. Presently, a lot of life-saving drugs are produced in E. coli. But till now, heterologous gene expression in E. coli is not that easy task as it looks.

Extracellular production of recombinant protein is highly desirable in order to reduce the complexity of a bioprocess as well as to improve the product quality. Use of E. coli expression system is limited due to the lack of secretion systems for efficient release of proteins into the growth medium, limited ability to facilitate extensive disulfide-bond formation, inefficient cleavage of the amino terminal methionine, protein inactivity, its toxicity and inability to confer posttranslational modifications which can result in lowered protein stability and increased immunogenicity [7], [8]. Leaky expression is another major problem associated with various expression systems in this organism. With advancement in rDNA technology, numerous engineered plasmid vectors and hosts have been developed to express these difficult proteins [9], [10]. But still, it completely relies on appropriate selection and availability of strains. Researcher have used various molecular strategies to express difficult proteins in available expression system by optimizing various parameters such as, cultivation temperature, inducer concentrations, cell growth and co-expression with chaperons at the time of induction and use of additives in culture medium [11], [12], [13].

Though in the last 25 years, several advanced systems were developed for heterogenous proteins and various reviews were published in this area [7], [14], [15], [16], [17], no comprehensive detail is available at one place. Therefore, an attempt has been made to summarize the available information in this area till date. This review describes various vectors/hosts/methods/strategies used for the high level expression of recombinant protein in E. coli.