With the increasing demand for commercially important proteins, it has become essential to develop more improved and efficient methods for heterologous protein production to meet the requirements of society. Although currently there are different host systems available for the production of proteins heterologously, still the selection of a perfect host system becomes a tedious task if the aim is to get fully functional protein in adequate quantity. Different types of hosts are commonly used for heterologous protein production include Escherichia coli, Saccharomyces cerevisiae, insect cell expression systems and various mammalian systems. Different host systems that are used for heterologous protein expression have their own advantages and disadvantages as in the case of bacterial systems, a good amount of protein is produced but the quality of the protein may not be appropriate. Similarly, eukaryotic hosts also suffer from specific merits and demerits. Different promoter systems that are used for gene expression also influence protein production in case of both prokaryotic and eukaryotic hosts. The selection of the appropriate host depends on the amount of the protein, quality of the protein in terms of functional activity and its application. In this chapter, we will discuss the principle of heterologous gene expression, different types of hosts organism available for heterologous protein production. We will also present the current developments and modifications which are being done in various host systems to improve the process of protein production. We will elaborate on the different types of vectors that are used for recombinant protein production, their contrasting features and different promoter systems used in the vectors, importance or promoters with respect to protein production. In addition to this, we will also elaborate on different aspects related to the overexpression and purification of recombinant heterologous protein and the type of tags and chromatographic techniques used for the purification of different types of proteins.