Utilization of protein splicing for purification of the human growth hormone

Abstract

Protein splicing is a post-translational autocatalystic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). The high specificity of excision ensured by intein makes it possible to use a phenomenon of protein splicing for the biotechnology purposes. The aim of this work was optimization of obtaining and purification of the recombinant human growth hormone using the protein splicing. It was experimentally demonstrated that the use of modified intein as auto-removal affine marker makes it possible to perform the rapid and cheap isolation of the recombinant protein Hgh.Furthermore, this approach allows to obtain the human growth hormone with native Nterminus, without formyl-metionine.Protein splicing is a post-translational autocatalystic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). The high

High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor inEscherichia coli

“…The Mxe GyrA intein system consisting of 198 amino acids was first reported as in-frame insertion for protein splicing nearly 20 years ago [ 18 ]. This self-chimeric system is capable of producing a native target protein with an unmodified C-terminal thioester and facilitating the expression and purification of interested proteins [ 19 , 20 ].…”

Application of magnetic iron oxide nanoparticles prepared from microemulsions for protein purification

“…Protein is the central dogma of cell function and study of proteins is essential to a better understanding of the cell function. Protein purification has been a fundamental requisite in advances made in biotechnology 18, 20. Pure protein helps to resolve the basic questions about the functional characteristics of protein while the development of apt techniques is essential where more than one step is required for protein purification 1, 16, 18.…”

“…Pure protein helps to resolve the basic questions about the functional characteristics of protein while the development of apt techniques is essential where more than one step is required for protein purification 1, 16, 18. Recombinant protein technologies are being developed in order to produce protein in large quantities; however, many problems remain unresolved, such as host contamination, solubility and structural integrity 20. In biotechnology, affinity protein purification using antibody‐based separation or a matrix with specific tags for binding target protein are commonly used methods 16–18.…”

Expression of intein‐tagged fusion protein and its applications in downstream processing

“…Mxe GyrA intein (the Mycobacterium xenopi GyrA enzyme) system is a self‐chimeric system capable of generating a target protein with an unmodified N‐terminus and C‐terminus thioester, resulting in a higher expression level and yield. As a self‐removable affinity tag, histidine tag or cellulose‐binding domain was used 44, 45. A remarkably high yield (90%) was reported for recombinant human growth hormone having the native N‐terminus with minimal losses 45.…”

“…As a self‐removable affinity tag, histidine tag or cellulose‐binding domain was used 44, 45. A remarkably high yield (90%) was reported for recombinant human growth hormone having the native N‐terminus with minimal losses 45. An elastin‐like polypeptide (ELP) tag was also used in conjunction with self‐cleaving inteins.…”