Recombinant human ITIH2 production can be achieved in multiple expression systems, including yeast, E. coli, in vivo biotinylation in E. coli, baculovirus, and mammalian cells. The generation process starts with cloning the gene of interest (55-702aa of human ITIH2) into an expression vector, which is transformed into the expression system. If needed, any tag can be added to the target gene. The cells are cultured under conditions that induce protein expression. Once adequate growth is achieved, the cells are lysed to release the recombinant human ITIH2 protein. Purification is done by affinity chromatography. The purity of the recombinant human ITIH2 protein is confirmed using SDS-PAGE, exceeding 85%.
Human ITIH2 is a serum protein that responds to tissue injury by binding to hyaluronan. ITIH2 is part of the inter-alpha-trypsin inhibitor family, which consists of serine protease inhibitors assembled from precursor proteins including a light chain and one or two heavy chains. Specifically, ITIH2 is a heavy chain protein that, in humans, is typically cross-linked to bikunin and contributes to extracellular matrix stability through covalent linkage to hyaluronan [1][2]. The inter-alpha-trypsin inhibitor family comprises four heavy chains (ITIH1, ITIH2, ITIH3, and ITIH4) and one light chain, bikunin, which are involved in inflammation, tumorigenesis, and metastasis processes [3][4]. Furthermore, ITIH2 is known to be a serine protease inhibitor and is part of the ITI family, which includes heavy chains H1, H2, and H3, associated with bikunin [5-7].
References:
[1] L. Almeida, D. Young, L. Chow, J. Nicholas, A. Lee, M. Poonet al., Proteomics and metabolomics profiling of platelets and plasma mediators of thrombo-inflammation in gestational hypertension and preeclampsia, Cells, vol. 11, no. 8, p. 1256, 2022. https://doi.org/10.3390/cells11081256
[2] A. Toledo, J. Nilsson, F. Noborn, C. Sihlbom, & G. Larson, Positive mode lc-ms/ms analysis of chondroitin sulfate modified glycopeptides derived from light and heavy chains of the human inter-α-trypsin inhibitor complex*, Molecular & Cellular Proteomics, vol. 14, no. 12, p. 3118-3131, 2015. https://doi.org/10.1074/mcp.m115.051136
[3] R. Geisert, Expression of inter-alpha-trypsin inhibitor heavy chains in endometrium of cyclic and pregnant gilts, Reproduction, vol. 126, no. 5, p. 621-627, 2003. https://doi.org/10.1530/rep.0.1260621
[4] A. Hamm, J. Veeck, N. Bektas, P. Wild, A. Hartmann, U. Heindrichset al., Frequent expression loss of inter-alpha-trypsin inhibitor heavy chain (itih) genes in multiple human solid tumors: a systematic expression analysis, BMC Cancer, vol. 8, no. 1, 2008. https://doi.org/10.1186/1471-2407-8-25
[5] S. Choi, S. Park, D. Kwak, S. Phark, M. Lee, J. Limet al., Proteomic analysis of proteins secreted by hepg2 cells treated with butyl benzyl phthalate, Journal of Toxicology and Environmental Health Part A, vol. 73, no. 21-22, p. 1570-1585, 2010. https://doi.org/10.1080/15287394.2010.511583
[6] W. Morelle, C. Capon, M. Balduyck, P. Sáutière, M. Kouach, C. Michalskiet al., Chondroitin sulphate covalently cross‐links the three polypeptide chains of inter‐α‐trypsin inhibitor, European Journal of Biochemistry, vol. 221, no. 2, p. 881-888, 1994. https://doi.org/10.1111/j.1432-1033.1994.tb18803.x
[7] L. Ødum and H. Nielsen, Human protein hc (α1microglobulin) and inter-α-trypsin inhibitor in connective tissue, The Histochemical Journal, vol. 26, no. 10, p. 799-803, 1994. https://doi.org/10.1007/bf00188078
