Cysteine cathepsins, a class of proteinaceous enzymes, regulate a wide variety of metabolic processes in human including protein breakdown and turnover and immune functions. Eleven cysteine cathepsins have been identified so far and a wide array of studies related to identifying their specific functions, regulation and distribution patterns in tissues have been conducted.However, in recent past, the association of cysteine cathepsins in occurrence and progression of cancers have been identified and this has caused unrest in scientists triggering them to investigate the physiology, biochemical pathways and interactions of these cathepsins in cancer metastasis and therefore has become a noteworthy topic of intensive research. This review focusses and collects together the published work on molecular functional and structural characterization studies that have been done so far on in vitro expression of genes encoding for cysteine cathepsins in the Escherichia coli bacterial expression system. Accordingly, it was found out that all cathepsins except for cathepsins K, C, H, X and W have been expressed this way and the majority of them were found to be expressed in E. coli BL21(DE3) pLysS expression host via pET3 expression vector. In addition, it was also noted that in most of the expression studies, the substrate that was used to validate the enzymatic activity of the recombinant enzyme that was produced was a cysteine residue along with a benzyloxy-carbonyl salt. Through this review, the authors suggest that there is a very high need that all cysteine cathepsins need to be characterized both structurally and functionally on a molecular platform to better understand their interactions including the biochemical pathways. It is also momentous that the mass production of the recombinant forms of these enzymes are facilitated via expression in such bacterial expression systems and in turn, would also provide a strong platform for the development and progression of studies related to human physiology including oncological studies such as cancer metastasis. Moreover, as per biochemical features of the enzymes that could be identified, the production of efficient inhibitors or inducers as per the necessity to improve health and promote wellbeing among the mankind could be facilitated.

The effect of proteolytic enzymes including Cathepsin K, a cysteine cathepsin, in onset and progression of cancers in human has been research intensive. Cathepsin K involves in many aspects and stages of cancers including apoptosis, cell proliferation, cancer immunology, inflammatory cell recruitment to tumors and aiding in the process of mobilization of normal healthy cells from their tissue compartments assisting in metastasis and angiogenesis. The objective of this review is to collect together and summarize and analyze the biochemical and physiological pathways of how cathepsin K is involved in onset and progression of cancers with more emphasis on breast and prostate cancers and cathepsin K regulated mechanisms underlying metastasis of such cancers to bones. Information for the review was gathered through published literature from global databases such as Google Scholar, PUBMED and NCBI on different studies on physiological interactions between enzymatic activity of cathepsin K with cancers and metastasis to bones. Analysis of published studies reveal that immunohistochemical studies of breast cancer cells indicate that they overexpress cathepsin K resulting in induction of aberrant mechanisms of cell signaling in breast cancers, creating a higher tendency for their metastasis to bones. Immunohistochemical, immunoprecipitation and fluorgenic assays of several studies done on the association of the same enzymatic activity on prostate cancers shows elevated levels of cathepsin K. Lesions derived from prostate cancer cell masses were observed to undergo increased bone formation and resorption levels. Such resorption levels cause secretion of biological factors promoting tumor expansion. In addition, studies indicate that Cathepsin K was observed to be a key component promoting higher bone resorption levels in patients suffering from cancer. Authors suggest that, to completely understand the association of cathepsin K on cancerous cells and their mechanism in metastasis, distributary patterns of cathepsin K in healthy human tissues needs to be extensively studied initially. It is also suggested that metastasis of breast and prostate cancers to bone could be terminated and overcome by successful production of efficient and precise inhibitory therapeutics targeting the enzymatic activity of Cathepsin K with minimum unintended adverse health effects.

Cathepsin K (CatK), encoded by CtsK gene in human, is involved in bone remodeling through ossification. The objective of the work conducted here was to express catalytic domains of CtsK gene in bacterial expression system as an initial step, facilitating recombinant production of human CatK for downstream applications in pharmacology. Four healthy human blood samples were collected. Genomic DNA was extracted using FlexiGene® whole blood DNA extraction kit. Upon quantification of DNA through NanodropTM spectrophotometer, sufficient quantity and quality was observed. CtsK gene was amplified by Polymerase Chain Reaction (PCR) using two pairs of primers tagged with restriction endonuclease sites of Sal1 and HindIII facilitating molecular cloning and visualized by Agarose Gel Electrophoresis (AGE). Two different bands of size 545bp and 265bp were observed. The bands were dissected and gel purified using GenaxxonTM gel purification kit and sequentially double digested by restriction enzymes; SalI and HindIII.  Vector PBS was also subjected to sequential double digestion using same enzymes and visualized via AGE. Double digested insert of size 265bp and vector were ligated using T4 DNA Ligase (all enzymes from PromegaTM). On another trail, ligation of the PCR product with band size 265bp to pGEM-TTM easy vector system (from PromegaTM) was also done and transformed to Top10 Escherichia coli competent cells for expression separately. Cells were grown in LB media in presence of XGAL, IPTG and Ampicillin and transformed cells were screened. In the restriction enzyme digestion and ligation setup, since the insert and vector were both double digested, it is confirmed that white colonies obtained were Escherichia coli cells were transformed with the desired recombinant vector and is therefore confirmatory. In the case of pGEM-TTM ligation, a colony PCR was done using the white colonies obtained and product size was confirmed via AGE. In conclusion, the objective of study was successfully achieved, by expressing a catalytic domain of CtsK. Developments and improvements could be made for expression of entire CatK gene and downstream production of the Cathepsin K protein for effective therapeutic purpose.