Krishnakumar V

• Expression of human protein kinases of therapeutic importance in eukaryotic, prokaryotic system and purification for crystallization, biochemical and biophysical assays.
• Training new team members, performance management, collaboration, strategic planning, and a focus on continuous improvement.

• Creation of stable mammalian cell lines for cell-based assays, expression, and purification of proteins using vectors like pEF4/V5-His series, pcDNA4/V5-His series, and pGene/V5-His series.
• Good hands-on experience in generating mutant proteins using site-directed mutagenesis, co-expression, in vitro dephosphorylation of proteins, and protein crystallization.

• Exposure to different FPLC purification systems, such as AKTA's prime plus and explorers, etc.
• Chromatography techniques like affinity (NiNTA, GST, Flag), ion exchange, hydrophobic interactions, and size exclusion chromatography.
• The protein purified for crystallization purposes typically had four steps: step 1, Ni affinity; step 2, His tag removal; step 3, ion exchange; and step 4, size exclusion chromatography.

• Cloning, expression of Human kinases in Prokaryotic expression system involves the domains from kinase targets cloned into T7, tac promoter vectors and co expressed with protein phosphatases in E.coli BL21DE3 series and E.coli K12 derivatives

• Cloning, expression of human genes related to diseases from cDNA libraries, expressing kinase domains, and complete ORF using baculovirus expression system in insect cells like Sf9, Sf21, and High five.
• Expression of the target protein from insect cells was quantified by specific antibody western blotting and by reporter genes, like EGFP.

• Project Title: 'An evaluation of endemism of herpetofaunal assemblages of the Western Ghats using molecular techniques.'
• I worked on phylogenetic and phylogeographic patterns of endemic frog lineages using mitochondrial and nuclear DNA regions. As an outcome of the programme, we identified a new frog species from the Central Western Ghats of India.
• I was also involved in working on the structure and arrangement of the Hox gene cluster in an ancient frog. Study of the Hox gene cluster may enable us to know about its evolution in the frog family and may give an idea about changes in the body plan development and axis determination through the frog lineage.
• Publications:

1. Gururaja, K. V., Aravind, N. A., Ali, S., Ramachandra, T. V., Velavan, T. P., Krishnakumar, V., & Aggarwal, R. K. (2007). A new frog species from the central Western Ghats of India, and its phylogenetic position. Zoological Science, 24(5), 525-534.

2. Aggarwal RK, Hendre PS, Varshney RK, Bhat PR, Krishnakumar V, Singh L. Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species. Theor Appl Genet. 2007 Jan;114(2):359-72. doi: 10.1007/s00122-006-0440-x. Epub 2006 Nov 18. PMID: 17115127.

• Project Title: "DNA fingerprinting of Coffee (Coffea species) Germplasm and development of molecular linkage map and development of EST for abiotic stress tolerance ”
• Iworked with the Coffee system in the area of EST development and analysis of Resistance Gene Analogues (RGAs) with respect to the molecular basis for abiotic and biotic stress tolerance.
• Developing ESTs: A cDNA library was constructed from leaf samples of coffee plants subjected to drought and oxidative stress, which consists of about 7,000 clones. Many clones with SOD, catalase, and peroxidase activity, usually involved in oxidative stress response, were found and identified for further study.
• RGAs: Degenerate primers were designed to amplify the NBS-LRR region in the coffee genome, and further primer conditions were standardized. The amplified product was cloned into the pMOS vector, and more than 500 clones were picked, sequenced, and analyzed bioinformatically.
• Analysis of lens and cornea specific transcripts: A cDNA library was constructed from the lens and cornea of the Indian mugger (Crocodylus palustris). Around 1,200 cDNA clones were sequenced and annotated. The library has yielded some transcripts of interest that are crystalline-specific and novel transcripts, which are being further characterized in the lab.
• Publications:

1. Prasanna R.Bhat, V. Krishnakumar and Ramesh K. Aggrwal, 2005, Development and characterization of gene-derived (EST) SSR-markers in Coffee (Coffea sp.). Molecular Ecology Notes (2005) 5: 80-83.

2. Kathiresan T, Krishnan K, Krishnakumar V, Agrawal R, Anand A, Muralidhar D, Mishra AK, Dhople VM, Aggrawal RK, Sharma Y. Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development. FEBS J. 2006 Jul;273(14):3370-80. doi: 10.1111/j.1742-4658.2006.05344.x. PMID: 16857018.

• Project Assistantship under the guidance of Prof. P. Gunasekaran, In the project "Cloning, nucleotide sequencing of Penicillin acylase encoding gene from a Bacillus sp.”
• Our interest was focused on identifying the PAC gene. A genomic library was constructed in E. coli DH5α, and about 2000 recombinant clones were obtained. These clones were screened for PAC activity using the Serratia marcescens overlay technique, but no PAC positive clone was found. When the library was screened for other markers of the Bacillus sp., one out of 2,000 was found to be urease positive. Further, a probe was designed based on the conserved sequences of the PAC gene, and a part of the gene has been amplified. This partial gene has been used to screen a genomic library to obtain a complete gene.