PRAKASH KUMAR SINHA

E.Mail ID: acbclk@r.postjobfree.com

Skype ID: prakashsinha02

Current status

I am working as a Postdoctoral Fellow (since 9th July, 2013 - till date) in

the department of Structural Biology, Weizmann Institute of Science,

Rehovot, Israel 76100

Work experience

I worked for 1 year and 10 months (01-Aug-2011 to 07-June-2013)

as a Job contract/ Senior project assistant in an industry

sponsored project entitled "Fourth generation clot bluster" at

Institute of Microbial Technology (IMTECH), Chandigarh, India 160036

Education

Ph.D. Aug, 2006 - March, 2013, Division of Protein Science and Engineering,

Institute of Microbial Technology (IMTECH), Chandigarh, India 160036, Ph.D.

thesis entitled "Directed evolution approach to gain insights on

streptokinase mediated plasminogen activation".

M.Sc. Med. Biotechnology (Aug, 2004-July, 2006), Post Graduate Institute of

Medical Education and Research (PGIMER), Chandigarh, India 160012

B.Sc. (Hons) Human Biology (Aug, 2001-July, 2004), All India Institute of

Medical Sciences (AIIMS), New Delhi, India 110029

Research Area

Protein science (expression of recombinant proteins in E. coli, P. pastoris

and mammalian cells, in vitro refolding and purification of recombinant

proteins, exploring structure and function of proteins, mutagenesis of

protein, biochemical/ biophysical analysis of proteins,), Molecular biology

(gene cloning, mutagenesis of gene).

Relevant Experience

Molecular Biology:

PCR, gene cloning, random mutagenesis, site-directed mutagenesis, site

saturation mutagenesis

Biochemistry and protein sciences:

Phenotypic selection of mutants, high throughput screening of mutants,

protein engineering, optimization of expression of genes in E. coli,

isolation of inclusion bodies, in vitro refolding of proteins, purification

of proteins (blood-derived as well as recombinant proteins) with various

chromatographic techniques, keen user of FPLC system, biochemical analysis

of proteins, activity assay, heterologous protein expression and

purification from Pichia pastoris, protein analysis with gel

electrophoresis and western blotting

Biophysics:

Circular dichroism (CD), Dynamic light scattering (DLS), Small angle X-ray

scattering (SAXS)

and X-ray crystallography

Animal cell culture:

Culturing/ maintenance of CHO and HEK 293 cell line, recombinant protein

expression in CHO

and HEK 293 cell line

Peer Reviewed Publications

1. Prakash Kumar Sinha, Yogendra S. Rathore, Amin Sagar, Kishore Joshi,

Shekhar Kumar, Ashish and Girish Sahni. Visualizing large scale domain

repositioning during streptokinase-plasminogen interactions

(manuscript submitted).

2. Kishore K. Joshi, Jagpreet S. Nanda, Prakash Kumar, Girish Sahni.

Substrate kringle-mediated catalysis by the streptokinase-plasmin

activator complex:Critical contribution of kringle-4 revealed by the

mutagenesis approaches. Biochim Biophys Acta. 2012 1824: 326-333.

3. Suman Yadav*, Rachna Aneja*, Prakash Kumar*, Manish Datt, Sonali Sinha

and Girish Sahni. Identification through Combinatorial Random and

Rational Mutagenesis of a Substrate-interacting Exosite in the y

Domain of Streptokinase. J. Biol. Chem. 2011 286: 6458-6469. (* First

authors/ authors contribute equally).

4. Eshu Singhal, Prakash Kumar and Pradip Sen. A novel role for Bruton's

tyrosine kinase in hepatocyte growth factor mediated immunoregulaion

of dendritic cells. J. Biol. Chem. 2011 286: 320**-*****.

Awards/Scholarships

1. Department of Biotechnology Junior Research Fellowship (DBT JRF year

2007) Ref. No. DBT-JRF/07-08/ 57

2. Indian Council of Medical Research Junior Research Fellowship (ICMR

JRF year 2006) No.3/1/3/JRF-2006/MPD

3. Council of Scientific and Industrial Research-University Grants

Commission National Eligibility Test (CSIR-UGC NET year 2006) F.No.10-

2(5)/2U06(i)-E.U.II

4. Graduate Aptitude Test in Engineering (GATE year 2006) Registration

No. XL 309327

5. Council of Scientific and Industrial Research-University Grants

Commission Junior Research Fellowship National Eligibility Test (CSIR-

UGC JRF NET year 2005) F.No.10-2(5)/2005(ii)-E.U.II

Symposiums Attended

1. Symposium on "Advanced Techniques in Basic Research: Tools of 21st

Century" held on May 1, 2010 at Postgraduate Institute of Medical

Education and Research (PGIMER), Chandigarh, India 160012

2. National Symposium on Cellular and Molecular Biophysics, January 22-

24, 2009, at Centre for Cellular and Molecular Biology (CCMB),

Hyderabad, India - 500 007

3. National Workshop on Trends in Protein Modeling and Cheminformatics,

January 14-16, 2009, at Institute of Microbial Technology, Chandigarh,

India 160036

Summer Training

1. M.Sc. Summer training (June-July 2005) Under the Supervision of Dr

Kalpana Luthra, (Dept. of Biochemistry) All India Institute of Medical

Sciences New-Delhi 110029

2. B.Sc. Summer training (May-July 2003) Under the Supervision of Dr Rima

Dada, (Dept. of Anatomy) All India Institute of Medical Sciences New-

Delhi 110029

Computer Skills

Microsoft Office, Gene Runner, SigmaPlot, Pymol, Swiss-PDB viewer,

OriginLab, Ubuntu linux

Strength

Team spirit, Team leader capability, Can work independently, Hardworking,

Adaptive Nature, Optimist, Patient, and Punctual

Summary of Ph.D. Work

During Ph.D., my research work was focused on studying the structure-

function relation of Streptokinase (SK, a thrombolytic agent) and

Plasminogen (HPG) proteins. Using random mutagenesis and site-directed

mutagenesis, one new hot-spot (exosite) in SK was found that interacts with

the substrate HPG. To further decipher the detailed insights behind the SK-

HPG interactions, recombinant HPG (rHPG) was required. Being a 7 domain

protein having 24 di-sulfide bonds, rHPG was difficult to be generated in

E. coli. Therefore, after expressing HPG as inclusion bodies inside E.

coli, several thousand buffer conditions were tried for the in vitro

refolding of rHPG. Subsequently, purification of rHPG were optimized to get

the zymogen form of rHPG. The E. coli derived rHPG's activity was found to

be comparable to that of blood-derived HPG. Notably, during protein-protein

interactions conformational changes occur and SK causes lysis of HPG.

Hence, inactive rHPG were prepared to get the stable complex of SK-rHPG. To

delineate the structural interactions of SK and HPG, and their binary and

ternary complexes inside solution, small angle X-ray crystallography (SAXS)

technique was used. SAXS was further used to study the rearrangement in the

domain organizations in SK and HPG in the complex state.

References

1. Dr. Girish Sahni, Director, Institute of Microbial Technology, Sector

39A, Chandigarh, India 160036. Telephone +91-172-******* Email:

acbclk@r.postjobfree.com, acbclk@r.postjobfree.com

2. Dr. Pradip Sen, Senior Scientist, Institute of Microbial Technology,

Sector 39A, Chandigarh, India 160036. Telephone +91-172-******* Email:

acbclk@r.postjobfree.com

3. Dr. G S Prasad, Senior Principal Scientist, Institute of Microbial

Technology, Sector 39A, Chandigarh, India 160036. Telephone +91-172-

2636680 Email: acbclk@r.postjobfree.com

Personal Information

Date of Birth 25th Oct 1982

Marital Status Married

Nationality Indian

Corresponding address Room no 561, Kimmelman Building, Weizmann

Institute of

Science, Rehovot, Israel 76100

Permanent Address S/o Sh. D.P. Sinha, Road no-4, 52-Bigha, near

Manasthali Public

School, Laxmisagar, Darbhanga, Bihar, India 846009

(Note: I have changed my name from Prakash Kumar to Prakash Kumar Sinha

from 26th July 2012).

Declaration

I hereby declare that the information furnished above is true and correct

to the best of my knowledge and belief, and that no related information is

concealed.

Prakash Kumar Sinha

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