SBNet - Research Reports 1998

PI: A. Liljas (LU)

Project: Crystallography at MAX II

I have started my service for SBNet since April 23, 1998. Part of my job (20%-30%) is user support for the beam line BL711 at MAX lab.

Besides the user support, I have been taking part in teaching and setting up my own research group here in department of Molecular Biophysics, Lund University. I have advised two summer students in last summer and one project student (examenarbetare) to do project in molecular biology work since last fall. I am also a co-advisor of a Ph.D. student and lab instructor of KEM103 (Physical Chemistry) at Lund University. The teaching activities took about 30-50% of my time.

Research activities: The rest 30% time I have mainly spent on starting up a few my own research projects detailed as following. No paper yet, but I have a few manuscripts in preparation, hopefully coming out 1999. Please note that most of the work on hemolin and neuroglian was done during my postdoc at Caltech.

Hemolin (Caltech & Lund Univ., 1995-present)

Hemolin is a secreted, putative glycoprotein, belonging to Ig superfamily. The H. cecropia hemolin contains one conserved N-linked glycosylation site, hemolins from other species may contain more N-linked glycosylation sites and at least one other hemolin (M. sexta) has been reported to be glycosylated in vivo.

I have over-produced the wild type and a Se-met substituted version of hemolin for MAD (multiple-wavelength anomalous dispersion) phasing experiments from baculovirus expression system. Using Sf-9 or High 5 (Tn-5) cells, the production level of hemolin could be well above 100mgs per litre of culture medium. During purification, both dimer and monomer forms have been obtained and crystallised. Since the crystals of monomer hemolin could diffract to better resolution, the structure of the monomer was solved first (The structure is published as Su, X-D; Gastinel, L.N; Vaughn, D.E; Faye, I; Poon, P. & Bjorkman, P.J. Crystal structure of hemolin: A horseshoe shape with implications for homophilic adhesion. (1998) Science 281 991-995).

Nrg-4D (Caltech & Lund Univ., 1997-present)

It is the first four N-terminal Ig-like domains of neuroglian, a Drosophila L1-CAM (cell adhesion molecule), that shares 38% sequence identity with hemolin. It contains three N-linked glycosylation sites; and Mass spectroscopy data showed that the recombinant Nrg-4D expressed in High 5 cells is glycosylated non-homogeneously, with 5-6 KD molecular weight difference from the calculated non-glycosylated value.

Starting from a cDNA of the entire neuroglian, the Nrg-4D with a C terminal His-tag was PCR-ed and subcloned into the transfer vector, pVL1393. The recombinant pVL1393 was co-transfected with BaculoGoldreg. linearised baculovirus DNA (PharMingen) using a Ca++ or lipofectin based protocol in Sf-9 or Sf-21 cells. The recombinant baculovirus producing Nrg-4D was plaque-purified and amplified then the protein was over-expressed using a high titer virus stock in either Sf-9 or High 5 cells. The production level in this case was only about 3-6 mgs per litre of culture medium. The Nrg-4D has been purified in reasonable amount here in Lund University. In addition to the ultracentrifugation results published on Science 281 991-995, (1998), more biochemical analysis and crystallisation trials are underway, and micro-crystals of Nrg-4D have been observed.

Parallel to the baculovirus expression system, E. coli expression is also under study to see if we can refold in vitro the non-glycosylated neuroglian fragments. The final goals are to obtain both glycosylated and non-glycosylated structure of neuroglian fragments to compare the structural and functional differences. This part of work has been carried out by the project student, so far she has got good level of inclusion body over-expression of the first six Ig domains as 3 x two domains in E. coli using a pET 28a vector. I will continue the refolding, purification and crystallisation work.

BV-RTP (Lund Univ., 1998)

BV-RTP is a baculovirus encoded RNA 5'-triphosphatase (RTP) which performs the first step in the eukaryotic mRNA capping reaction. It was characterised earlier as a putative PTP (protein tyrosine phosphatase) due to its homology to the PTP family proteins, particularly in the loop region of the active site, the so called PTP-loop which was discovered by me and co-workers during my thesis work (Su, X-D; Taddei, N.; Stefani, M.; Ramponi, G. & Nordlund, P. "The crystal structure of a low-molecular-weight phosphotyrosine protein phosphatase". (1994) Nature 370 546-568). It is very interesting for me to see how this enzyme uses the same active site and possibly the same catalytic mechanism as PTPs. Therefore, I have cloned BV-RTP from baculovirus and over-expressed it in E. coli BL21 (DE3) cells. The over-expression has high yield and the expressed protein is highly active. Since the catalytic mechanism of RTP is a brand new field, further functional analysis is underway. The goal of this project is to have a detailed study of the structure and function of BV-RTP and use it to serve as a model for other eukaryotic RTPs.