Wei Luo Chunxia Guo Jing Zheng and Marvin L Tanzer 1 Introduction

Target proteoglycan gene(s) can be rearranged to fulfill special designs for expression, purification, or characterization purposes. These are achieved through several procedures, including polymerase chain reaction (PCR) amplification of a target gene, cloning of PCR products into cloning vectors, subcloning of a correct gene fragment into an expression vector, purification of target gene DNA via a large preparation, transfection into host cells for expression, purification of candidate proteins, and identification of the proteins. Characterization can be done of core proteins or of the glycosaminoglycan chains attached to the core proteins. A summary of the expression of an engineered protien is shown in Fig. 1.

1.1. Construct Assembly of Target Proteoglycans

Gene structure arrangements are accomplished through PCR with specifically designed oligonucleotides and target template(s) (1). Appropriate PCR products are engineered into a suitable eukaryotic expression vector using appropriate polylinkers. The vector containing target gene(s) can express (transiently) proteoglycans through transfection into candidate eukaryotic cell lines (see Subheading 1.2.).

1.2. Expression of Target Proteoglycans

Large amounts of DNA with molecular-level purity are needed for transfection and are prepared by a maxiprep kit (Qiagen). The maxiprep-prepared DNA is then used to transfect eukaryotic cell lines to express/obtain the corresponding proteins. Cell lines are chosen based on two major criteria: (1) the cell lines themselves do not express the same target endogenous genes; (2) the cell lines have the capability (e.g., machinery to add glycosaminoglycan chains) to express proteoglycans.

Fig 1. Summary diagram of expression of an engineered proteoglycan.

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Fig 1. Summary diagram of expression of an engineered proteoglycan.

1.3. Purification of Target Proteoglycans

Expressed products are purified using a nickel column with stringent washing procedures to eliminate background proteins. A histidine-6 (His6) tag is engineered in the construct assembly (see Subheading 1.1.) to allow such a purification procedure.

1.4. Identification of Target Proteoglycans

The purified proteins can be run on sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) directly to separate the products from background proteins or processed further for other experiments. Purified products can be characterized by various procedures, e.g., quantitation by radioactivity, classification of polysacchrides by enzymatic digests, and identification of core proteins by immunocapture.

1.5. Characterization of Expressed Proteoglycans

Proteoglycans have unique features that can be individually characterized. Chon-droitin sulfate, keratan sulfate, dematan sulfate, and heparan sulfate can be digested with appropriate enzymes to confirm their presence. The example protocol is for chon-droitin sulfate digestion (see Subheading 3.5.).

1.6. Detection of Proteoglycan Interaction with Heat-Shock Proteins Part I

Expressed proteoglycans may interact with certain types of heat shock proteins (Hsp) (2). For example, aggrecan's G3 domain interacts with Hsp25. The example protocol is for immunocapture of Hsp25 using anti-Hsp25 antibody (see Subheading 3.6.).

I.7. Detection of Proteoglycan Interaction with Heat-Shock Proteins Part II

Interaction between heat-shock proteins and proteoglycans can also be detected by in situ cross-linking. This method is suitable only when the interacting candidate Hsp and proteoglycan are adjacent to each other and they have suitable amino group(s) in proximity that can be cross-linked. The procedure here uses 3,3'-dithiobis sulfosuccinimidylpropionate (DTSSP) as a cross-linking reagent that requires lysines or an N-terminal amino group for reaction. Other reagents may be used for other chemical groups.

2. Materials (see Note 1)

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