Cloning, expression and purification of the SRCR domains of glycoprotein 340

Abstract

Glycoprotein 340 (gp340), an innate immunity molecule is secreted luminally by monolayered epithelia and associated glands within the human oral cavity. Gp340 contains 14 scavenger receptor cysteine rich (SRCR) domains, two CUB (C1r/C1s Uegf Bmp1) domains and one zona pellucida (ZP) domain. Oral streptococci are known to adhere to the tooth immobilized gp340 via its surface protein Antigen I/II (AgI/II), which is considered to be the critical first step in pathogenesis that eventually results in colonization and infection. In order to decipher the interactions between gp340's domains and oral streptococcal AgI/II domains, we undertook to express human gp340's first SRCR domain (SRCR1) and the first three tandem SRCR domains (SRCR123) in Drosophila S2 cells. While our initial attempts with human codons did not produce optimal results, codon-optimization for expression in Drosophila S2 cells and usage of inducible/secretory Drosophila expression system (DES) pMT/BiP/V5-HisA vector greatly enhanced the expression of the SRCR domains. Here we report the successful cloning, expression, and purification of the SRCR domains of gp340. Recognition of expressed SRCRs by the conformational dependent gp340 antibody indicate that these domains are appropriately folded and furthermore, surface plasmon resonance studies confirmed functional adherence of the SRCR domains to AgI/II.

Keywords: DMBT1; SAG; SRCR; Streptococcus mutans; gp340.

Figure 1

Primary sequence layout of Gp340 which contains fourteen SRCR domains, two CUB domains and one ZP domain.

Figure 2. Expression of SRCR genes in stably transfected S2 cells

RT-PCR of SRCRs recombinantly cloned in Drosophila expression vector pMT/V5-HisA. Lane 1) Generuler 1 kb plus DNA Ladder (Fermentas), Lane 2) RT-PCR product of SRCR₁ transfected S2 cells, Lane 3) RT-PCR product of SRCR₁₂₃ transfected S2 cells.

Figure 3

Plasmid constructs map showing the elements and schematic representations of the gene constructs (A) SRCR₁ and (B) SRCR₁₂₃.

Figure 4. Expression of codon-optimized SRCR genes in stably transfected S2 cells

(A) RT-PCR of SRCRs recombinantly cloned in Drosophila expression vector pMT/BiP/V5-HisA. Lane 1) Generuler 1 kb plus DNA Ladder (Fermentas), Lane 2) RT-PCR product of SRCR₁₂₃ transfected S2 cells, Lane 3) RT-PCR product of SRCR₁ transfected S2 cells.

Figure 5. Purification of codon-optimized SRCR₁

A) Affinity chromatogram of SRCR₁ over the 3 × Histrap™ 5 ml column [Milli absorbance unit (mAU) = A₂₈₀, CV = Column volume, % B = gradient elution]. B) Ladder: Page Ruler™ pre-stained protein ladder (Fermentas), Lanes 1-10: Eluted SRCR₁ peak fractions (5 ml each) from affinity purification and electrophoretically separated on a 12.5% SDS-PAGE gel C) 12.5% SDS-PAGE gels displaying SRCR₁ purified over Mono Q HR10/10 and Superdex 200 10/300 GL columns.

Figure 6. Purification of codon-optimized SRCR₁₂₃

A) Affinity chromatogram of SRCR₁₂₃ separated over a 3 × Histrap™ 5 ml column [Milli absorbance unit (mAU) = A₂₈₀, CV = Column volume, %B = gradient elution]. B) Ladder: Page Ruler™ pre-stained protein ladder (Fermentas), Lanes 1-7: Eluted SRCR₁₂₃ peak fractions (5 ml each) from affinity purification and electrophoretically separated on a 12.5% SDS-PAGE gel. C) 10% SDS-PAGE gels displaying SRCR₁₂₃ purified over Mono Q HR10/10 and Superdex 200 10/300 GL columns.

Figure 7

Immunoblot of SRCRs probed using the SAG mAb 213-06 confirmed the expression of SRCR₁ and SRCR₁₂₃.

Figure 8

Sensorgrams from surface plasmon resonance studies showing interaction of AgI/II with immobilized (A) SRCR₁ and (B) SRCR₁₂₃ on CM5 sensor chip. Adherence to labeled chip was measured in resonance unit (RU), where 1 RU = 1 picogram of protein.