Preparation Of Cross-linked Protein Crystal Seeds And Their Effects On Protein Crystallization

The probability of obtaining protein crystals from the high purity protein is less than 20%.Hence,it is required to develop methods to increase the chance of nucleation process to start the develop of a protein crystal.This nucleation is usually of two types including homogeneous nucleation and heterogeneous nucleation.In the metastable region,proteins cannot crystallize by homogenous nucleation.However,utilization of modified interface materials or solid particles as nucleating agents can induce protein nucleation in the metastable region thus obtain larger crystals suitable for diffraction.Therefore,it's significant to perform heterogeneous nucleation using the interfaces of various materials.Similar to the nucleants,seeds which are small fragments of protein crystals have also showed impressive effects to facilitate protein crystallization process.In this work,we proposed the use of cross-linked protein crystals(CLPCs)as seeds to increase the efficiency of heterogeneous nucleation in protein crystallization.CLPCs are protein crystals that are chemically cross-linked with a cross-linker in their crystalline state.Importantly,CLPCs are insoluble in water and organic solvents;possess excellent mechanical and thermal stability.Furthermore,CLPCs possess three-dimensionally ordered structures and their diffraction patterns are very similar to those of native crystals.In summary,CLPCs may be a very desirable material for inducing protein crystallization,and we study the effect of cross-linked crystals on protein crystallization.Here we first studied the preparation of high-quality cross-linked protein crystals.Natural protein crystals can only maintain the complete structure in the mother liquor.Once separating from the mother liquor,they will crack quickly accompanying dissolution.One way to solve this problem is cross-link the protein crystals using glutaraldehyde,which is an efficient way to confine protein molecules in the crystal lattice and hence greatly improve crystal stability.However,a problem is often encountered.That is,cross-linking induces cracking of the crystals.To find solutions to this problem,we initiated an investigation into the phenomenon,i.e.,cross-link induced cracking of protein crystals using an orthogonal experiment design.Experimental parameters including crystal growth time,temperature,cross-linking time,solution pH,and different concentrations of glutaraldehyde were used as factors.The major goal was to obtain suitable conditions for preparing cross-linked lysozyme crystals free of cracks.At present,the mechanism involved in the formation of cross-linked protein crystals(CLPCs)by using glutaraldehyde is unclear.The cross-linking reaction between crystal and glutaraldehyde is an external-inside process.The external protein molecules preferentially react with glutaraldehyde molecules,while the internal protein molecules are post-cross-linked.Therefore,the cross-links inside and outside the crystal may not be completely uniform.Quality of cross-linked lysozyme protein crystals was investigated at different time intervals such as after 12 h,24 h,36 h,48 h,60 h,84 h,and 108 h of incubation.Furthermore,to explain the mechanism of cross-linking between glutaraldehyde and protein crystals different parameters i.e.resolution,mosaicity,B factors,and unit cell were analyzed.After obtaining high-quality CLPCs,the experiment using CLPCs as seeds in protein crystallization was carried out.At first,we studied the effect of CLPCs seeds on the reproducibility study of lysozyme crystallization.Results showed that in the presence of CLPCs the rate of lysozyme crystallization was significantly increased over the control(crystallization in the absence of CLCPs).In addition,the lower the initial concentration of the protein was,the more significant the difference in the crystallization success rate.Secondly,we conducted screening study of protein crystallization.It showed the more types of proteins used as seeds,the bigger improvement of the screening hits.In addition,seeding using the CLPCs of a protein could most significantly improve the screening hits of that same protein.The third,the effect of CLPCs seeds on the quality of protein crystals was investigated.It showed that adding the CLPCs seeds can not only improved the morphology of protein crystals,but also enhanced the quality of protein crystals.In summary,we used an orthogonal array to investigate the cracking of lysozyme crystals cross-linked by glutaraldehyde and proposed the best cross-linking conditions.By preparing lysozyme crystals with different cross-linking times,the mechanism between the glutaraldehyde and protein crystals was studied.Finally,the screening hits and crystal quality were studied by using the CLPCs as the seeds,aiming to provide a new method for preparing high-quality protein crystals.