| Objective:Streptavidin(SA) was first produced and isolatedfrom the culture broth of Streptomyces avidinii by Stapley et al.and later Chaiet and Wolf established its chemical nature anddetermined its amino acid composition. It has been supposedthat this bacterial protein is secreted by Streptomyces avidiniiand functions as an antibiotic by reducing the free biotinconcentration.SA is a non-glycosylated, neutral bacterial protein which isremarkably similar in its biotin-binding properties to thepositively charged egg-white glycoprotein, avidin. The strengthof the biotin-binding affinity which these two proteins exhibitis the highest recorded between a protein and its ligand. Severalstructural characteristics are also shared. For example, both SAand avidin are of similar molecular size and are composed offour identical subunits, each containing a single biotin-bindingsite. The major problem of using avidin in some applicationssuch as ELISA and Immuno histo-chemitry is the highnonspecific binding, which is attributed to both the presence ofthe sugars and the high pI. There no exist these flaws in SAwhich contains no carbohydrate and whose pI is lower. So SA iswidely used in place of avidin because of its lower nonspecificbinding. Applications of SA in various biological fields arenumerous, such as TRFIA, quantifying PCR, isolation of single-stranded nucleotide, purification of biomolecule, monoclonalantibody production as well.Hitherto, there is not SA made, on a large scale, in China.Most of the internal laboratory have to employ imports of SA. Aneed has arisen for production of this substance at a moderatecost in order to make it available to most investigators. Liu L etal., members of our research group, picked out a strain ofstreptomyces which secretes SA from 860 strains of soil bacteriaand named as L-183. Song SX et al. explored primarily cultureconditions for L-183 and achieved a SA production amount of111mg/L.One target of our experiment is to examine further into thenature of SA and refine the protocol of purification of SA.Another is to improve fermentation conditions for L-183 and toobtain mass production of SA to provision the internallaboratory.Methods1 Purification and identification of SA①Fermentation culture of L-183 was carried out utilizing amethod described by Song SX et al.. After fermentation culture,streptomyces cells and insoluble material were removed fromthe culture supernatant liquid by centrifugation at 5000×g for15min at 4℃. Solid ammonium sulfate was added to thesupernatant liquid with constant stirring for 10min until 80%saturation was achieved. Stirring was continued for 2h at 4℃.The precipitate that formed was pelleted by centrifugation at10000×g for 15min at 4℃. The supernatant fluid was discarded,and the precipitate was dissolved in approximately 100ml ofdistilled water/liter of supernatant liquid. The dissolvedprecipitate was denatured at -20℃for 48h. After melted atroom temperature, the solution was centrifugated at 10000×g for15min at 4 ℃. The supernatant liquid, which containedself-made SA, was collected and was brought to pH11 with 6NNaOH. The precipitate was re-dissolved in distilled water andrepeated the above-described procedure. The affinity columnwhich contained 2-iminobiotin Sephrose 4B was washed withpH11 0.05M carbonate buffer and closed. Then a appropriatevolume of the SA-containing supernatant liquid was loaded intothe affinity column and kept still for 5min at 37℃before theoutlet was opened. The Sephrose was washed with pH11 0.05Mcarbonate buffer until the absorbance at 280nm of the effluentfractions was zero. Subsequently, the adsorbed protein waseluted with 0.01M acetate buffer and collected until theabsorbance at 280nm of the effluent fractions was zero. Theprotein-containing fractions were pooled into the dialysis bagand concentrated with polyethylene glycol 6000 in 1/10 volume.The contents were dialyzed for 24h at 4℃against distilledwater which was refreshed 5～6 times and then subjected toexamination. ②In order to analyze the molecular weight andthe subunit composition and the purification degree of aself-made SA sample, discontinuous SDS-PAGE was performedand gel imaging analysis system was also employed. ProteinaseK was used to convert self-made SA to the truncated form. Theactivity of self-made SA was obtained with the ultravioletspectrum method. A self-made SA sample was combined withincreasing quantities of biotin. The absorbance increment at233nm of the mixture reflects the activity of self-made SA.Determination of pI of self-made SA was carried out withcellulose acetate membrane electrophoresis.2 Improvement of fermentation conditions for StreptomycesL-183①Preparation of spore suspending fluid and seed culture ofL-183 proceeded with a method described by Song SX et al..Improvement of fermentation conditions was firstly performedin several groups of experiment, which respectively aimed toselected essential carbon source, essential nitrogen source,secondary carbon or nitrogen source, range of concentration ofsodium chloride, range of concentration of primary carbon andnitrogen source. Thereafter five factors, including starch, soycake meal, glucose, sodium chloride, shaking speed, was laid onthree levels and arranged into orthogonal experiment which wasrepeated twice. The culture liquid was sampled from 7 day to 10day after inoculating, when the relative activity of SA reached aplateau. After fermentation culture, the relative activity ofsamples of every group was detected and the highest one waskept down. According to the results of the orthogonalexperiment, a best culture medium was prepared to conductflask fermentation which repeated once. Linear regressionequation was deduced from the standard curve of relativeactivity of SA to calculate the highest production amount. ②The relative activity of SA in the culture liquid was determinedwith a three-step method. The SA sample was mixed with thebiotin-labeled horseradish peroxidase liquid to form a complex.Than the mixture was added into wells of microtiter plate, whichhad been coated by the rabbit anti-SA serum, and anantibody-SA-enzyme complex was brought into existence.Again the substrate buffer was instilled and reacted in dark.After the reaction was terminated, the absorbance at 492nm wasread and the absorbance average of every sample (two wells persample) was treated as a reflection of production amount of SA.Results1 Identification of SA①Both the elution curve and the relative activity curve ofself-made SA sample exhibited one absorbance peak, and thesetwo curve are in the thoroughly same trend. In addition, it couldbe seen from SDS-PAGE that there existed only one SA band.So self-made SA had been purified at an affinitychromatography degree. ②the molecule weight of self-madeSA lay between 66kD and 97kD. It was determined to be76.8kD and approximated to 75kD of original SA described bydocuments and larger than Promega core SA. ③After beingtreated by proteinase K, a new band ,whose molecular weightwas similar with that of core SA, was brought out. It indicatedthat self-made SA had been converted into core SA. ④Because2.5mg self-made SA was saturated with 28μl 1μg/μl biotinsolution, the activity of self-made SA was 11.2u/mg and lowerthan 14u/mg of biotin-free SA and indicated self-made SA waspartly saturated by biotin. ⑤The pI of self-made SA wasaround 7.4 and slightly lower than 7.5 of biotin-free SAdescribed by documents. ⑥After self-made SA being saturatedwith biotin, the absorbance at 233nm was obviously amplifiedas described by documents.2 Improvement of fermentation conditions for StreptomycesL-183①The relative activity of SA in the culture medium 1A(0.581±0.108) consisting of starch was significantly increasedcompared to control (0.380±0.100) and 1B (0.466±0.115) (р﹤0.05). It indicated starch was the desirable essential carbonsource. ②The relative activity of SA in 2A (0.581±0.108)consisting of soy cake meal was significantly increasedcompared to control (0.249±0.075) and 2B (0.435±0.112) (р﹤0.05). It indicated soy cake meal was the desirable essentialnitrogen source. ③The relative activity of SA in 3B(0.599±0.112) consisting of glucose was significantly increasedcompared to control (0.464±0.107), 3A (0.407±0.102) and 3C(0.385±0.099) (р﹤0.05). So glucose was selected as secondarycarbon source. ④The relative activity of SA in 4A(0.565±0.091) consisting 5g/L sodium chloride was significantlyincreased compared to the others (р﹤0.05). There was nostatistical difference in the relative activity of SA between 4B(0.498±0.077) consisting 10g/L sodium chloride and control(0.445±0.067) without odium chloride. But the relative activityof SA in both was significantly increased compared to 4C(0.089±0.016) consisting 20g/L sodium chloride and 4D(0.078±0.019) consisting 30g/L sodium chloride (р﹤0.05).Three sodium chloride levels of 0g/L, 5g/L, 10g/L werearranged into the orthogonal experiment. ⑤There was nostatistical difference in the relative activity of SA among 5A(0.488±0.115) consisting of starch 10g/L, soy cake meal 10g/L,5B (0.604±0.108) consisting of 20g/L, 20g/L, 5C (0.593±0.105)consisting of 40g/L, 40g/L. But the relative activity of SA in 5A,5B, 5C was significantly increased compared to 5D(0.420±0.091) consisting of 60g/L, 60g/L and 5E (0.390±0.109)consisting of 80g/L, 80g/L (р﹤0.05). Three starch and soy cakemeal levels of 10g/L, 10g/L; 20g/L, 20g/L; 40g/L, 40g/L werearranged into the orthogonal experiment. ⑥The results of theorthogonal experiment showed the relative activity of SA wassignificantly affected by soy cake meal (р﹤0.05) and glucose (р﹤0.01). Also pH of culture medium was significantlyinfluenced by soy cake meal (р﹤0.0001), glucose (р﹤0.05)and shaking speed (р﹤0.05). The best composition of culturemedium was A3B3C1D1E3, that is, starch 40g/L, soy cake meal40g/L, glucose 5g/L, sodium chloride 5g/L, shaking speed...
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